Hydrogen production by fermentation of a composite microbial inoculant and applications thereof

Abstract

The application provides a composite microbial inoculum for hydrogen production by fermentation and application thereof. The composite microbial inoculum comprises Priestia aryabhattai YLS2, Niallia nealsonii YLS4 and Lysinibacillus boronitolerans S5; wherein the preservation number of the Priestia aryabhattai YLS2 is CGMCC NO.32138, the preservation number of the Niallia nealsonii YLS4 is CGMCC NO.32137, and the preservation number of the Lysinibacillus boronitolerans S5 is CGMCC NO.32139. The hydrogen production process is optimized by the synergistic effect of multiple microorganisms, the composite microbial inoculum for hydrogen production by fermentation can improve the hydrogen production and the stability in the gas production process, and has significant environmental benefits and economic potential.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound bacterial agent for hydrogen production through fermentation and its application. Background Technology

[0002] Existing hydrogen production methods mainly fall into three categories: hydrogen production from fossil fuel reforming, hydrogen production from industrial by-products, and hydrogen production from renewable energy combined with water electrolysis. While these technologies address hydrogen demand to some extent, they also consume significant amounts of energy. Biohydrogen production, a technology utilizing biomass and microorganisms to produce hydrogen, holds great promise as a hydrogen energy production method. The biohydrogen production process produces virtually no greenhouse gases or harmful substances, contributing to climate change mitigation. Furthermore, by utilizing waste materials to produce hydrogen, it promotes resource recycling and reduces environmental burden. Microbial fermentation is an important form of biohydrogen production; therefore, selecting and constructing suitable and efficient microbial populations is a crucial research direction for those skilled in the art.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a compound microbial agent for hydrogen production through fermentation and its application, which can improve hydrogen production and stability during the gas production process.

[0005] This invention provides a compound microbial agent for hydrogen production through fermentation, comprising *Priestiaaryabhattai* YLS2, *Niallia nealsonii* YLS4, and boron-resistant lysine-tolerant *Lysinibacillus boronitolerans* S5; wherein *Priestiaaryabhattai* YLS2 has the accession number CGMCC NO.32138, *Niallia nealsonii* YLS4 has the accession number CGMCC NO.32137, and boron-resistant lysine-tolerant *Lysinibacillus boronitolerans* S5 has the accession number CGMCC NO.32139.

[0006] The taxonomous species *Priestia aryabhattai* YLS2 was selected from a water sample of Yanqi Lake in Huairou District, Beijing. It was deposited on September 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 32138.

[0007] The taxonomous bacillus YLS4, named Niallia nealsonii, was screened from water samples of Yanqi Lake in the Yanqi Lake Scenic Area, Huairou District, Beijing. It was deposited on September 30, 2024, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32137.

[0008] The boron-tolerant lysine-resistant Bacillus S5, classified as *Lysinibacillus boronitolerans*, was screened from anaerobic activated sludge from a wastewater treatment plant in Huairou District, Beijing, and deposited on September 30, 2024, at the China General Microbiological Culture Collection Center (CGMCC), No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 32139.

[0009] This invention does not impose strict limitations on the viable counts of *Primatex auriculata* YLS2, *Bacillus niger* YLS4, and boron-resistant lysine-tolerant *Bacillus niger* S5 in the compound bacterial agent. For example, the ratio of viable counts of *Primatex auriculata* YLS2, *Bacillus niger* YLS4, and boron-resistant lysine-tolerant *Bacillus niger* S5 in the compound bacterial agent can be 1:(0.8-1.2):(0.8-1.2).

[0010] This invention does not impose strict restrictions on the compounding method of compound microbial agents. *Primatex auriculata* YLS2, *Bacillus niger* YLS4, and *Bacillus boron-resistant lysine-tolerant* S5 can exist in single microbial agent form or in mixed microbial agent form.

[0011] The present invention also provides a method for hydrogen production by fermentation, which uses the above-mentioned compound bacterial agent. The method includes: inoculating Precipitella aristata YLS2, Bacillus niger YLS4 and boron-resistant lysine-tolerant Bacillus spp. S5 into a hydrogen production medium, anaerobic treating the inoculated hydrogen production medium, and then carrying out hydrogen production by fermentation under anaerobic and light-free conditions.

[0012] Furthermore, prior to inoculation, the following steps were taken: activation culture of *Primatex auriculata* YLS2, *Bacillus niger* YLS4, and boron-resistant lysine-tolerant *Bacillus* S5, respectively.

[0013] Specifically, 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; the activation culture temperature was 28-37℃, and the activation culture time was 24-48 h.

[0014] Anaerobic treatment includes: purging with nitrogen gas until oxygen is completely removed from the hydrogen-producing medium and then sealing it; there is no strict limit to the nitrogen purging time, as long as oxygen can be completely removed from the hydrogen-producing medium, the purging time is, for example, 5-8 minutes.

[0015] The hydrogen-producing medium consists of: 8-12 g / L anhydrous glucose, 4-6 g / L beef extract, 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 of 7±0.2; the fermentation temperature is 28-37℃, preferably 36-37℃; and the fermentation time is 12-240 h, preferably 48-96 h.

[0016] In the above-described fermentation hydrogen production method of the present invention, the hydrogen production amount per 100 mL of hydrogen-producing medium is 7-35 mL, preferably 20-30 mL; the mixed gas production is 11-62 mL, preferably 33-51 mL; and the volume percentage of hydrogen in the mixed gas is 52-61%.

[0017] The compound microbial agent of this invention is composed of different types of hydrogen-producing strains. This compound microbial agent can produce hydrogen through fermentation using organic matter as a substrate under specific nutrient conditions and temperatures. By adjusting the culture conditions of different strains during fermentation, such as pH, temperature, culture time, and substrate concentration, hydrogen production can be significantly increased while ensuring a stable gas production process. The compound microbial agent of this invention and its application not only improve hydrogen production efficiency but also reduce the emission of organic waste, exhibiting significant environmental benefits and economic potential. It is suitable for large-scale industrial hydrogen production, wastewater treatment, and the comprehensive utilization of agricultural waste. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The images show the plate growth of the strains; (a) is Precipitella aristata YLS2, (b) is Bacillus niger YLS4, and (c) is Boron-resistant Lysine-tolerant Bacillus S5.

[0020] Figure 2 The trends of cumulative mixed gas production, cumulative hydrogen production, and hydrogen percentage over time are shown.

[0021] Figure 3A graph showing the increment of mixed gas production at different times;

[0022] Figure 4 This is a graph showing the increase in hydrogen production at different times. Detailed Implementation

[0023] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. 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 application pertains.

[0024] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this description, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1

[0027] I. Compound microbial agents

[0028] The compound bacterial agent in this embodiment consists of Priestia aryabhattai YLS2, Niallia nealsonii YLS4 and Lysinibacillus boronitolerans S5; strain information is shown in Table 1.

[0029] Table 1. Strain Information

[0030]

[0031]

[0032] II. Preparation of Culture Medium

[0033] Prepare LB agar medium according to the composition in Table 2, and prepare hydrogen production medium (pH 7 ± 0.2) according to the composition in Table 3.

[0034] Table 2 Composition of LB agar medium

[0035] composition concentration trypsin 10g / L Yeast extract 5g / L Sodium chloride 10g / L Agar 15-20g / L

[0036] Table 3 Hydrogen-producing culture medium

[0037] composition concentration Anhydrous glucose 10g / L Beef 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

[0038] III. Fermentation for Hydrogen Production

[0039] Precipitella aristata YLS2, Bacillus niger YLS4, and boron-resistant lysine-tolerant Bacillus spp. S5 were inoculated into LB agar medium for activation culture at a temperature of 28-37℃ for 24-48 hours.

[0040] Wait until healthy, clear, single, and uniformly grown colonies appear on the LB agar medium (e.g. Figure 1 As shown in the figure, single, pure colonies were gently inoculated into hydrogen-producing medium using a sterile inoculation loop, with a culture volume of 100 mL. The reaction system was anaerobic by purging with 99.9% nitrogen gas for 5-8 minutes to ensure complete removal of oxygen from the bottle. After purging, the bottle was immediately sealed with a rubber stopper. The anaerobic bottle was tightly wrapped with aluminum foil to block light, ensuring the compound bacterial agent reacted under anaerobic and light-free conditions. The culture temperature was adjusted to 37℃ using an incubator. During the reaction period, gas samples were taken at 12h, 24h, 36h, 48h, 60h, 72h, 96h, 120h, 144h, 168h, 192h, 216h, and 240h, and the gas yield and proportion were analyzed using gas chromatography.

[0041] IV. Testing

[0042] Quantitative analysis of the gas was performed using a gas chromatograph (GC-7860D) equipped with a thermal conductivity detector (TCD) column, manufactured by Shanghai Youke Instrument Co., Ltd. Before starting the detection, the baseline needed to be leveled to ensure the accuracy of the results. 1 mL of the mixed gas was drawn using a 1 mL syringe and injected into the TCD detection port of the gas chromatograph to begin detection. The detection was stopped when the peak line completely disappeared and the baseline remained horizontal, and then analyzed using gas chromatography. The results are shown in Table 4. Figures 2-4 .

[0043] Table 4. Cumulative Mixed Gas Production, Cumulative Hydrogen Production, and Hydrogen Percentage

[0044]

[0045]

[0046] The data analysis is as follows:

[0047] 1) Analyze the cumulative mixed gas production:

[0048] The largest increase occurred in the early stage of the reaction, indicating that the mixed microbial community could rapidly degrade organic matter and produce gas; subsequently, the increase gradually decreased and tended to stabilize, indicating that the rate of increase in the system's yield had reached a relatively stable state.

[0049] 2) Analysis of cumulative hydrogen production:

[0050] Hydrogen production increased continuously over time, showing a rapid initial increase followed by a gradual stabilization. The largest increase (8.81 mL) was observed between 12 and 24 hours, after which the increase gradually decreased and stabilized (approximately 0.7 mL). This was attributed to the high concentrations of organic matter and nutrients in the initial stage of the system, which kinetically drove the reaction rate. Temperature and pressure parameters were at their optimal levels, promoting rapid hydrogen formation. In the middle stage, the main reactants were gradually consumed, causing the reaction rate to decrease. In the later stage, as reactant consumption neared completion, the system entered the final reaction phase, and pressure and temperature conditions within the system ceased to change, indicating that hydrogen production was approaching saturation.

[0051] 3) Analyze the percentage of hydrogen:

[0052] In the initial stage, reactants are consumed rapidly, resulting in a high hydrogen production rate and a temporary increase in the hydrogen percentage. The hydrogen proportion in the mixed gas is high, with noticeable fluctuations in percentage initially. As time progresses, other gases (such as carbon dioxide or methane) increase, diluting the relative proportion of hydrogen. The system gradually approaches saturation, and the hydrogen production rate decreases, but the total cumulative hydrogen volume continues to rise. In the later stage (144-240 hours), the decrease in the hydrogen percentage stabilizes, indicating that the mixed gas composition of the system has approached dynamic equilibrium.

[0053] In summary, the compound microbial agent for hydrogen production by fermentation of the present invention and its application optimize the hydrogen production process through the synergistic effect of multiple microorganisms. By rationally designing and optimizing the composition of the microbial agent, fermentation conditions and related production processes, hydrogen production can be effectively increased. It can be widely used in energy, environmental protection and other fields, and has broad market prospects and application value.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 compound microbial agent for hydrogen production through fermentation, characterized in that, Including Primate ( Priestia aryabhattai YLS2, Bacillus niger ( Niallia nealsonii YLS4 and boron-resistant lysine-tolerant Bacillus ( Lysinibacillus boronitolerans S5; among them, *Primatex auriculata* YLS2, *Bacillus niger* YLS4, and *Bacillus boron-lysine-resistant* S5 were all deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 30, 2024. The accession number of *Primatex auriculata* YLS2 is CGMCC NO. 32138, the accession number of *Bacillus niger* YLS4 is CGMCC NO. 32137, and the accession number of *Bacillus boron-lysine-resistant* S5 is CGMCC NO. 32139. The ratio of viable counts of *Primatex auriculata* YLS2, *Bacillus niger* YLS4, and *Bacillus boron-lysine-resistant* S5 in the compound microbial agent is 1:(0.8-1.2):(0.8-1.2).

2. A method for hydrogen production through fermentation, characterized in that, The method for hydrogen production by fermentation using the compound microbial agent described in claim 1 includes: inoculating *Primatex auriculata* YLS2, *Bacillus niger* YLS4, and boron-resistant lysine-tolerant *Bacillus lysine* S5 into a hydrogen production medium, subjecting the inoculated hydrogen production medium to anaerobic treatment, and then carrying out hydrogen production by fermentation under anaerobic and light-free conditions.

3. The fermentation method for hydrogen production according to claim 2, characterized in that, Prior to inoculation, the following steps were taken: activation culture of *Primus aestivus* YLS2, *Bacillus nisei* YLS4, and boron-resistant lysine-tolerant *Bacillus* S5, respectively.

4. The fermentation method for hydrogen production according to claim 3, characterized in that, Activation was performed using LB medium; the composition of LB medium included: 8-12 g / L tryptone, 4-6 g / L yeast extract, and 8-12 g / L sodium chloride.

5. The fermentation method for hydrogen production according to claim 3, characterized in that, The activation culture temperature is 28-37 ℃, and the activation culture time is 24-48 h.

6. The fermentation method for hydrogen production according to claim 2, characterized in that, Anaerobic treatment includes: purging with nitrogen until oxygen is completely removed from the hydrogen-producing medium and then sealing it.

7. The fermentation method for hydrogen production according to claim 2, characterized in that, The hydrogen-producing medium consists of: 8-12 g / L anhydrous glucose, 4-6 g / L beef extract, 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.

8. The fermentation method for hydrogen production according to claim 2, characterized in that, The fermentation temperature is 28-37 ℃, and the fermentation time is 12-240 h.

9. The fermentation method for hydrogen production according to claim 2, characterized in that, The hydrogen production rate during fermentation is 7-35 mL per 100 mL of hydrogen-producing medium.

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