Method for producing methane by using artificial mixed bacteria assisted fermentation

Abstract

The invention discloses a method for producing methane by using artificial mixed bacteria assisted fermentation, and relates to the technical field of biogas fermentation processes, and the method comprises the following steps: selecting a fermentation substrate and an inoculum; mixing the fermentation substrate and the inoculum with animal urine to establish a liquid fermentation system; carrying out flora screening by utilizing the liquid fermentation system to obtain an artificial mixed inoculant; and adding the artificial mixed inoculant into the liquid fermentation system, and carrying out anaerobic fermentation to obtain methane. According to the method, the liquid fermentation system is used for carrying out flora screening to obtain the artificial mixed inoculant, and then the artificial mixed inoculant is added into the screened liquid fermentation system for anaerobic fermentation to obtain methane, so that the composition of inoculated bacterial colonies is effectively simplified, and the purpose of reducing inter-bacterial substrate and ecological niche competition is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of biogas fermentation technology, and in particular to a method for producing methane by utilizing artificial mixed bacteria-assisted fermentation. Background Art

[0002] Corn stalks are a high-quality biomass energy. If they are used for energy, they can reduce the consumption of fossil energy such as coal and oil to a certain extent, and can also reduce carbon dioxide emissions. In addition, using crop waste for anaerobic digestion to produce methane is a clean and efficient way to use corn stalks for energy. At the same time, with the development of my country's animal husbandry and the improvement of large-scale farms, the manure produced by livestock and poultry has increased, which has led to an increasingly prominent agricultural pollution problem. Therefore, how to effectively combine agricultural waste with manure and produce methane has great application potential and value.

[0003] At present, it is common to use mixed bacterial systems such as livestock manure and activated sludge to synthesize methane. However, although mixed bacterial systems have strong methane synthesis capabilities, they also have problems that need to be solved urgently, such as redundancy of microbial flora, strong competition for substrates, and interference with metabolic pathways. Studies have shown that microbial methane production often requires the use of metabolites of other strains as electron donors, so mixed bacterial methane synthesis is a mutualistic production model between bacteria. Summary of the invention

[0004] The invention simplifies the composition of inoculated bacterial colonies through a directed evolution strategy to solve the problems of redundant microbial flora, large substrate competition and interference of metabolic pathways in the prior art of methane synthesis using mixed bacterial systems such as livestock manure and activated sludge.

[0005] The technical solution of the present invention is: A method for producing methane by artificial mixed bacteria-assisted fermentation comprises the following steps: Select fermentation substrate and inoculum; mixing the fermentation substrate and the inoculum with animal urine to establish a liquid fermentation system; Using the liquid fermentation system to screen bacterial flora to obtain an artificial mixed bacterial inoculum; The artificial mixed bacteria inoculum is added to the liquid fermentation system to obtain methane through anaerobic fermentation.

[0006] As one of the preferred schemes, the fermentation substrate includes at least one of corn straw, rice straw, and wheat straw; the inoculant includes at least one of biogas residue, pig manure, and cow manure; and the animal urine includes mammalian urine.

[0007] As one of the preferred solutions, the steps further include: before establishing the liquid fermentation system, pre-treating the fermentation substrate with alkali; wherein, The reaction temperature of the pretreatment is 120-140° C., and the reaction time is 25-35 min.

[0008] As one of the preferred solutions, the liquid fermentation system is biogas residue-alkali pretreated corn straw-pig urine.

[0009] As one of the preferred embodiments, the artificial mixed bacterial inoculum includes Cronobacter sp. , Bacteroides sp. , Enterobacter sp. and Enterococcus sp. At least one of; Among them, the genus Enterobacter sp. The name of the strain is (Enterobacter), the depository is (General Microbiology Center of China Microbiological Culture Collection Administration), the deposit address is (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the deposit date is (August 26, 2024), and the deposit registration number is (CGMCCNO.31757); Belongs to the genus Enterococcus sp. The strain name is (Enterococcus), the depository is (General Microbiology Center of China Microbiological Culture Collection Administration), the deposit address is (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the deposit date is (August 26, 2024), and the deposit registration number is (CGMCC NO.31758); Belongs to the genus Cronobacter sp. The name of the strain is (Cronobacter), the depository is (General Microbiology Center of China Microbiological Culture Collection Administration), the deposit address is (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the deposit date is (August 26, 2024), and the deposit registration number is (CGMCCNO.31759); Belongs to the genus Bacteroides sp. The name of the strain is (Bacteroides), the preservation unit is (General Microbiology Center of China Microbiological Culture Collection Administration), the preservation address is (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the preservation date is (August 26, 2024), and the preservation registration number is (CGMCC NO.31760).

[0010] As one of the preferred solutions, the liquid fermentation system for bacterial screening includes one of the first liquid fermentation system, the current liquid fermentation system and the previous liquid fermentation system for producing methane.

[0011] As one of the preferred solutions, the liquid fermentation system to which the artificial mixed bacterial inoculum is added and the liquid fermentation system for bacterial flora screening belong to the same system.

[0012] As one of the preferred solutions, the liquid fermentation system to which the artificial mixed bacterial inoculum is added is established under the same system conditions as those in the liquid fermentation system for bacterial population screening.

[0013] As one of the preferred solutions, the biogas residue includes the remaining substrate of the previous liquid fermentation system for producing methane.

[0014] As one of the preferred solutions, in the liquid fermentation system, the volume proportions of the biogas residue and corn straw are 1-3% and 17-19% respectively, the inoculation volume of the artificial mixed inoculum is 1-3 mL, and the OD in the liquid fermentation system after inoculation is 600 It is 0-5 (excluding 0), and the cycle of anaerobic fermentation is 15-20 days.

[0015] Compared with the prior art, this application has the following advantages: The embodiment of the present invention first optimizes the type of inoculum in the methane liquid fermentation process to obtain a liquid fermentation system with excellent performance, and then uses the optimized liquid fermentation system to screen the bacterial community to obtain an artificial mixed bacterial inoculum, and then adds the artificial mixed bacterial inoculum to the liquid fermentation system for auxiliary liquid fermentation experiments. This process can effectively streamline the composition of the inoculated colony through a directed evolution strategy, and achieve the purpose of reducing substrate and niche competition between bacteria. By optimizing the bacterial community structure to construct a new artificial mixed bacterial system, the problems of low substrate utilization and product preparation in traditional mixed bacterial systems for methane fermentation are solved, thereby improving the relative abundance of the dominant strains in the liquid fermentation system and increasing the product synthesis capacity.

[0016] The strains screened in the present invention belong to Cronobacter sp., Bacteroides sp. , Enterobacter sp. and Enterococcus sp. The genus was added to the liquid fermentation body to assist anaerobic fermentation, and the CH4 content produced was increased by at least 4.30%. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the description of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0018] Figure 1 It is a state diagram of gas volume produced by different liquid fermentation systems in Comparative Examples 1-3 of the present invention; Figure 2This is a state diagram of methane content in the gas produced by different liquid fermentation systems in Comparative Examples 1-3 of the present invention; Figure 3 This is a comparison chart of the reproductive capacity of the strains screened in the examples of the present invention. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0020] The present application proposes a method for producing methane by artificial mixed bacteria-assisted fermentation, comprising the following steps: S1, select fermentation substrate and inoculum; S2, mixing the fermentation substrate and the inoculum with animal urine to establish a liquid fermentation system; S3, using the liquid fermentation system to screen bacterial flora to obtain an artificial mixed bacterial inoculum; S4, adding the artificial mixed bacteria inoculum into the liquid fermentation system to obtain methane through anaerobic fermentation.

[0021] In the embodiment of the present invention, different fermentation substrates and inocula can be selected to mix with animal urine to construct different liquid fermentation systems, and then by characterizing the anaerobic fermentation properties in different liquid fermentation systems, a specific type of liquid fermentation system with the strongest methane production capacity can be selected. Therefore, by optimizing the type of inoculum, an efficient fermentation system is first established, which provides a basis for subsequent bacterial screening.

[0022] Since the optimized liquid fermentation system is more conducive to the growth of methane-synthesizing microorganisms, it is easier to identify and screen the dominant bacterial flora that can effectively grow and function in the system during the bacterial flora screening process. Therefore, the liquid fermentation system selected in step S2 is used to screen the bacterial flora, and an artificial mixed bacterial inoculum with higher biomass degradation and methane synthesis capabilities is obtained in the optimized fermentation environment.

[0023] Furthermore, the liquid fermentation system is used to screen the bacterial flora to obtain an artificial mixed bacterial inoculum, including: Bacterial screening can use a liquid fermentation system to domesticate the strains, and then separate, purify and identify the microorganisms in the liquid fermentation system to screen out target strains that can effectively grow and function in the system. Further, by comparing the growth of multiple target strains screened, dominant strains with higher reproduction rates are obtained. Finally, the dominant strains with good biomass degradation and methane synthesis capabilities are randomly combined to eventually construct an artificial mixed inoculum. Subsequently, the artificial mixed inoculum is applied to additional liquid fermentation systems to verify its ability to degrade biomass and synthesize methane in actual operations.

[0024] In some embodiments, the additional liquid fermentation system can be the same type of liquid fermentation system or a different type of liquid fermentation system.

[0025] Therefore, we first optimized the inoculum type in the methane liquid fermentation process to obtain a liquid fermentation system with excellent performance, and then used the optimized liquid fermentation system to screen the bacterial community to obtain an artificial mixed inoculum. Through systematic step-by-step optimization, the artificial mixed inoculum and the constructed liquid fermentation system can achieve efficient methane fermentation. Then, the artificial mixed inoculum was added to the new liquid fermentation system for auxiliary liquid fermentation experiments, so that the final anaerobic fermentation experiment can maximize the methane production.

[0026] Preferably, the additional liquid fermentation system is a liquid fermentation system of the same type. That is, the liquid fermentation system to which the artificial mixed bacteria inoculum is added belongs to the same system as the liquid fermentation system for flora screening, and more preferably, the system is established under the same conditions. Therefore, when flora screening is performed, an efficient and stable liquid fermentation system has been established. When the screened artificial mixed bacteria inoculum is added to a new liquid fermentation system, since the new fermentation system and the system for flora screening previously belong to the same optimized system, the new system can therefore continue and superimpose the advantages brought by the previous optimization, maximize the methane production and the stability of the fermentation system, and ensure the reliability and repeatability of the results.

[0027] This example is used to illustrate a specific implementation method for obtaining a liquid fermentation system with excellent performance.

[0028] The fermentation substrate includes at least one of corn straw, rice straw, and wheat straw, preferably corn straw; the inoculant includes at least one of biogas residue, pig manure, and cow manure; and the animal urine includes mammalian urine.

[0029] Specifically, corn straw is rich in cellulose and hemicellulose and is a good raw material for bioenergy production. Rice straw contains high organic matter and silicates and can be used as a fermentation substrate. Wheat straw also contains rich cellulose and hemicellulose and is suitable for anaerobic fermentation.

[0030] Of course, in other embodiments, the choice of fermentation substrate is not limited to the above three types, and can also be corn cobs, etc. These crop straws are usually used as agricultural waste after harvest. If they are not used, they will burden the environment. Using them as fermentation substrates can achieve the recycling of resources.

[0031] Among them, biogas residue is the solid residue from biogas fermentation, which contains a variety of microorganisms and incompletely decomposed organic matter, and can be used as an inoculum to provide microbial populations. Pig manure and cow manure contain a large amount of organic matter and microorganisms, and are used as common inoculants.

[0032] The present invention first optimizes the type of inoculant in the methane liquid fermentation process and establishes liquid fermentation systems corresponding to different inocula. And by characterizing the fermentation properties exhibited by different liquid fermentation systems, it is obtained that the methane production content in the liquid fermentation system when the biogas residue is used as the inoculant is higher than that in other liquid fermentation systems. Therefore, the preferred inoculant is biogas residue, and the biogas residue contains microbial communities that have undergone anaerobic digestion processes. The microbial communities have adapted to the anaerobic environment and can efficiently participate in the decomposition of organic matter and the production of methane. At the same time, the incompletely decomposed organic matter contained in the biogas residue can serve as a fermentation substrate as a solid inoculant, providing an energy source for methane production.

[0033] Mammalian urine contains urea, electrolytes and other nutrients. For example, it can be one or a combination of pig urine, cow urine and human urine, which is not limited here and is not limited to the above three types of urine. Mammalian urine can be used as a nitrogen source to promote microbial growth, while providing a liquid fermentation environment for the entire liquid fermentation system and realizing resource utilization of animal waste.

[0034] Therefore, the liquid fermentation system constructed in the embodiment of the present invention can promote the growth and metabolic activities of microorganisms, and at the same time convert agricultural waste and animal manure into renewable energy, thereby reducing the accumulation of various wastes and environmental pollution at a low cost.

[0035] In a further technical solution, the steps further include: S5. Before establishing the liquid fermentation system, the fermentation substrate is pretreated with an alkali, which may be sodium hydroxide, potassium hydroxide, etc., and is not limited here; the reaction temperature of the pretreatment is 120-140° C., and the reaction time is 25-35 min.

[0036] Specifically, pre-treating the fermentation substrate with alkali can promote the hydrolysis and conversion of organic matter in the substrate, so that it is easier for microorganisms to use it in the subsequent anaerobic fermentation process. In the anaerobic fermentation stage, microorganisms can more effectively convert organic matter into methane and other useful byproducts, and the anaerobic fermentation reaction is more thorough.

[0037] Combined with the above embodiments, the liquid fermentation system is biogas residue-alkali pretreated corn stalks-pig urine The selection criteria of biogas residue can refer to the above. Alkaline pretreatment can destroy the structure of cellulose and lignin in corn stalks, making them easier to be decomposed by microorganisms, and thus easier to be used by microorganisms in the subsequent anaerobic fermentation process. Pig urine contains nutrients such as nitrogen, phosphorus, and potassium, which can be used as a source of nutrients for microbial growth.

[0038] Therefore, by using the sludge-alkali pretreated corn straw-pig urine liquid fermentation system to domesticate the strains, and separating, purifying and identifying the microorganisms in the above system, an artificial mixed bacterial inoculum with strong methane synthesis and biomass degradation capabilities can be obtained, thereby maximizing the potential for mixed bacteria to synthesize methane.

[0039] Furthermore, the artificial mixed bacterial inoculum includes Cronobacter sp. , Bacteroides sp. , Enterobacter sp. and Enterococcus sp. At least one of; Among them, belonging to the genus Enterobacter sp. The name of the strain is (Enterobacter), the depository is (General Microbiology Center of China Microbiological Culture Collection Administration), the deposit address is (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the deposit date is (August 26, 2024), and the deposit registration number is (CGMCCNO.31757); Belongs to the genus Enterococcus sp. The strain name is (Enterococcus), the depository is (General Microbiology Center of China Microbiological Culture Collection Administration), the deposit address is (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the deposit date is (August 26, 2024), and the deposit registration number is (CGMCC NO.31758); Belongs to the genus Cronobacter sp. The name of the strain is (Cronobacter), the depository is (General Microbiology Center of China Microbiological Culture Collection Administration), the deposit address is (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the deposit date is (August 26, 2024), and the deposit registration number is (CGMCCNO.31759); Belongs to the genus Bacteroides sp.The name of the strain is (Bacteroides), the preservation unit is (General Microbiology Center of China Microbiological Culture Collection Administration), the preservation address is (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the preservation date is (August 26, 2024), and the preservation registration number is (CGMCC NO.31760).

[0040] In this embodiment, the above-mentioned strains are all extracted in a liquid fermentation system, and therefore are strains related to biomass degradation and methane synthesis and have a relatively high reproduction rate.

[0041] Among them, Cronobacter can be used for biomass degradation and methane synthesis during fermentation, and has strong metabolic capacity and tolerance. Strains of the genus Bacteroides can effectively decompose complex organic matter, such as cellulose and hemicellulose. Strains of the genus Enterobacter can utilize a variety of carbon sources during fermentation, including sugars and organic acids in biomass, which helps to increase methane production in anaerobic fermentation. Strains of the genus Enterococcus can produce a variety of enzymes during fermentation, which helps to degrade biomass.

[0042] Furthermore, the liquid fermentation system for bacterial screening includes one of the first liquid fermentation system, the current liquid fermentation system and the previous liquid fermentation system for producing methane.

[0043] It should be noted that the first liquid fermentation system is the initially established liquid fermentation system for bacterial screening. In the initial fermentation process, after the superior bacterial colonies that perform well under specific fermentation conditions are screened out, the artificial mixed bacterial inoculum composed of the superior bacterial colonies can be directly used for each subsequent fermentation test. Therefore, in each subsequent liquid fermentation system for anaerobic fermentation, the obtained artificial mixed bacterial inoculum can be used, and no additional bacterial colony screening is required, that is, step S3 is omitted.

[0044] The previous liquid fermentation system for producing methane includes one or more of the previous liquid fermentation system for producing methane, the second liquid fermentation system for producing methane, the Nth liquid fermentation system for producing methane, etc. In the current fermentation process, the existing (the previous liquid fermentation system for producing methane) or the liquid fermentation system being established (the current liquid fermentation system) can be used for multiple rounds of artificial screening to obtain a new artificial mixed bacterial inoculum, which is convenient to source, saves cost and time, and is easy to be consistent with the newly established liquid fermentation system that needs to add the artificial mixed bacterial inoculum later.

[0045] As a preferred embodiment of the present invention, the liquid fermentation system to which the artificial mixed bacterial inoculum is added belongs to the same system as the liquid fermentation system for bacterial screening. That is to say, the two liquid fermentation systems use the same substrate, such as corn stalks, rice stalks, wheat stalks, etc., as fermentation raw materials. The same inoculum, such as biogas residue, pig manure, cow manure, etc., is used as a source of microorganisms. The same animal urine is used, such as pig urine, cow urine, horse urine, etc.

[0046] Exemplarily, if the liquid fermentation system to which the artificial mixed bacterial inoculum is added is biogas residue-alkali pretreated corn stalks-pig urine, then the liquid fermentation system for bacterial flora screening should also be biogas residue-alkali pretreated corn stalks-pig urine. In other words, if the liquid fermentation system to which the artificial mixed bacterial inoculum is added is pig manure-alkali pretreated corn stalks-pig urine, then the liquid fermentation system for bacterial flora screening should also be pig manure-alkali pretreated corn stalks-pig urine.

[0047] Therefore, two liquid fermentation systems of the same type are used for bacterial screening and anaerobic fermentation respectively. The fermentation environment is consistent, which reduces the competition of non-target microorganisms. The artificial mixed inoculum can adapt quickly and exert the best effect, so that the screened dominant bacterial flora can show the same or even better degradation and metabolic capabilities as in the screening environment in the actual fermentation process, thereby improving the conversion efficiency and methane production of artificial mixed inoculum-assisted fermentation.

[0048] More preferably, the liquid fermentation system to which the artificial mixed bacteria inoculum is added has the same system establishment conditions as the liquid fermentation system for flora screening. When flora screening and anaerobic fermentation are respectively carried out in two identical liquid fermentation systems, when the artificial mixed bacteria inoculum screened in the liquid fermentation system is added to another identical liquid fermentation system for anaerobic fermentation, it can fully adapt to the actual anaerobic fermentation process and further improve the yield and quality of the fermentation product.

[0049] It should be noted that the establishment conditions include the type, content, concentration, volume, fermentation conditions (pH, temperature, time), etc. of each component.

[0050] According to the screening results, the screened microbial strains were randomly combined into a number of different artificial mixed inoculums, including Cronobacter sp., Bacteroides sp., Enterobacter sp. and Enterococcus sp. A combination of one or more of .

[0051] In this embodiment, the growth conditions of several screened microbial strains can be compared to obtain a dominant strain with a higher reproduction rate, and then the multiple dominant strains can be artificially combined to obtain an optimized artificial mixed inoculum.

[0052] Furthermore, in order to further improve the reuse of resources, in an embodiment of the present invention, the biogas residue includes the remaining substrate in the liquid fermentation system of the previous methane production. In this embodiment, by reusing the biogas residue, the resource utilization rate of agricultural waste and livestock and poultry manure can be improved to achieve a circular economy. At the same time, the biogas residue carries a part of the microbial flora. Although most of the active microorganisms may have completed their fermentation, there is still a part of the microbial flora in the biogas residue.

[0053] In addition, the repeated use of biogas residue as inoculum will, to a certain extent, lead to multiple iterative directed evolution of the bacterial community. Each fermentation process is actually a screening process, in which microorganisms that adapt to the fermentation environment will have an advantage in the next round of fermentation. Therefore, after repeated use, the residual bacterial community in the biogas residue gradually adapts to the specific substrate type, fermentation conditions and the production of target products, producing an artificial mixed bacterial inoculum that is more adapted to the current fermentation environment. In subsequent fermentations, the bacterial community can make more full use of these substrates for methane synthesis.

[0054] Furthermore, in the liquid fermentation system, the volume proportions of the biogas residue and corn straw are 1-3% and 17-19% respectively, the inoculation volume of the artificial mixed bacteria inoculum is 1-3 mL, and the OD in the liquid fermentation system after inoculation is 600 It is 0-5 (excluding 0), and the cycle of anaerobic fermentation is 15-20 days.

[0055] The present invention is described in detail with reference to the above embodiments.

[0056] Example 1 The embodiment of the present invention provides a method for producing methane by using artificial mixed bacteria assisted fermentation, comprising the following steps: The fermentation substrate was selected as alkali-pretreated corn straw, and the inoculum was biogas residue. The conditions of alkali pretreatment were: 10 g corn straw, 9 mL sodium hydroxide, reaction temperature 130 ℃, and reaction time 30 min. Alkali pretreated corn stover and biogas residue were mixed with pig urine to establish a liquid fermentation system with a volume of 300 mL and a fermentation period of 17 days. The solid inoculation ratio was 20%, the biogas residue ratio was 2%, and the remaining reaction system was supplemented by pig urine. The liquid fermentation system was used to domesticate the strains, and the microorganisms in the system were separated, purified and identified to screen out an artificial mixed bacterial inoculum. The screened strains were randomly combined to construct an artificial mixed bacterial system, wherein the combined artificial mixed bacterial inoculum included strain 1, strain A1, strain A2 and strain C3, strain 1 being Enterococcus sp. (Enterococcus spp.), strain A1 is Cronobacter sp. (Cronobacter spp.), strain A2 is Bacteroides sp.(Bacteroides genus), strain C3 is Enterobacter sp. (Enterobacter spp.); The artificial mixed inoculum was added to the same type of liquid fermentation system (i.e., strain 1, strain A1, strain A2 and strain C3 were added to the system at the same time) for anaerobic fermentation to obtain methane. The liquid fermentation system was 300 mL, the fermentation cycle was 17 days, and the solid inoculation ratio was 20%; the inoculation volume ratios of biogas residue and corn straw were 2% and 18%, respectively, and the inoculation volume of the above strains was 1 mL (OD 600 is 1.5).

[0057] Example 2 The embodiment of the present invention provides a method for producing methane by using artificial mixed bacteria assisted fermentation, comprising the following steps: The fermentation substrate was selected as alkali-pretreated corn straw, and the inoculum was biogas residue. The conditions of alkali pretreatment were: 10 g corn straw, 9 mL sodium hydroxide, reaction temperature 130 ℃, and reaction time 30 min. The alkali-pretreated corn stover and biogas residue (product of Example 1) were mixed with pig urine to establish a liquid fermentation system. The liquid fermentation system was 300 mL, the fermentation period was 17 days, the solid inoculation ratio was 20%, the biogas residue ratio was 2%, and the remaining reaction system was supplemented by pig urine; The liquid fermentation system was used to domesticate the strains, and the microorganisms in the system were separated, purified and identified to screen out an artificial mixed bacterial inoculum. The screened strains were randomly combined to construct an artificial mixed bacterial system, wherein the combined artificial mixed bacterial inoculum included strain 1, strain A1 and strain C3, strain 1 being Enterococcus sp. (Enterococcus spp.), strain A1 is Cronobacter sp. (Cronobacter spp.), strain C3 Enterobacter sp. (Enterobacter spp.); The artificial mixed inoculum was added to the same type of liquid fermentation system (i.e., strain 1, strain A1 and strain C3 were added to the system at the same time) for anaerobic fermentation to obtain methane. The liquid fermentation system was 300 mL, the fermentation cycle was 17 days, the solid inoculum volume ratio was 20%, the biogas residue and corn straw inoculation volume ratios were 2% and 18%, respectively, and the inoculation volume of the above strains was 1 mL (OD 600 is 1.5).

[0058] Example 3 Different from Example 1, the artificial mixed bacterial inoculum obtained by screening and combining includes strain 1, strain A2 and strain C3, strain 1 is Enterococcus sp. (Enterococcus spp.), strain A2 is Bacteroides sp. (Bacteroides genus), strain C3 is Enterobacter sp. (Enterobacter spp.).

[0059] Example 4 The difference from Example 1 is that the inoculation volume ratios of biogas residue and corn straw are 1% and 19% respectively.

[0060] Example 5 The difference from Example 1 is that the inoculation volume ratios of biogas residue and corn straw are 2% and 18% respectively, and the OD 600 is 2.

[0061] Comparative Example 1 A method for producing methane by fermentation is provided, comprising the following steps: The fermentation substrate was selected as alkali-pretreated corn straw, and the inoculum was biogas residue. The conditions of alkali pretreatment were: 10 g corn straw, 9 mL sodium hydroxide, reaction temperature 130 ℃, and reaction time 30 min. Alkali pretreated corn stover and biogas residue were mixed with pig urine to establish a liquid fermentation system with a volume of 300 mL. Methane was obtained by anaerobic fermentation with a fermentation cycle of 17 days and a solid inoculation volume ratio of 20%, of which the inoculation volume ratios of biogas residue and corn straw were 2% and 18%, respectively.

[0062] Comparative Example 2 A method for producing methane by fermentation is provided, comprising the following steps: The fermentation substrate was selected as corn stalks and the inoculum was selected as pig manure; Corn stalks, pig manure and pig urine were mixed to establish a liquid fermentation system with a volume of 300 mL; Methane was obtained by anaerobic fermentation with a fermentation cycle of 17 days and a solid inoculation volume ratio of 20%, of which the inoculation volume ratios of biogas residue and corn straw were 2% and 18%, respectively.

[0063] Comparative Example 3 A method for producing methane by fermentation is provided, comprising the following steps: The fermentation substrate was selected as corn stalks and the inoculum was selected as pig manure; Alkali pretreated corn stover and pig feces were mixed with pig urine to establish a liquid fermentation system with a volume of 300 mL; Methane was obtained by anaerobic fermentation with a fermentation cycle of 17 days and a solid inoculation volume ratio of 20%, of which the inoculation volume ratios of biogas residue and corn straw were 2% and 18%, respectively.

[0064] Test results: The gas during the fermentation process of Comparative Examples 1-3 was collected, and the methane content of the collected gas was detected. The volume change of the collected gas was as follows: Figure 1 As shown in Figure 2, the detection of methane content is as follows:Figure 2 shown.

[0065] Depend on Figure 1 It can be seen that the gas production rate in the anaerobic fermentation process of the liquid fermentation system of biogas residue-alkali pretreated corn straw-pig urine is the fastest, and the amount of gas produced is the largest after 17 days of fermentation. At 17 days of fermentation, the gas production no longer increases, indicating that the liquid fermentation body of biogas residue-alkali pretreated corn straw-pig urine can be completely reacted in an anaerobic environment for 17 days.

[0066] The difference between Comparative Example 3 and Comparative Example 1 is that the inoculant is pig manure. In comparison, the gas production rate of Comparative Example 3 is slower, and the volume of the final gas obtained is smaller, the reaction cycle is also longer, and the total amount of gas increases significantly after 17 days. Even after 31 days of reaction, the fermentation system is still reacting, indicating that the sludge as an inoculant contains microbial communities that have undergone anaerobic digestion. These microorganisms have adapted to the anaerobic environment and can improve the efficiency of the fermentation reaction.

[0067] As for Comparative Example 2, the difference from Comparative Example 3 is that the fermentation substrate is not pretreated with alkali, so the reaction efficiency is slower and the reaction thoroughness is low.

[0068] Depend on Figure 2 It can be seen that the methane content in the gas produced during the anaerobic fermentation of the liquid fermentation system of biogas residue-alkali pretreated corn straw-pig urine will gradually increase with the longer the anaerobic reaction cycle. When the fermentation cycle reaches 17 days, the methane content reaches the maximum value, and the methane content may decrease as time goes by. The methane content in the liquid fermentation system of biogas residue-alkali pretreated corn straw-pig urine is always the highest during the fermentation process, and its maximum methane content is 3.6%.

[0069] Compared with Example 1, in Example 2 and Example 3, not only the total amount of gas produced by fermentation is small, but also the methane content is relatively low, indicating that the sludge can be used as an inoculum to improve the methane conversion efficiency. The final methane content in Example 2 and Example 3 reaches a relatively constant state, indicating that pretreatment can improve the efficiency and thoroughness of the fermentation reaction.

[0070] Based on the liquid fermentation system of Comparative Example 1, strain screening is performed, and the screened artificial mixed bacteria inoculum is added to the liquid fermentation system for anaerobic fermentation, which is the scheme of Examples 1 to 5 of the present invention.

[0071] In an embodiment of the present invention, a biogas residue-alkali pretreated corn straw-pig urine liquid fermentation system is used to screen strains, and the microorganisms are separated, purified and identified; the screening method and principle are the same as the existing microbial screening technology; 7 strains related to biomass degradation and methane synthesis are screened out, namely strain 1, strain 2, strain A1, strain A2, strain B1, strain B2 and strain C3, and the above strains are tested for reproduction rate. The testing method includes culturing the 7 strains in a suitable culture medium to obtain an active microbial population, estimating the bacterial concentration by measuring the absorbance of the culture solution at a specific wavelength (600 nanometers) to reflect the growth of each strain, centrifuging the culture solution, removing the supernatant, and drying and weighing the precipitated bacteria to determine the biomass of the bacteria, and during the culture process, sampling is regularly taken and the number of microorganisms is determined, and then the growth curve of the microorganism is plotted with time (days) as the horizontal axis and OD value as the vertical axis.

[0072] The results are as follows Figure 3 As shown. Figure 3 It can be seen that by comparing the growth of the above strains, strain 1, strain A1, strain A2 and strain C3 have a higher reproduction rate. At the same time, the identification of strain 1, strain A1, strain A2 and strain C3 shows that strain 1 is Enterococcus sp. (Enterococcus spp.), strain A1 is Cronobacter sp. (Cronobacter spp.), strain A2 is Bacteroides sp. (Bacteroides genus), strain C3 is Enterobacter sp. (Enterobacter genus). Therefore, after obtaining the dominant strains, the four dominant strains are artificially combined to obtain an optimized artificial mixed bacterial inoculum.

[0073] The difference between Example 1, Example 2 and Example 3 is that the strain composition of the artificial mixed inoculum is different, wherein the artificial mixed inoculum of Example 1 includes strain 1, strain A1, strain A2 and strain C3, the artificial mixed inoculum of Example 2 includes strain 1, strain A1 and strain C3, and the artificial mixed inoculum of Example 3 includes strain 1, strain A2 and strain C3.

[0074] The methane content of the gas obtained by the final fermentation of Examples 1-3 was detected. The methane content of Example 1 was 4.30%, the methane content of Example 2 was 1.05%, and the methane content of Example 3 was 0.22%. By comparing Example 1 with the comparative example, it can be seen that the artificial mixed bacteria combination method adopted in the embodiment of the present invention can improve the conversion efficiency of methane. By comparing Example 1 with Example 2 and Example 3, it can be seen that the methane content is significantly improved.

[0075] The difference between Example 4 and Example 1 is that the inoculation volume ratio of biogas residue and corn straw in the liquid fermentation system is different, and the difference between Example 5 and Example 1 is that the inoculation amount of artificial mixed bacteria inoculated in the liquid fermentation system is different.

[0076] The methane content of the gases prepared in Example 4 and Example 5 was tested respectively. The test results showed that the methane content of Example 4 was 3.65%, and the methane content of Example 5 was 5.08%. By comparing Example 4 with Example 1, it can be seen that a reduction in the inoculum content can easily lead to a decrease in the methane synthesis efficiency. By comparing Example 5 with Example 1, it can be seen that increasing the number of microbial inoculations can promote methane accumulation.

[0077] It should be noted that the various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0078] It should also be noted that, in this article, the orientation or position relationship indicated by the terms "upper", "lower", "left", "right", "inside", "outside", etc. is based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation of the present invention. In addition, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations, nor can they be understood as indicating or implying relative importance. Moreover, the term "include" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device.

[0079] The above is a detailed introduction to the method for producing methane by artificial mixed bacteria assisted fermentation provided by the present application. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the present application, and the content of this specification should not be understood as a limitation on the present application. At the same time, for those of ordinary skill in the art, according to the present application, there will be different forms of changes in the specific implementation methods and application scopes. It is not necessary and impossible to list all the implementation methods here, and the obvious changes or modifications derived therefrom are still within the scope of protection of the present application.

Claims

1. A method for producing methane using artificial mixed bacteria assisted fermentation, It is characterized in that The following steps are involved: Select fermentation substrate and inoculum; mixing the fermentation substrate and the inoculum with animal urine to establish a liquid fermentation system; Using the liquid fermentation system to screen bacterial flora to obtain an artificial mixed bacterial inoculum; The artificial mixed bacteria inoculum is added to the liquid fermentation system to obtain methane through anaerobic fermentation.

2. A method for producing methane by artificial mixed bacteria assisted fermentation according to claim 1, It is characterized in that The fermentation substrate includes at least one of corn straw, rice straw and wheat straw; the inoculant includes at least one of biogas residue, pig manure and cow manure; and the animal urine includes mammalian urine.

3. A method for producing methane by artificial mixed bacteria assisted fermentation according to claim 2, It is characterized in that The steps also include: before establishing the liquid fermentation system, pre-treating the fermentation substrate with alkali; wherein, The reaction temperature of the pretreatment is 120-140° C., and the reaction time is 25-35 min.

4. A method for producing methane by artificial mixed bacteria assisted fermentation according to claim 3, It is characterized in that The liquid fermentation system is biogas residue-alkali pretreated corn stalks-pig urine.

5. The method for producing methane by artificial mixed bacteria-assisted fermentation according to claim 1, It is characterized in that The artificial mixed bacterial inoculum includes Cronobacter sp. , Bacteroides sp. , Enterobacter sp. and Enterococcus sp. At least one of; Among them, the genus Enterobacter sp. The name of the strain is (Enterobacter), the depository is (General Microbiology Center of China Microbiological Culture Collection Administration), the deposit address is (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the deposit date is (August 26, 2024), and the deposit registration number is (CGMCC NO.31757); Belongs to the genus Enterococcus sp. The strain name is (Enterococcus), the depository is (General Microbiology Center of China Microbiological Culture Collection Administration), the deposit address is (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the deposit date is (August 26, 2024), and the deposit registration number is (CGMCC NO.31758); Belongs to the genus Cronobacter sp. The strain name is (Cronobacter), the depository is (General Microbiology Center of China Microbiological Culture Collection Administration), the deposit address is (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the deposit date is (August 26, 2024), and the deposit registration number is (CGMCC NO.31759); Belongs to the genus Bacteroides sp. The name of the strain is (Bacteroides), the preservation unit is (General Microbiology Center of China Microbiological Culture Collection Administration), the preservation address is (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the preservation date is (August 26, 2024), and the preservation registration number is (CGMCC NO.31760).

6. The method for producing methane by artificial mixed bacteria-assisted fermentation according to claim 1, It is characterized in that The liquid fermentation system for bacterial colony screening includes one of the first liquid fermentation system, the current liquid fermentation system and the previous liquid fermentation system for producing methane.

7. The method for producing methane by artificial mixed bacteria-assisted fermentation according to claim 1, It is characterized in that The liquid fermentation system to which the artificial mixed bacterial inoculum is added and the liquid fermentation system for bacterial flora screening belong to the same system.

8. A method for producing methane by artificial mixed bacteria assisted fermentation according to claim 7, It is characterized in that The liquid fermentation system to which the artificial mixed bacterial inoculum is added has the same system establishment conditions as those in the liquid fermentation system for bacterial population screening.

9. The method for producing methane by artificial mixed bacteria-assisted fermentation according to claim 4, It is characterized in that The digestate comprises the remaining substrate from a previous liquid fermentation system for producing methane.

10. The method for producing methane by artificial mixed bacteria assisted fermentation according to claim 4, It is characterized in that In the liquid fermentation system, the volume proportions of the biogas residue and corn straw are 1-3% and 17-19% respectively, the inoculation volume of the artificial mixed bacteria inoculum is 1-3 mL, and the OD in the liquid fermentation system after inoculation is 600 It is 0-5 (excluding 0), and the cycle of anaerobic fermentation is 15-20 days.

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