Synchronous carbon sequestration and denitrification method and application

Abstract

The invention provides a synchronous carbon sequestration and denitrification method and application, and belongs to the technical field of solid waste resource utilization. The invention provides a synchronous carbon sequestration and denitrification method. The method comprises the following steps: enriching microbial populations mainly containing hydrogen oxidizing bacteria in soil; microbial populations mainly containing sulfur oxidizing bacteria in the soil are enriched; microbial populations mainly containing methane-oxidizing bacteria in the soil are enriched; the methane-oxidizing bacteria enriched flora, the sulfur-oxidizing bacteria enriched flora and the hydrogen-oxidizing bacteria enriched flora are mixed and inoculated into a domestication culture medium for domestication culture, and then the mixture is inoculated into a domestication culture medium containing biogas slurry for synchronous carbon sequestration and denitrification. According to the method disclosed by the invention, a synergistic metabolism system is formed by enriching and screening a compound microbial metabolism group in which a plurality of microbial populations coexist mainly including methane-oxidizing bacteria. The synergistic metabolism system has H2S tolerance, can metabolize methane and carbon dioxide in biogas and N and P nutrient substances in biogas slurry, and achieves the purpose of synchronous carbon sequestration and denitrification.

Description

Technical Field

[0001] The present invention belongs to the technical field of solid waste resource utilization, and specifically relates to a method and application of simultaneous carbon fixation and nitrogen removal. Background Art

[0002] At present, anaerobic digestion is one of the most important methods for organic waste treatment. Anaerobic digestion is a process in which microorganisms decompose organic matter under anaerobic conditions to produce biogas. The main components of biogas are methane (CH4) and carbon dioxide (CO2), of which methane accounts for about 50%~75%, carbon dioxide accounts for about 25%~50%, and the rest are small amounts of other gases, such as hydrogen (H2), hydrogen sulfide (H2S) and trace amounts of nitrogen and oxygen. As a renewable bioenergy, the efficient use of biogas can replace the use of part of fossil energy, which is conducive to the reduction of greenhouse gas emissions. However, since biogas contains a large amount of carbon dioxide, trace amounts of hydrogen sulfide and other gas impurities, it needs to be purified before it meets the transmission requirements of the gas pipeline network. Traditional biogas utilization methods, such as power generation and fuel substitution, emit a large amount of carbon dioxide again and have limited profitability. In addition, the biogas produced by anaerobic digestion is usually discharged as sewage after drug degradation. Some biogas, such as the anaerobic digestion of livestock and poultry manure, contains a large amount of ammonia nitrogen that has not been effectively degraded or utilized. Appropriate recovery of ammonia nitrogen can effectively reduce ammonia volatilization and nitrogen oxide emissions, and avoid pollution to the atmospheric environment. In addition, ammonia nitrogen in biogas slurry can be converted into other high-value-added chemicals, such as ammonium bicarbonate, for industrial or agricultural nitrogen fertilizers through technical processing. Therefore, it is urgent to explore efficient ways to comprehensively utilize biogas and biogas slurry.

[0003] The production of microbial protein (MP) from anaerobic digestion products will become a promising approach to solve the above problems. Microbial protein refers to the production of high-protein food and feed by microorganisms. Since most microorganisms have strong adaptability to the environment (such as biogas and digestate), they can also purify pollutants while producing MP, which provides an emerging path for waste recycling and biocarbon capture. Compared with traditional protein production models (such as soybean cultivation), MP production requires less land resources and is more beneficial to human health and ecosystems. In addition, the protein content of MP is usually between 30% and 70%, depending on the microbial species and the environment. This will alleviate the global food crisis caused by excessive population growth and meet the demand for high-protein consumption in some regions. Methane and carbon dioxide in biogas can be converted into MP by microorganisms, which is a carbon sequestration process that can effectively avoid carbon dioxide emissions. Microorganisms can effectively utilize the remaining nutrients in the digestate and purify them to meet emission requirements. In recent years, bacterial production of MP has gradually been widely reported, due to its fast growth rate and rich protein content in its chemical structure, it has the ability to produce a large amount of MP. Methanogenic bacteria such as hydrogen oxidizing bacteria or methanotrophic bacteria may be suitable bacteria that can not only fill the protein gap, but also utilize methane and carbon dioxide to solve the solid waste problem caused by population growth. Both bacteria require an aerobic environment for metabolism. Hydrogen oxidizing bacteria usually require hydrogen and carbon dioxide as initial fermentation raw materials, while methanotrophic bacteria use methane as the main carbon source. Both also require a certain nitrogen source to produce microbial protein. Studies have shown that the protein content of methanotrophic bacteria is higher than that of fungi and yeast, and the digestibility of their cell walls is also better than that of algae.

[0004] However, reports indicate that H2S in biogas can significantly inhibit the growth of methanotrophic bacteria. H2S is a weak acid that can easily permeate membranes and inhibit a variety of processes, such as aerobic respiration by binding to cytochrome C oxidase. How to efficiently utilize methanotrophic bacteria to metabolize methane, carbon dioxide, and N and P nutrients in biogas or slurry to achieve the goal of simultaneous carbon fixation and nitrogen removal still faces great challenges. Summary of the invention

[0005] In view of the defects in the prior art, the object of the present invention is to provide a method for simultaneous carbon fixation and denitrification, which can efficiently utilize methanotrophic bacteria to metabolize methane, carbon dioxide and N and P nutrients in biogas or biogas slurry to achieve the purpose of simultaneous carbon fixation and denitrification.

[0006] The objective of the present invention is achieved through the following technical solutions: The present invention provides a method for simultaneous carbon fixation and nitrogen removal, comprising the following steps: The hydrogen-oxidizing bacteria mixed gas is used to enrich the microbial population dominated by hydrogen-oxidizing bacteria in the soil to obtain a hydrogen-oxidizing bacteria-enriched bacterial population; The sulfur-oxidizing bacteria mixed gas is used to enrich the microbial population dominated by sulfur-oxidizing bacteria in the soil to obtain a sulfur-oxidizing bacteria enriched bacterial population; The methane-oxidizing bacteria mixed gas is used to enrich the microbial population dominated by methane-oxidizing bacteria in the soil to obtain a methane-oxidizing bacteria-enriched bacterial population; The methane oxidizing bacteria enriched bacterial group, the sulfur oxidizing bacteria enriched bacterial group and the hydrogen oxidizing bacteria enriched bacterial group are mixed and inoculated into an acclimation medium for acclimation culture to obtain a composite microbial metabolite group; The composite microbial metabolite group is inoculated into an acclimation medium containing biogas slurry and biogas is introduced to perform simultaneous carbon fixation and nitrogen removal; The hydrogen oxidizing bacteria mixed gas comprises H2, O2 and CO2, and the volume ratio of H2, O2 and CO2 is (50-70): (30-20): (20-10); The sulfur oxidizing bacteria mixed gas includes H2, O2, CO2 and H2S, and the volume ratio of H2, O2, CO2 and H2S is (30-50): (20-30): (20-30): (20-30); The methane oxidizing bacteria mixed gas includes CH4 and O2, and the volume ratio of CH4 to O2 is (50-80): (50-20).

[0007] Preferably, the soil enriched with hydrogen oxidizing bacteria includes soil 2 to 10 cm below the surface of a water-retaining paddy field and / or compost residues of livestock and poultry manure; Soils rich in sulfur-oxidizing bacteria include 2-10 cm of soil below the surface of swamps and / or sludge from oxidation ponds in sewage treatment plants; Soils enriched with methane-oxidizing bacteria include soil 2 to 10 cm below the surface of impounded rice fields and / or sludge from oxidation ponds in sewage treatment plants.

[0008] Preferably, the method for enriching the microbial population mainly composed of hydrogen oxidizing bacteria in the soil comprises: Inoculate the microorganisms in the soil into the primary seed culture medium of hydrogen oxidizing bacteria and introduce the hydrogen oxidizing bacteria mixed gas for cultivation until the H2 consumption is more than 50% or the CO2 concentration no longer decreases, thereby obtaining the primary seed solution of hydrogen oxidizing bacteria; The hydrogen oxidizing bacteria primary seed liquid is inoculated into the hydrogen oxidizing bacteria secondary seed culture medium, and a hydrogen oxidizing bacteria mixed gas is introduced for culturing to obtain a hydrogen oxidizing bacteria enriched bacterial population; The composition of the hydrogen oxidizing bacteria primary seed culture medium includes: Na2HPO4: 2200-2800 mg / L, KH2PO4: 500-1500 mg / L, (NH4)2SO4: 450-500 mg / L, MgCl2: 45-60 mg / L, CaCl2·2H2O: 40-50 mg / L, disodium EDTA: 0.2-0.7 mg / L, FeSO4·7H2O: 0.1-0.5 mg / L, ZnSO4·7H2O: 0.01-0.02 mg / L, MnCl2·4H2O: 0.002-0.003 mg / L, H3BO3: 0.01-0.04 mg / L, CoCl2·6H2O: 0.01-0.02 mg / L, CuCl2·2H2O: 0.001-0.01 mg / L, NiCl2·6H2O: 0.002~0.004 mg / L, Na2MoO4·2H2O: 0.001~0.003 mg / L; The composition of the hydrogen oxidizing bacteria secondary seed culture medium is based on the hydrogen oxidizing bacteria primary seed culture medium and further includes: 1%~3% glucose, 0.6%~2% yeast powder and 1.5%~3.2% casein peptone.

[0009] Preferably, the method for enriching the microbial population mainly composed of sulfur-oxidizing bacteria in the soil comprises: Inoculating soil microorganisms into a primary seed culture medium for sulfur-oxidizing bacteria and introducing a sulfur-oxidizing bacteria mixed gas for culturing until the H2S concentration no longer decreases, thereby obtaining a primary seed solution for sulfur-oxidizing bacteria; The primary seed liquid of sulfur-oxidizing bacteria is inoculated into the secondary seed culture medium of sulfur-oxidizing bacteria, and a sulfur-oxidizing bacteria mixed gas is introduced for culturing to obtain a sulfur-oxidizing bacteria enriched bacterial population; The composition of the sulfur-oxidizing bacteria primary seed culture medium includes: phenol red: 0.001-0.003 g / L, 1,4-piperazine diethanesulfonic acid: 4.5-8.5 g / L, sodium chloride: 10-25.000 g / L, magnesium sulfate heptahydrate: 1.1-2.9 g / L, potassium chloride: 0.1-1 g / L, ammonium chloride: 0.015-0.3 g / L, sodium thiosulfate pentahydrate: 1.5-3.2 g / L, calcium chloride dihydrate: 0.05-0.2 g / L, magnesium chloride hexahydrate: 2.5-4.5 g / L, potassium hydrogen phosphate: 0.05-0.15 g / L, ammonium ferrous sulfate hexahydrate: 0.001-0.002 g / L, trace element solution: 1 mL / L and vitamin solution: 10 mL / L; The composition of the secondary seed culture medium for sulfur-oxidizing bacteria is based on the primary seed culture medium for sulfur-oxidizing bacteria and further includes: 1% to 3% glucose, 0.6% to 2% yeast powder and 1.5% to 3.2% casein peptone.

[0010] Preferably, the method for enriching the microbial population dominated by methanotrophic bacteria in the soil comprises: Inoculating soil microorganisms into a primary seed culture medium for methane oxidizing bacteria and introducing a mixed gas of methane oxidizing bacteria for cultivation until the CH4 concentration no longer decreases, thereby obtaining a primary seed solution for methane oxidizing bacteria; The primary seed liquid of methanotrophic bacteria is inoculated into the secondary seed culture medium of methanotrophic bacteria, and a mixed gas of methanotrophic bacteria is introduced for cultivation to obtain a methanotrophic bacteria enriched bacterial population; The composition of the primary seed culture medium of methanotrophic bacteria includes: KNO3: 100-200 mg / L, KH2PO4: 100-200 mg / L, MgSO4·7H2O: 25-55 mg / L, CaCl2·2H2O: 5-20 mg / L, EDTA: 2-5 mg / L, CuCl2·5H2O: 0.05-0.1 mg / L, FeSO4·7H2O: 1-2 mg / L, ZnSO4·7H2O: 0.1-0.2 mg / L, NiCl2·6H2O: 0.008-0.02 mg / L, CoCl2·6H2O: 0.1-0.2 mg / L and Na2MoO4: 0.01-0.03 mg / L; The composition of the secondary seed culture medium for methanotrophic bacteria is based on the primary seed culture medium for methanotrophic bacteria and further includes: 1% to 3% glucose, 0.6% to 2% yeast powder and 1.5% to 3.2% casein peptone.

[0011] Preferably, a mixed gas is introduced during the acclimation culture; the composition of the mixed gas is H2, O2, CO2, CH4 and H2S; the volume ratio of H2, O2, CO2, CH4 and H2S is (0.1~1): (10~20): (15~25): (50~60): (0.1~0.3).

[0012] Preferably, the composition of each 1L acclimatization medium includes: 0.5~1.00 g MgSO4·7H2O, 1.00~2.5 g KNO3, 0.15~0.3 g KH2PO4, 0.2~0.45 gNa2HPO4, 0.1~0.15 g CaCl2·2H2O and 1 mL trace element solution.

[0013] Preferably, the acclimation culture is continuously subcultured 3 to 4 times; the acclimation medium also includes Na2S and ammonium chloride; during the first acclimation culture, the mass concentration of Na2S in the acclimation medium is 1 to 2 mg / L, and the mass concentration of ammonium chloride is 100 to 300 mg / L; during each subculture, the mass concentration of Na2S and ammonium chloride in the acclimation medium is 1.5 to 2 times the mass concentration of Na2S and ammonium chloride in the previous culture.

[0014] The present invention provides application of the method described in the above technical solution in resource utilization of biogas slurry and / or biogas.

[0015] The present invention provides application of the method described in the above technical solution in preparing protein.

[0016] Beneficial effects of the present invention: The present invention provides a method for simultaneous carbon fixation and denitrification, comprising the following steps: enriching a microbial population dominated by hydrogen oxidizing bacteria in soil by hydrogen oxidizing bacteria mixed gas to obtain a hydrogen oxidizing bacteria enriched population; enriching a microbial population dominated by sulfur oxidizing bacteria in soil by sulfur oxidizing bacteria mixed gas to obtain a sulfur oxidizing bacteria enriched population; enriching a microbial population dominated by methane oxidizing bacteria in soil by methane oxidizing bacteria mixed gas to obtain a methane oxidizing bacteria enriched population; inoculating the methane oxidizing bacteria enriched population, the sulfur oxidizing bacteria enriched population and the hydrogen oxidizing bacteria enriched population in an acclimation medium for acclimation and culturing to obtain a composite microbial metabolic population; and inoculating the composite The microbial metabolites are inoculated into an acclimated culture medium containing biogas, and biogas is introduced to perform synchronous carbon fixation and nitrogen removal; the hydrogen oxidizing bacteria mixed gas includes H2, O2 and CO2, and the volume ratio of H2, O2 and CO2 is (50-70): (30-20): (20-10); the sulfur oxidizing bacteria mixed gas includes H2, O2, CO2 and H2S, and the volume ratio of H2, O2, CO2 and H2S is (30-50): (20-30): (20-30): (20-30); the methane oxidizing bacteria mixed gas includes CH4 and O2, and the volume ratio of CH4 and O2 is (50-80): (50-20). The method of the present invention enriches and screens a composite microbial metabolite group in which multiple microbial groups mainly composed of methane oxidizing bacteria coexist to form a synergistic metabolic system. The synergistic metabolic system has H2S tolerance, can metabolize methane and carbon dioxide in biogas and N and P nutrients in biogas, and achieves the purpose of synchronous carbon fixation and nitrogen removal. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 1 This is a diagram showing the growth of bacteria in the third round of culture in step S3 in Example 1; Figure 2 This is a graph showing the detection results of cell dry weight and protein content in step S4 in Example 1. DETAILED DESCRIPTION

[0019] The present invention provides a method for simultaneous carbon fixation and nitrogen removal, comprising the following steps: The hydrogen-oxidizing bacteria mixed gas is used to enrich the microbial population dominated by hydrogen-oxidizing bacteria in the soil to obtain a hydrogen-oxidizing bacteria-enriched bacterial population; The sulfur-oxidizing bacteria mixed gas is used to enrich the microbial population dominated by sulfur-oxidizing bacteria in the soil to obtain a sulfur-oxidizing bacteria enriched bacterial population; The methane-oxidizing bacteria mixed gas is used to enrich the microbial population dominated by methane-oxidizing bacteria in the soil to obtain a methane-oxidizing bacteria-enriched bacterial population; The enriched bacterial groups of methane oxidizing bacteria, the enriched bacterial groups of sulfur oxidizing bacteria and the enriched bacterial groups of hydrogen oxidizing bacteria are mixed and inoculated into an acclimation medium for acclimation and culture to obtain a composite microbial metabolic group; The composite microbial metabolites are inoculated into an acclimated culture medium containing biogas and biogas is introduced to perform simultaneous carbon fixation and nitrogen removal; The hydrogen oxidizing bacteria mixed gas comprises H2, O2 and CO2, and the volume ratio of H2, O2 and CO2 is (50-70): (30-20): (20-10); The sulfur oxidizing bacteria mixed gas includes H2, O2, CO2 and H2S, and the volume ratio of H2, O2, CO2 and H2S is (30-50): (20-30): (20-30): (20-30); The methane oxidizing bacteria mixed gas includes CH4 and O2, and the volume ratio of CH4 to O2 is (50-80): (50-20).

[0020] The present invention has no particular limitation on the order of enriching the microbial population dominated by hydrogen oxidizing bacteria in the soil, enriching the microbial population dominated by sulfur oxidizing bacteria in the soil, and enriching the microbial population dominated by methane oxidizing bacteria in the soil. The microbial population dominated by hydrogen oxidizing bacteria may be enriched first, or the microbial population dominated by sulfur oxidizing bacteria may be enriched first, or the microbial population dominated by methane oxidizing bacteria may be enriched first.

[0021] The limiting terms for mixed gas, culture medium or culture in the following technical scheme of the present invention have no limiting effect on the corresponding mixed gas, culture medium or culture, and are only used to distinguish different mixed gases, different culture media and different cultures.

[0022] The present invention enriches the microbial population dominated by hydrogen oxidizing bacteria in the soil to obtain a hydrogen oxidizing bacteria enriched population. In the present invention, the method for enriching the microbial population dominated by hydrogen oxidizing bacteria in the soil comprises: inoculating the microorganisms in the soil into a primary seed culture medium and introducing a mixed gas for cultivation until H2 consumption is more than 50% or the CO2 concentration no longer decreases, thereby obtaining a primary seed solution of hydrogen oxidizing bacteria. In the present invention, the primary seed culture medium is referred to as the first primary seed culture medium or the primary seed culture medium of hydrogen oxidizing bacteria in order to distinguish it from other primary seed culture media in the following text; the mixed gas is referred to as the first hydrogen oxidizing bacteria mixed gas in order to distinguish it from other mixed gases in the following text; the culture is referred to as the first culture in order to distinguish it.

[0023] In the present invention, the composition of the first primary seed culture medium includes: Na2HPO4: 2200~2800 mg / L, KH2PO4: 500~1500 mg / L, (NH4)2SO4: 450~500 mg / L, MgCl2: 45~60 mg / L, CaCl2·2H2O: 40~50 mg / L, disodium EDTA: 0.2~0.7 mg / L, FeSO4·7H2O: 0.1~0.5 mg / L, ZnSO4·7H2O: 0.01~0.02 mg / L, MnCl2·4H2O: 0.002~0.003 mg / L, H3BO3: 0.01~0.04 mg / L, CoCl2·6H2O: 0.01~0.02 mg / L, CuCl2·2H2O: 0.001~0.01 mg / L, NiCl2·6H2O: 0.002~0.004 mg / L, Na2MoO4·2H2O: 0.001~0.003 mg / L. As an optional embodiment of the present invention, the composition of the first primary seed culture medium is: Na2HPO4: 2800 mg / L, KH2PO4: 1000 mg / L, (NH4)2SO4: 500 mg / L, MgCl2: 53 mg / L, CaCl2·2H2O: 50 mg / L, disodium EDTA: 0.5 mg / L, FeSO4·7H2O: 0.2 mg / L, ZnSO4·7H2O: 0.01 mg / L, MnCl2·4H2O: 0.003 mg / L, H3BO3: 0.03 mg / L, CoCl2·6H2O: 0.02 mg / L, CuCl2·2H2O: 0.001 mg / L, NiCl2·6H2O: 0.002 mg / L, Na2MoO4·2H2O: 0.003 mg / L. In the present invention, the pH of the first primary seed culture medium may be 6.8 to 7.4. In the present invention, the pH is preferably adjusted using a phosphate buffer. The first primary seed culture medium of the present invention provides an optimal culture medium according to the metabolic conditions of hydrogen oxidizing bacteria.

[0024] In the present invention, the hydrogen oxidizing bacteria mixed gas includes H2, O2 and CO2; the volume ratio of H2, O2 and CO2 is (50-70): (30-20): (20-10). As an optional embodiment of the present invention, the volume ratio of H2, O2 and CO2 can be 50:30:20, 50:20:10, 50:25:15, 60:30:20, 60:20:10, 60:25:15, 70:30:20, 70:20:10 or 70:25:15. In the present invention, the hydrogen oxidizing bacteria mixed gas can also be referred to as the first hydrogen oxidizing bacteria mixed gas. In the present invention, the first hydrogen oxidizing bacteria mixed gas can meet the metabolic growth needs of hydrogen oxidizing bacteria and help to enrich hydrogen oxidizing bacteria, wherein the hydrogen oxidizing bacteria use CO2 as a carbon source, H2 as an electron donor, and O2 as an electron acceptor to carry out metabolic cycles. The composition ratio setting of the first mixed gas of hydrogen oxidizing bacteria provided by the present invention can significantly shorten the enrichment time of hydrogen oxidizing bacteria. If the gas ratio of the first mixed gas is changed, for example, the volume of CO2 is increased or the ratio of H2 is increased, the enrichment time of hydrogen oxidizing bacteria will be significantly prolonged.

[0025] After obtaining the primary seed liquid of hydrogen oxidizing bacteria, the present invention inoculates the primary seed liquid of hydrogen oxidizing bacteria into a secondary seed culture medium and introduces the first mixed gas of hydrogen oxidizing bacteria for cultivation to obtain a hydrogen oxidizing bacteria enriched flora. In the present invention, the cultivation includes a second cultivation and a sub-cultivation. In the present invention, the secondary seed culture medium is referred to as a first secondary seed culture medium or a hydrogen oxidizing bacteria secondary culture medium in order to distinguish it from other secondary seed culture media described below.

[0026] The composition of the first secondary seed culture medium is based on the first primary seed culture medium and further includes: 1% to 3% glucose, 0.6% to 2% yeast powder and 1.5% to 3.2% casein peptone. In the present invention, the pH of the first secondary seed culture medium can be 6.8 to 7.4.

[0027] In the present invention, different hydrogen oxidizing bacteria may use CO2 only as a carbon source, or may use organic matter in the culture medium as a carbon source. The purpose of adding is to provide different forms of carbon sources and nutrients to the microorganisms as much as possible, so as to create a suitable culture environment as much as possible.

[0028] In the present invention, the soil includes 2-10 cm soil below the surface of the water-retaining paddy field and / or livestock and poultry manure compost residues. The present invention has found through research that the 2-10 cm soil below the surface of the water-retaining paddy field and the livestock and poultry manure compost residues are rich in hydrogen oxidizing bacteria. After obtaining the corresponding soil, the present invention preferably mixes the soil with water to obtain a soil-water mixture. In the present invention, the mass volume ratio of the soil to water can be (5-10) g: (100-120) mL. As an optional embodiment of the present invention, the mass volume ratio of the soil to water can be 5g:100mL, 10g:100mL, 5g:120mL, 10g:100mL, 8g:100mL, 8g:120mL, 5g:110mL, 8g:110mL or 8g:120mL. After obtaining the soil-water mixture, the present invention preferably shakes the soil-water mixture on a shaker at 30°C and 120rpm for 1-2h. After the oscillation is completed, the present invention preferably mixes the oscillated soil-water mixture with the first primary seed culture medium to complete the inoculation; the volume ratio of the soil-water mixture to the first primary seed culture medium is 1: (1-2). After the inoculation is completed, the present invention obtains a soil culture medium mixed system. After obtaining the soil culture medium mixed system, the present invention preferably stirs the soil culture medium mixed system with a magnetic stirrer at 250-450rpm for 1-2h. After the stirring is completed, the present invention places the mixed soil culture medium mixed system in a culture container; the volume ratio of the soil culture medium mixed system to the culture container is 1: (5-10), and can also be 1:5, 1.1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. After the soil culture medium mixed system is placed in the culture container, the first mixed gas is introduced. The present invention introduces the hydrogen oxidizing bacteria first mixed gas until the culture container is filled. Before the hydrogen oxidizing bacteria first mixed gas is introduced, nitrogen is preferably introduced to exhaust the original air. After the original air is exhausted, the hydrogen oxidizing bacteria first mixed gas is introduced. After the first mixed gas is introduced, the present invention performs a first culture. In the present invention, the temperature of the first culture is 25-35°C. As an optional embodiment of the present invention, the temperature of the first culture can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C. In the present invention, the rotation speed of the first culture can be 150-210rpm. As an optional embodiment of the present invention, the rotation speed can be 150, 160, 170, 180, 190, 200 or 210rpm. The first culture of the present invention is preferably cultured until H2 consumption is more than 50% or CO2 concentration no longer decreases to obtain a primary seed solution of hydrogen oxidizing bacteria.

[0029] After obtaining the primary hydrogen oxidizing bacteria seed solution, the present invention inoculates the primary hydrogen oxidizing bacteria seed solution into the first secondary seed culture medium and introduces the first hydrogen oxidizing bacteria mixed gas for cultivation to obtain a hydrogen oxidizing bacteria enriched flora. The cultivation of the present invention includes a second cultivation and a sub-cultivation.

[0030] When the hydrogen oxidizing bacteria primary seed liquid is inoculated into the first secondary seed culture medium, preferably 5 to 10 mL of the hydrogen oxidizing bacteria primary seed liquid is inoculated into every 100 to 200 mL of the first secondary seed culture medium to obtain a secondary culture system. As an optional embodiment of the present invention, the volume ratio of the secondary culture system to its culture container can be 1: (5 to 10), or 1: 5, 1.1: 5, 1: 6, 1: 7, 1: 8, 1: 9 or 1: 10. After the hydrogen oxidizing bacteria primary seed liquid is inoculated into the first secondary seed culture medium, nitrogen is preferably introduced first to exhaust the original air. After exhausting the original air, the first mixed gas of the hydrogen oxidizing bacteria is introduced to fill the culture container. In the present invention, the temperature of the second culture is 25 to 35 ° C. As an optional embodiment of the present invention, the temperature of the second culture can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 ° C. In the present invention, the rotation speed of the second culture can be 150 to 210 rpm. As an optional embodiment of the present invention, the rotation speed can be 150, 160, 170, 180, 190, 200 or 210 rpm. In the second culture process of the present invention, it is preferred to culture until H2 consumption is more than 50% or CO2 concentration no longer decreases to obtain a second culture seed solution. As an optional embodiment of the present invention, the second culture time is shorter than the first culture time.

[0031] After the second culture is completed, the present invention preferably further comprises inoculating the second culture seed liquid into the first secondary seed culture medium for subculture. The subculture of the present invention is mainly to enrich the bacterial community and enhance and stabilize its carbon fixation capacity. When the present invention performs subculture, it is preferred to inoculate 5 to 10 mL of the second culture seed liquid into every 100 to 200 mL of the first secondary seed culture medium to obtain a subculture system. As an optional embodiment of the present invention, the volume ratio of the subculture system to its culture container can be 1: (5 to 10), or 1: 5, 1.1: 5, 1: 6, 1: 7, 1: 8, 1: 9 or 1: 10. In the process of subculture of the present invention, it is preferred to replace the secondary seed culture medium and the first mixed gas of hydrogen oxidizing bacteria every 2 to 10 days, and the interval time can be 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. The present invention preferably replaces the culture medium and the mixed gas 5 to 10 times continuously during sub-culturing, which may be 5, 6, 7, 8, 9 or 10 times; the present invention preferably replaces the mixed gas while replacing the culture medium, and each time the culture medium is replaced, the volume ratio of the culture system to the culture container remains unchanged. In the sub-culturing process of the present invention, after continuously replacing the first mixed gas of hydrogen oxidizing bacteria 5 to 10 times, the last culturing is preferably carried out until H2 consumption is more than 50% or CO2 concentration no longer decreases, to obtain a hydrogen oxidizing bacteria enriched flora.

[0032] The hydrogen oxidizing bacteria enriched bacterial community obtained by the above method of the present invention can efficiently utilize hydrogen and carbon dioxide: hydrogen oxidizing bacteria can use hydrogen (H2) as an electron donor and carbon dioxide (CO2) as a carbon source to generate organic matter through photosynthesis or chemosynthesis. The hydrogen oxidizing bacteria enriched by the specific culture medium and gas conditions of the present invention have a high hydrogen and carbon dioxide utilization capacity and can effectively convert these gases into biomass. The hydrogen oxidizing bacteria enriched bacterial community can improve the carbon fixation efficiency: in the process of utilizing hydrogen and carbon dioxide for metabolism, the hydrogen oxidizing bacteria can fix a large amount of carbon as biomass and generate useful substances such as microbial protein. This helps to improve the carbon fixation efficiency of the entire system, reduce carbon dioxide emissions, and reduce the greenhouse effect. The hydrogen oxidizing bacteria enriched bacterial community can enhance the stability of the system: hydrogen oxidizing bacteria play an important role in the composite microbial metabolic community, and can synergize with other microbial communities to enhance the stability and anti-interference ability of the entire system. For example, in the process of synchronous carbon fixation and denitrification, hydrogen oxidizing bacteria can work together with methane oxidizing bacteria and sulfur oxidizing bacteria to improve the tolerance and adaptability of the system. The hydrogen oxidizing bacteria enriched flora can promote the recycling of resources: the hydrogen oxidizing bacteria can utilize the hydrogen and carbon dioxide in the biogas to realize the recycling of resources, which not only reduces the dependence on fossil energy, but also improves the utilization efficiency of resources, which is in line with the development concept of circular economy.

[0033] The method for enriching a microbial population mainly composed of sulfur-oxidizing bacteria in soil to obtain a sulfur-oxidizing bacteria enriched bacterial population comprises: inoculating microorganisms in the soil into a primary seed culture medium for sulfur-oxidizing bacteria, introducing a first mixed gas of sulfur-oxidizing bacteria for first culture, until the H2S concentration no longer decreases, and obtaining a primary seed solution of sulfur-oxidizing bacteria. In the present invention, the primary seed culture medium for sulfur-oxidizing bacteria may also be referred to as a second primary seed culture medium.

[0034] In the present invention, the composition of the second primary seed culture medium includes: phenol red: 0.001-0.003 g / L, 1,4-piperazine diethanesulfonic acid: 4.5-8.5 g / L, sodium chloride: 10-25.000 g / L, magnesium sulfate heptahydrate: 1.1-2.9 g / L, potassium chloride: 0.1-1 g / L, ammonium chloride: 0.015-0.3 g / L, sodium thiosulfate pentahydrate: 1.5-3.2 g / L, calcium chloride dihydrate: 0.05-0.2 g / L, magnesium chloride hexahydrate: 2.5-4.5 g / L, potassium hydrogen phosphate: 0.05-0.15 g / L, ammonium ferrous sulfate hexahydrate: 0.001-0.002 g / L, trace element solution: 1 mL / L and vitamin solution: 10 mL / L. The pH of the second primary seed culture medium can be 6.8-7.4. In the present invention, the pH is preferably adjusted using a phosphate buffer. As an optional embodiment of the present invention, the composition of the second primary seed culture medium is: phenol red: 0.003 g / L, 1,4-piperazine diethanesulfonic acid: 6.500 g / L, sodium chloride: 25.000 g / L, magnesium sulfate heptahydrate: 2.700 g / L, potassium chloride: 0.500 g / L, ammonium chloride: 0.250 g / L, sodium thiosulfate pentahydrate: 2.480 g / L, calcium chloride dihydrate: 0.140 g / L, magnesium chloride hexahydrate: 4.300 g / L, potassium hydrogen phosphate: 0.140 g / L, ammonium ferrous sulfate hexahydrate: 0.002 g / L, trace element solution: 1 mL / L and vitamin solution: 10 mL / L.

[0035] In the present invention, each liter of trace element solution includes: MgSO4·7H2O 1.00~3.00g, MnSO4·H2O 0.1~1g, NaCl 0.5~1.00g, FeSO4·7H2O 0.10~0.20g, CoSO4·7H2O 0.15~0.2g, CaCl2·2H2O 0.10~0.2g, ZnSO4·7H2O 0.15~0.18g, CuSO4·5H2O 0.01~0.02g, AlK(SO4)2·12H2O 0.01~0.02g, H3BO3 0.01~0.02g, Na2MoO4·2H2O 0.01~0.05g, NiCl2·6H2O 0.01~0.05g, Na2SeO3·5H2O 0.01~0.50mg and Na2WO4·2H2O 0.02~0.40mg. As an optional embodiment of the present invention, the composition of the trace element solution per liter is: MgSO4·7H2O 2.00g, MnSO4·H2O 0.5g, NaCl 0.8g, FeSO4·7H2O 0.15g, CoSO4·7H2O 0.18g, CaCl2·2H2O 0.15g, ZnSO4·7H2O 0.16g, CuSO4·5H2O 0.015g, AlK(SO4)2·12H2O 0.015g, H3BO3 0.015g, Na2MoO4·2H2O 0.03g, NiCl2·6H2O 0.03g, Na2SeO3·5H2O 0.2mg and Na2WO4·2H2O 0.2mg.

[0036] In the present invention, the vitamin solution includes: vitamin B7 1-2 mg / L, folic acid 1-2 mg / L, pyridoxine hydrochloride 5-10 mg / L, vitamin B1 2.5-5.5 mg / L, riboflavin 2-5 mg / L, vitamin B3 1.5-6 mg / L, vitamin B5 2.5-5.5 mg / L, vitamin B 12 0.1~0.2mg / L, p-aminobenzoic acid 2~5mg / L and (DL)-α-lipoic acid 2~5mg / L. As an optional embodiment of the present invention, the composition of the vitamin solution is: vitamin B7 1.5mg / L, folic acid 1.5mg / L, pyridoxine hydrochloride 8mg / L, vitamin B14mg / L, riboflavin 4mg / L, vitamin B34mg / L, vitamin B54mg / L, vitamin B 12 0.15 mg / L, p-aminobenzoic acid 4 mg / L and (DL)-α-lipoic acid 4 mg / L. In the present invention, the second primary seed culture medium is an optimal culture medium according to the metabolic conditions of sulfur oxidizing bacteria.

[0037] The sulfur-oxidizing bacteria mixed gas includes H2, O2, CO2 and H2S; the volume ratio of H2, O2, CO2 and H2S is (30-50): (20-30): (20-30): (20-30). As an optional embodiment of the present invention, the volume ratio of H2, O2, CO2 and H2S can be 30:20:20:20, 30:30:30:30, 50:20:20:20, 50:30:30:30, 30:20:30:20, 30:20:30:30, 50:20:30:20 or 50:20:30:30. In the present invention, the sulfur-oxidizing bacteria mixed gas can also be referred to as the first sulfur-oxidizing bacteria mixed gas. The composition ratio setting of the first mixed gas of sulfur-oxidizing bacteria provided by the present invention can significantly shorten the enrichment time of sulfur-oxidizing bacteria. If the gas ratio of the first mixed gas is changed, the enrichment time of sulfur-oxidizing bacteria will be significantly prolonged.

[0038] After obtaining the primary seed liquid of sulfur-oxidizing bacteria, the present invention inoculates the primary seed liquid of sulfur-oxidizing bacteria into the secondary seed culture medium of sulfur-oxidizing bacteria and introduces the first mixed gas of sulfur-oxidizing bacteria for cultivation to obtain a sulfur-oxidizing bacteria enriched bacterial population. In the present invention, the secondary seed culture medium of sulfur-oxidizing bacteria can be referred to as a second secondary seed culture medium. In the present invention, the cultivation includes a second cultivation and a sub-cultivation.

[0039] In the present invention, the composition of the second secondary seed culture medium is based on the second primary seed culture medium, and further includes: 1% to 3% glucose, 0.6% to 2% yeast powder and 1.5% to 3.2% casein peptone. The pH of the second secondary seed culture medium can be 6.8 to 7.4.

[0040] In the process of enriching the microbial population dominated by sulfur-oxidizing bacteria according to the present invention, the sulfur-oxidizing bacteria use H2S as an electron donor, O2 as an electron acceptor, and CO2 as a carbon source for metabolic circulation. The components in the culture medium provide necessary nutrients and a buffer system to maintain pH stability, which is conducive to the growth of sulfur-oxidizing bacteria. In the process of enriching the sulfur-oxidizing bacteria, a higher H2S ratio (20% to 30%) ensures that the sulfur-oxidizing bacteria have sufficient electron donors for metabolism, and the ratios of H2, O2 and CO2 meet their respiration and carbon fixation needs. When the microbial population dominated by sulfur-oxidizing bacteria is enriched according to the present invention, the pH value of the culture medium is adjusted between 6.8 and 7.4, which is suitable for the growth of sulfur-oxidizing bacteria. The present invention creates a micro-oxygen environment suitable for the growth of sulfur-oxidizing bacteria by introducing a mixed gas and exhausting the original air, which is conducive to enriching the microbial population dominated by oxidizing bacteria.

[0041] In the present invention, the soil includes 2-10 cm soil below the surface of the swamp and / or sludge from oxidation ponds in sewage treatment plants. The present invention has found through research that the 2-10 cm soil below the surface of the swamp and / or sludge from oxidation ponds in sewage treatment plants are rich in sulfur-oxidizing bacteria. After obtaining the corresponding soil, the present invention preferably mixes the soil with water to obtain a soil-water mixture. In the present invention, the mass volume ratio of the soil to water can be (5-10) g: (100-120) mL. As an optional embodiment of the present invention, the mass volume ratio of the soil to water can be 5g:100mL, 10g:100mL, 5g:120mL, 10g:100mL, 8g:100mL, 8g:120mL, 5g:110mL, 8g:110mL or 8g:120mL. After obtaining the soil-water mixture, the present invention preferably oscillates the soil-water mixture on a shaker at 30°C and 120rpm for 1-2h. After the oscillation is completed, the present invention preferably mixes the oscillated soil-water mixture with the second primary seed culture medium to complete the inoculation; the volume ratio of the soil-water mixture to the second primary seed culture medium is 1: (1-2). After the inoculation is completed, the present invention obtains a soil culture medium mixed system. After obtaining the soil culture medium mixed system, the present invention preferably stirs the soil culture medium mixed system with a magnetic stirrer at 250-450rpm for 1-2h. After the stirring is completed, the present invention places the mixed soil culture medium mixed system in a culture container; the volume ratio of the soil culture medium mixed system to the culture container is 1: (5-10), and can also be 1:5, 1.1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. After the soil culture medium mixed system is placed in the culture container, the first mixed gas of sulfur-oxidizing bacteria is introduced. The present invention introduces the first mixed gas of sulfur-oxidizing bacteria until the culture container is filled. Before the first mixed gas of sulfur-oxidizing bacteria is introduced, the present invention preferably introduces nitrogen first to exhaust the original air, and then introduces the first mixed gas of sulfur-oxidizing bacteria after exhausting the original air. After the first mixed gas is introduced, the present invention performs a first culture. In the present invention, the temperature of the first culture is 25-35°C. As an optional embodiment of the present invention, the temperature of the first culture can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C. In the present invention, the rotation speed of the first culture can be 150-210rpm. As an optional embodiment of the present invention, the rotation speed can be 150, 160, 170, 180, 190, 200 or 210rpm. The first culture of the present invention is preferably cultured until the H2S concentration no longer decreases to obtain a primary seed solution of sulfur-oxidizing bacteria.

[0042] After obtaining the primary seed liquid of sulfur oxidizing bacteria, the present invention inoculates the primary seed liquid of sulfur oxidizing bacteria into a second secondary seed culture medium and introduces the first mixed gas of sulfur oxidizing bacteria to perform second culture and subculture to obtain an enriched bacterial population of sulfur oxidizing bacteria.

[0043] When the primary seed liquid of sulfur-oxidizing bacteria is inoculated into the second secondary seed culture medium, 5-10 mL of the primary seed liquid of sulfur-oxidizing bacteria is preferably inoculated into 100-200 mL of the second secondary seed culture medium to obtain a secondary culture system. As an optional embodiment of the present invention, the volume ratio of the secondary culture system to its culture container can be 1: (5-10), or 1: 5, 1.1: 5, 1: 6, 1: 7, 1: 8, 1: 9 or 1: 10. After the primary seed liquid of sulfur-oxidizing bacteria is inoculated into the second secondary seed culture medium, nitrogen is preferably introduced first to exhaust the original air. After exhausting the original air, the first mixed gas of sulfur-oxidizing bacteria is introduced to fill the culture container. In the present invention, the temperature of the second culture is 25-35°C. As an optional embodiment of the present invention, the temperature of the second culture can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C. In the present invention, the rotation speed of the second culture can be 150-210rpm. As an optional embodiment of the present invention, the rotation speed can be 150, 160, 170, 180, 190, 200 or 210 rpm. In the second culture process of the present invention, it is preferred to culture until the H2S concentration no longer decreases to obtain a second culture seed solution. As an optional embodiment of the present invention, the second culture time is shorter than the first culture time.

[0044] After the second culture is completed, the present invention preferably further comprises inoculating the second culture seed liquid into the second secondary seed culture medium for subculture. The subculture of the present invention is mainly to enrich bacteria and enhance and stabilize their sulfur oxidation ability. When the present invention performs subculture, it is preferred to inoculate 5-10 mL of the second culture seed liquid into 100-200 mL of the second secondary seed culture medium to obtain a subculture system. As an optional embodiment of the present invention, the volume ratio of the subculture system to its culture container can be 1: (5-10), or 1: 5, 1.1: 5, 1: 6, 1: 7, 1: 8, 1: 9 or 1: 10. In the process of subculture, the present invention preferably replaces the first mixed gas of sulfur-oxidizing bacteria every 2-10 days; the interval time can be 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. When performing subculture, the present invention preferably replaces the culture medium and the mixed gas continuously for 5-10 times, which can be 5, 6, 7, 8, 9 or 10 times; the present invention preferably replaces the mixed gas while replacing the culture medium, and the volume ratio of the culture system to the culture container remains unchanged each time the culture medium is replaced. In the subculture process of the present invention, after the first mixed gas of sulfur-oxidizing bacteria is replaced continuously for 5 to 10 times, the last culture is preferably cultured until the H2S concentration no longer decreases, thereby obtaining a sulfur-oxidizing bacteria enriched flora.

[0045] The sulfur-oxidizing bacteria enriched by the specific culture medium and gas conditions of the present invention have a high ability to degrade hydrogen sulfide, can use hydrogen sulfide (H2S) as an electron donor, and can generate sulfate and other substances by oxidizing hydrogen sulfide, thereby effectively reducing the concentration of hydrogen sulfide in the environment, and can treat a large amount of hydrogen sulfide gas in a short time. The sulfur-oxidizing bacteria enriched bacterial community has strong tolerance: the enriched sulfur-oxidizing bacteria can grow well under specific culture conditions and have strong tolerance. The sulfur-oxidizing bacteria enriched bacterial community can reduce environmental pollution: hydrogen sulfide is a toxic and harmful gas with a strong odor, which is harmful to the environment and human health. By degrading hydrogen sulfide, sulfur-oxidizing bacteria can effectively reduce its concentration in the environment, reduce pollution to the atmosphere and water bodies, and protect the ecological environment. The synergistic effect of the sulfur-oxidizing bacteria enriched bacterial community: sulfur-oxidizing bacteria and other microbial groups (such as methane oxidizing bacteria and hydrogen oxidizing bacteria) together constitute a composite microbial metabolic group, which can play a synergistic role. For example, in the process of synchronous carbon fixation and denitrification, sulfur-oxidizing bacteria can work together with methane oxidizing bacteria and hydrogen oxidizing bacteria to improve the metabolic efficiency and stability of the entire system and achieve the synchronous removal of multiple pollutants.

[0046] The method of enriching a microbial population mainly composed of methane oxidizing bacteria in soil of the present invention comprises: inoculating microorganisms in the soil into a primary seed culture medium of methane oxidizing bacteria, introducing a first mixed gas of methane oxidizing bacteria for first culture, and culturing until the CH4 concentration no longer decreases, thereby obtaining a primary seed solution of methane oxidizing bacteria. In the present invention, the primary seed culture medium of methane oxidizing bacteria may also be referred to as a third primary seed culture medium.

[0047] In the present invention, the composition of the third primary seed culture medium includes: KNO3: 100-200 mg / L, KH2PO4: 100-200 mg / L, MgSO4·7H2O: 25-55 mg / L, CaCl2·2H2O: 5-20 mg / L, EDTA: 2-5 mg / L, CuCl2·5H2O: 0.05-0.1 mg / L, FeSO4·7H2O: 1-2 mg / L, ZnSO4·7H2O: 0.1-0.2 mg / L, NiCl2·6H2O: 0.008-0.02 mg / L, CoCl2·6H2O: 0.1-0.2 mg / L and Na2MoO4: 0.01-0.03 mg / L. The pH of the third primary seed culture medium can be 6.8-7.4. In the present invention, the pH is preferably adjusted using phosphate buffer. As an optional embodiment of the present invention, the composition of the third primary seed culture medium is: KNO3: 100 mg / L, KH2PO4: 100 mg / L, MgSO4·7H2O: 50 mg / L, CaCl2·2H2O: 10 mg / L, EDTA: 5 mg / L, CuCl2·5H2O: 0.1 mg / L, FeSO4·7H2O: 2 mg / L, ZnSO4·7H2O: 0.1 mg / L, NiCl2·6H2O: 0.02 mg / L, CoCl2·6H2O: 0.2 mg / L and Na2MoO4: 0.03 mg / L. In the present invention, the third primary seed culture medium provides the best culture medium according to the metabolic conditions of methanotrophic bacteria.

[0048] In the present invention, the methane oxidizing bacteria mixed gas includes CH4 and O2; the volume ratio of CH4 and O2 is (50~80): (50~20), which can be (50~75): (50~25), or 50:50, 75:25, 80:20 or 60:40. In the present invention, the methane oxidizing bacteria mixed gas can also be referred to as the first methane oxidizing bacteria mixed gas. In the present invention, the first methane oxidizing bacteria mixed gas can meet the metabolic growth needs of methane oxidizing bacteria, wherein the metabolism of methane oxidizing bacteria uses methane as a carbon source for metabolic cycle. The composition ratio setting of the first methane oxidizing bacteria mixed gas provided by the present invention can significantly shorten the time for methane oxidizing bacteria to be enriched. If the gas ratio of the first mixed gas is changed, for example, the volume of CH4 is increased or the proportion of O2 is increased, the enrichment time of methane oxidizing bacteria will be significantly prolonged.

[0049] After obtaining the primary seed liquid of methanotrophic bacteria, the present invention inoculates the primary seed liquid of methanotrophic bacteria into the secondary seed culture medium of methanotrophic bacteria and introduces the first mixed gas of methanotrophic bacteria for cultivation to obtain a methanotrophic bacteria enriched bacterial population. In the present invention, the secondary seed culture medium of methanotrophic bacteria can also be referred to as the third secondary seed culture medium. The cultivation includes the second cultivation and the sub-cultivation.

[0050] In the present invention, the third secondary seed culture medium is based on the third primary seed culture medium and further comprises: 1% to 3% glucose, 0.6% to 2% yeast powder and 1.5% to 3.2% casein peptone. The pH of the third secondary seed culture medium may be 6.8 to 7.4.

[0051] In the process of enriching the microbial population dominated by methanotrophic bacteria in the soil of the present invention, the methanotrophic bacteria use CH4 as a carbon source and electron donor and O2 as an electron acceptor for metabolic circulation. The higher proportion of CH4 in the mixed gas (50% to 85%) ensures that the methanotrophic bacteria have sufficient carbon sources and electron donors for metabolism, while the proportion of O2 meets its respiratory needs. By introducing the mixed gas and exhausting the original air, a micro-oxygen environment suitable for the growth of methanotrophic bacteria is created. The culture medium for enriching the microbial population dominated by methanotrophic bacteria in the soil of the present invention contains KNO3, KH2PO4, MgSO4·7H2O, CaCl2·2H2O, EDTA, CuCl2·5H2O, FeSO4·7H2O and other components, which provide the nutrients and buffer system required by the methanotrophic bacteria, and the pH value of the culture medium is adjusted between 6.8 and 7.4, which is suitable for the growth of methanotrophic bacteria.

[0052] In the present invention, the soil includes: soil 2 to 10 cm below the surface of the water-reservoir paddy field and / or sludge from oxidation ponds of sewage treatment plants. The present invention has found through research that the soil collected from the soil 2 to 10 cm below the surface of the water-reservoir paddy field and / or sludge from oxidation ponds of sewage treatment plants is rich in methane oxidizing bacteria. After obtaining the corresponding soil, the present invention preferably mixes the soil with water to obtain a soil-water mixture. In the present invention, the mass volume ratio of the soil to water may be (5 to 10) g: (100 to 120) mL. As an optional embodiment of the present invention, the mass volume ratio of the soil to water may be 5 g: 100 mL, 10 g: 100 mL, 5 g: 120 mL, 10 g: 100 mL, 8 g: 100 mL, 8 g: 120 mL, 5 g: 110 mL, 8 g: 110 mL or 8 g: 120 mL. After obtaining the soil-water mixture, the present invention preferably oscillates the soil-water mixture on a shaker at 30° C. and 120 rpm for 1 to 2 hours. After the oscillation is completed, the present invention preferably mixes the oscillated soil-water mixture with the third primary seed culture medium; the volume ratio of the soil-water mixture to the third primary seed culture medium is 1: (1-2). After the mixing is completed, the present invention obtains a soil culture medium mixed system. After obtaining the soil culture medium mixed system, the present invention preferably stirs the soil culture medium mixed system with a magnetic stirrer at 250-450rpm for 1-2h. After the stirring is completed, the present invention places the mixed soil culture medium mixed system in a culture container; the volume ratio of the soil culture medium mixed system to the culture container is 1: (5-10), or can be 1:5, 1.1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. After the present invention places the soil culture medium mixed system in the culture container, the first mixed gas is introduced. Before the first mixed gas of methanotrophic bacteria is introduced, nitrogen is preferably introduced first to exhaust the original air. After the original air is exhausted, the first mixed gas of methanotrophic bacteria is introduced. In the present invention, the temperature of the first culture is 25-35°C. As an optional embodiment of the present invention, the temperature of the first culture can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C. In the present invention, the rotation speed of the first culture can be 150-210rpm. As an optional embodiment of the present invention, the rotation speed can be 150, 160, 170, 180, 190, 200 or 210rpm. The first culture of the present invention is preferably cultured until the CH4 concentration no longer decreases to obtain a primary seed solution of methanotrophic bacteria.

[0053] After obtaining the primary seed liquid of methanotrophic bacteria, the present invention inoculates the primary seed liquid of methanotrophic bacteria into a third secondary seed culture medium, introduces the first mixed gas of methanotrophic bacteria for second culture and subculture, and obtains a methanotrophic bacteria enriched bacterial population.

[0054] When the primary seed liquid of methanotrophic bacteria is inoculated into the third secondary seed culture medium, 5-10 mL of the primary seed liquid of methanotrophic bacteria is preferably inoculated into 100-200 mL of the third secondary seed culture medium to obtain a secondary culture system. As an optional embodiment of the present invention, the volume ratio of the secondary culture system to its culture container may be 1: (5-10), or may be 1: 5, 1.1: 5, 1: 6, 1: 7, 1: 8, 1: 9 or 1: 10. After the primary seed liquid of methanotrophic bacteria is inoculated into the third secondary seed culture medium, nitrogen is preferably introduced first to exhaust the original air. After exhausting the original air, the first mixed gas of methanotrophic bacteria is introduced to fill the culture container. In the present invention, the temperature of the second culture is 25-35°C. As an optional embodiment of the present invention, the temperature of the second culture may be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35°C. In the present invention, the rotation speed of the second culture can be 150-210 rpm. As an optional embodiment of the present invention, the rotation speed can be 150, 160, 170, 180, 190, 200 or 210 rpm. In the second culture process of the present invention, it is preferred to culture until the CH4 concentration no longer decreases to obtain a second culture seed solution. As an optional embodiment of the present invention, the time of the second culture is shorter than the time of the culture.

[0055] After the second culture is completed, the present invention preferably further comprises inoculating the second culture seed liquid into the third secondary seed culture medium for subculture. The subculture of the present invention is mainly to enrich bacteria and enhance and stabilize their methane oxidation ability. When the present invention performs subculture, it is preferred to inoculate 5-10 mL of the second culture seed liquid into 100-200 mL of the third secondary seed culture medium to obtain a subculture system. As an optional embodiment of the present invention, the volume ratio of the subculture system to its culture container can be 1: (5-10), or 1:5, 1.1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In the process of subculture of the present invention, it is preferred to replace the secondary seed culture medium and the first mixed gas of methane oxidizing bacteria every 2-10 days; the interval time can be 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. In the present invention, when performing poplar cultivation, the culture medium and the mixed gas are preferably replaced continuously for 5 to 10 times, which can be 5, 6, 7, 8, 9 or 10 times; the present invention preferably replaces the mixed gas while replacing the culture medium, and each time the culture medium is replaced, the volume ratio of the culture system to the culture container remains unchanged. In the subculture process of the present invention, after the first mixed gas of methane oxidizing bacteria is replaced continuously for 5 to 10 times, the last culture is preferably cultured until the CH4 concentration no longer decreases, so as to obtain a methane oxidizing bacteria enriched bacterial population.

[0056] In the present invention, the methane-oxidizing bacteria enriched by specific culture medium and gas conditions have a high methane degradation ability, can process a large amount of methane gas in a short time, and generate carbon dioxide and water by oxidizing methane. The methane-oxidizing bacteria enriched bacterial community has strong adaptability: the enriched methane-oxidizing bacteria can grow well under specific culture conditions and have strong adaptability. The methane-oxidizing bacteria enriched bacterial community can promote carbon fixation: in the process of degrading methane, methane-oxidizing bacteria can fix part of the carbon as biomass and generate useful substances such as microbial protein, which not only helps to reduce greenhouse gas emissions, but also can achieve the effective utilization of carbon resources, meeting the requirements of sustainable development. The methane-oxidizing bacteria enriched bacterial community has a synergistic effect: methane-oxidizing bacteria and other microbial groups (such as hydrogen oxidizing bacteria and sulfur oxidizing bacteria) together constitute a composite microbial metabolic group, which can play a synergistic role. For example, in the process of synchronous carbon fixation and denitrification, methane-oxidizing bacteria can work together with sulfur oxidizing bacteria and hydrogen oxidizing bacteria to improve the metabolic efficiency and stability of the entire system.

[0057] The present invention can effectively enrich hydrogen oxidizing bacteria, sulfur oxidizing bacteria and methane oxidizing bacteria through the optimization design of the above-mentioned culture medium, gas composition and culture conditions. The enrichment process of each microbial population is based on its specific metabolic needs and environmental conditions. By accurately controlling the culture medium composition and gas ratio, the efficient enrichment and activity maintenance of the target microbial population are ensured. These optimized conditions not only improve the enrichment efficiency of microorganisms, but also provide a stable microbial metabolic group for the subsequent synchronous carbon fixation and denitrification process.

[0058] After respectively obtaining the enriched bacterial population of methane oxidizing bacteria, the enriched bacterial population of sulfur oxidizing bacteria and the enriched bacterial population of hydrogen oxidizing bacteria, the present invention inoculates the enriched bacterial population of methane oxidizing bacteria, the enriched bacterial population of sulfur oxidizing bacteria and the enriched bacterial population of hydrogen oxidizing bacteria in a mixed manner into an acclimation medium for acclimation culture to obtain a composite microbial metabolite.

[0059] In the present invention, a third mixed gas is introduced during domestication and cultivation; the third mixed gas is composed of H2, O2, CO2, CH4 and H2S; the volume ratio of H2, O2, CO2, CH4 and H2S is (0.1-1): (10-20): (15-25): (50-60): (0.1-0.3). The third mixed gas in the present invention is closest to the real composition of biogas, in order to further domesticate the survival and digestion ability of the mixed bacteria under real biogas.

[0060] In the present invention, the composition of each 1L acclimation medium includes: 0.5-1.00g MgSO4·7H2O, 1.00-2.5g KNO3, 0.15-0.3g KH2PO4, 0.2-0.45g Na2HPO4, 0.1-0.15g CaCl2·2H2O and 1mL trace element solution. As an optional embodiment of the present invention, the composition of each 1L acclimation medium is: 1.00g MgSO4·7H2O, 1.00g KNO3, 0.27g KH2PO4, 0.28g Na2HPO4, 0.13g CaCl2·2H2O and 1mL trace element solution. In the present invention, the composition of each 1L trace element solution includes 1.00g EDTA-2Na, 0.50g FeSO4·7H2O, 0.07g ZnSO4·7H2O, 0.03g MnCl2·4H2O, 0.30g H3BO3, 0.60g CoCl2·6H2O, 0.02g NiCl2·6H2O, 0.03g Na2MoO4·2H2O and 0.01g CuCl2·H2O. In the present invention, the acclimation medium is a basic nitrate mineral medium, and the addition of nutrient elements can simultaneously meet the metabolic needs of the methane oxidizing bacteria enriched flora, the sulfur oxidizing bacteria enriched flora and the hydrogen oxidizing bacteria enriched flora.

[0061] When the enriched bacterial population of methane oxidizing bacteria, the enriched bacterial population of sulfur oxidizing bacteria and the enriched bacterial population of hydrogen oxidizing bacteria are mixed and inoculated into the acclimation medium, the corresponding enriched bacterial populations are preferably mixed into the acclimation medium at an inoculation ratio of 5% to 25% (v); the volume of the acclimation medium in the culture bottle is 10% to 25% (v). After the inoculation of the present invention is completed, an enriched bacterial population culture medium mixed system is obtained. After obtaining the enriched bacterial population culture medium mixed system, the present invention introduces a third mixed gas into the enriched bacterial population culture medium mixed system until the culture bottle is filled. Before the third mixed gas is introduced, nitrogen is preferably introduced first to exhaust the original air. After the original air is exhausted, the third mixed gas is introduced. The temperature of the acclimation culture of the present invention can be 28 to 35°C; intermittent oscillation is preferably performed during the acclimation culture; the rotation speed of the oscillation can be 120 to 280 rpm; the intermittent oscillation is 15 to 30 minutes of static and 10 to 20 minutes of oscillation. The acclimation culture of the present invention is preferably carried out in a shaking table. In the acclimation culture process of the present invention, it is preferred to replace the new acclimation medium and the new third mixed gas every 1 to 2 days. In the present invention, each time the new acclimation medium is replaced, it is preferred that the acclimation medium in the culture bottle is 10% to 25% (v). When the new acclimation medium is replaced for subculture in the present invention, it is preferred that 40% to 60% (v), or 50% of the bacterial liquid in the original culture system is inoculated into the new acclimation medium. In the present invention, the acclimation culture is subcultured continuously 3 to 4 times; the acclimation medium also includes Na2S and ammonium chloride; during the first acclimation culture, the mass concentration of Na2S in the acclimation medium is 1 to 2 mg / L, and the mass concentration of ammonium chloride is 100 to 300 mg / L; during each subculture, the mass concentration of Na2S and ammonium chloride in the acclimation medium is 1.5 to 2 times the mass concentration of Na2S and ammonium chloride in the previous culture. In the present invention, it is preferred to measure OD every 2 to 4 hours during each subculture. 600 The invention adds Na2S and ammonium chloride to the acclimation medium, mainly to improve the H2S tolerance and ammonia nitrogen degradation ability of the acclimated culture mixed bacteria.

[0062] After the domestication and cultivation are completed, the present invention obtains a composite microbial metabolite group.

[0063] After obtaining the composite microbial metabolite, the present invention preferably inoculates the composite microbial metabolite into an acclimation culture medium containing biogas slurry to perform simultaneous carbon fixation and nitrogen removal.

[0064] The present invention has no special limitation on the composition and source of the biogas slurry, and any conventional biogas slurry in the art can be used. To verify the effect, the biogas slurry used in the present invention is laboratory simulated biogas slurry; the composition of the biogas slurry includes: acetic acid: 1.5~2.5 g / L, propionic acid: 0.5~1.0 g / L, butyric acid: 0.2~0.5 g / L, glucose: 0.1~0.3 g / L, ammonium salt (NH4Cl): 0.3~0.5 g / L, potassium dihydrogen phosphate (KH2PO4): 0.05~0.1 g / L, potassium chloride (KCl): 0.05~0.1 g / L, sodium sulfate (Na2SO4): 0.02~0.05 g / L, ferric chloride (FeCl3): 0.001~0.005 g / L, magnesium sulfate (MgSO4·7H2O): 0.01~0.02 g / L, calcium chloride (CaCl2): 0.01~0.02 / L, and water is used as solvent. In the present invention, the amount of the biogas slurry added is 10% to 20% (v / v) of the culture container; the amount of the acclimation medium added is 10% to 25% of the culture container. In the present invention, when the composite microbial metabolite group is inoculated into the acclimation medium containing biogas slurry, the inoculation amount of the composite microbial metabolite group is 20% to 50% of the volume of the acclimation medium containing biogas slurry. After the inoculation is completed, the present invention obtains a microbial metabolite-biogas mixed system. The present invention introduces biogas into the microbial metabolite-biogas mixed system. The present invention does not specifically limit the source of the composition of the biogas, and conventional biogas in the field can be used. To verify the effect, the present invention uses the third mixed gas described in the above technical solution as biogas. Before the third mixed gas is introduced, the present invention preferably introduces nitrogen first to evacuate the original gas, and then introduces the third mixed gas until the third mixed gas fills the culture container. The temperature for the synchronous carbon fixation and denitrification of the present invention can be 28-35°C, or 28, 29, 30, 31, 32, 33, 34 or 35°C; the synchronous carbon fixation and denitrification process is preferably intermittent oscillation; the rotation speed of the oscillation can be 120-280rpm; the intermittent oscillation is 15-30min standing and 10-20min oscillation. The synchronous carbon fixation and denitrification of the present invention is preferably carried out in a shaking table; the time of the synchronous carbon fixation and denitrification can be 12-35d. The third mixed gas is preferably replaced every 1-2 days during the synchronous carbon fixation and denitrification process of the present invention. The purpose of replacing the third mixed gas every 1-2 days in this process of the present invention is that the composite microbial metabolic group can efficiently utilize gases such as H2, CO2, CH4 and H2S. If it is not replaced in time, the growth rate of the composite microbial metabolic period will be reduced, thereby reducing the protein yield during the synchronous carbon fixation and denitrification process.

[0065] The present invention can enable the enriched composite microbial metabolites to efficiently utilize methane and carbon dioxide in biogas and nutrients such as nitrogen and phosphorus in biogas slurry to generate microbial protein through the synchronous carbon fixation and denitrification process.

[0066] After the simultaneous carbon fixation and denitrification of the present invention is completed, the fermentation product is preferably centrifuged at 10000-12000 rpm for 10-30 minutes, and the supernatant is poured off; the centrifuged product is washed with phosphate buffer and centrifuged again, and the process is repeated 1-3 times; the fermentation product after multiple centrifugal washings is baked at 103-110° C. for 20-30 hours to obtain microbial protein.

[0067] The present invention provides application of the method described in the above technical solution in resource utilization of biogas slurry and / or biogas.

[0068] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0069] Example 1 A method for simultaneous carbon fixation and nitrogen removal, comprising the following steps: S1: Collect different soils at different sampling locations, and introduce the first mixed gas into different seed culture media for primary enrichment culture: Soil samples were collected from the soil 2~10cm below the surface of the impounded rice field, the compost residue of livestock and poultry manure, the soil 2~10cm below the surface of the swamp, and the sludge in the oxidation pond of the sewage treatment plant. Among them, the soil 2~10cm below the surface of the impounded rice field was taken from a rice field in Shunyi District, Beijing; the compost residue of livestock and poultry manure was taken from the composting site of the farm; the soil 2~10cm below the surface of the swamp was taken from the riverside swamp; the sludge in the oxidation pond of the sewage treatment plant was taken from the Xiaohongmen Sewage Treatment Plant in Beijing.

[0070] The reason for selecting these four soils is that sulfide oxidizing bacteria (abbreviated as sulfur oxidizing bacteria) are easily enriched in the soil 2~10cm below the surface of the swamp and / or the sludge in the oxidation pond of the sewage treatment plant. Similarly, hydrogen oxidizing bacteria are easily enriched in the soil 2~10cm below the surface of the water-retained rice field and / or the compost residue of livestock and poultry manure. Methane oxidizing bacteria are easily enriched in the soil 2~10cm below the surface of the water-retained rice field and / or the sludge in the oxidation pond of the sewage treatment plant.

[0071] The corresponding relationship of primary seed liquid culture medium: For ① the soil corresponding to the enrichment of hydrogen oxidizing bacteria is: soil 2-10 cm below the surface of the impounded paddy field and the compost residue of livestock and poultry manure. The culture medium corresponding to this soil is the first primary seed culture medium, and the composition of the first primary seed culture medium is: Na2HPO4: 2800 mg / L, KH2PO4: 1000 mg / L, (NH4)2SO4: 500 mg / L, MgCl2: 53 mg / L, CaCl2·2H2O: 50 mg / L, EDTA disodium: 0.5 mg / L, FeSO4·7H2O: 0.2 mg / L, ZnSO4·7H2O: 0.01 mg / L, MnCl2·4H2O: 0.003 mg / L, H3BO3: 0.03 mg / L, CoCl2·6H2O: 0.02 mg / L, CuCl2·2H2O: 0.001 mg / L, NiCl2·6H2O: 0.002 mg / L, Na2MoO4·2H2O: 0.003 mg / L.

[0072] The soil corresponding to the enrichment of ② sulfur-oxidizing bacteria is: soil 2~10cm below the surface of the swamp and sludge in the oxidation pond of the sewage treatment plant. The culture medium corresponding to this soil is the second primary seed culture medium. The composition of the second primary seed culture medium is: phenol red: 0.003g / L, 1,4-piperazine diethanesulfonic acid: 6.500g / L, sodium chloride: 25.000g / L, magnesium sulfate heptahydrate: 2.700g / L, potassium chloride: 0.500g / L, ammonium chloride: 0.250g / L, sodium thiosulfate pentahydrate: 2.480g / L, calcium chloride dihydrate: 0.140g / L, magnesium chloride hexahydrate: 4.300g / L, potassium hydrogen phosphate: 0.140g / L, ammonium ferrous sulfate hexahydrate: 0.002g / L, trace element solution: 1mL / L, vitamin solution: 10mL / L. The composition of the trace element solution is: MgSO4·7H2O 2.00g / L, MnSO4·H2O 0.5g / L, NaCl 0.8g / L, FeSO4·7H2O 0.15g / L, CoSO4·7H2O 0.18g / L, CaCl2·2H2O 0.15g / L, ZnSO4·7H2O 0.16g / L, CuSO4·5H2O 0.015g / L, AlK(SO4)2·12H2O 0.015g / L, H3BO3 0.015g / L, Na2MoO4·2H2O 0.03g / L, NiCl2·6H2O 0.03g / L, Na2SeO3·5H2O 0.2mg / L and Na2WO4·2H2O 0.2mg / L. The composition of the vitamin solution is: vitamin B7 1.5 mg / L, folic acid 1.5 mg / L, pyridoxine hydrochloride 8 mg / L, vitamin B1 4 mg / L, riboflavin 4 mg / L, vitamin B3 4 mg / L, vitamin B5 4 mg / L, vitamin B 12 0.15mg / L, p-aminobenzoic acid 4mg / L and (DL)-α-lipoic acid 4mg / L.

[0073] For ③ the soil corresponding to the enrichment of methanotrophic bacteria, the soil is 2-10 cm below the surface of the impounded rice field and the sludge in the oxidation pond of the sewage treatment plant. The culture medium corresponding to this soil is the third primary seed culture medium. The composition of the third primary seed culture medium is: KNO3: 100 mg / L, KH2PO4: 100 mg / L, MgSO4·7H2O: 50 mg / L, CaCl2·2H2O: 10 mg / L, EDTA: 5 mg / L, CuCl2·5H2O: 0.1 mg / L, FeSO4·7H2O: 2 mg / L, ZnSO4·7H2O: 0.1 mg / L, NiCl2·6H2O: 0.02 mg / L, CoCl2·6H2O: 0.2 mg / L, Na2MoO4: 0.03 mg / L.

[0074] The above culture media were dissolved in 1 L of distilled water, adjusted to pH 6.8-7.4 with phosphate buffer, and sterilized by high pressure at 121 °C for 30 min.

[0075] Enrichment process of hydrogen oxidizing bacteria, sulfur oxidizing bacteria and methane oxidizing bacteria: take the corresponding soil samples respectively, then take 10g of the corresponding soil and add it into a conical flask filled with 120mL of sterile distilled water, shake it in a shaker at 30℃ and 120rpm for 1.5h to obtain the soil-water mixture enriched with each bacteria.

[0076] 100 mL of the well-mixed soil sample (i.e., soil-water mixture) and 200 mL of the above culture medium were mixed in a conical flask and stirred at 250 rpm for 1 h using a magnetic stirrer.

[0077] 100 mL of the mixed solution was placed into a 500 mL sterile serum bottle, nitrogen was passed through for 5 minutes to exhaust the original air, and then the first mixed gas was passed through until the first mixed gas filled the sterile serum bottle.

[0078] The first mixed gas: For the above-mentioned ① hydrogen oxidizing bacteria enrichment, the corresponding first mixed gas I (referred to as the first hydrogen oxidizing bacteria mixed gas for the sake of distinction) is composed of H2, O2 and CO2, and the volume ratio of H2:O2:CO2 is 7:2:1; For the above-mentioned sulfur-oxidizing bacteria enrichment, the corresponding first mixed gas II (referred to as the first sulfur-oxidizing bacteria mixed gas for the sake of distinction) is composed of H2, O2, CO2, H2S, and the volume ratio of H2:O2:CO2:H2S is 3:3:2:2; For ③ methane oxidizing bacteria enrichment, the corresponding first mixed gas III (referred to as the first methane oxidizing bacteria mixed gas for easy distinction) is composed of CH4 and O2, and the volume ratio of CH4:O2 is 3:1.

[0079] After the first mixed gas was introduced into each of the above culture systems, the bacteria were cultured in a constant temperature shaker at 28° C. and 180 rpm.

[0080] For ① hydrogen oxidizing bacteria enrichment, when H2 consumption exceeds 50% or CO2 concentration no longer decreases, culture solution is obtained; For ② enrichment of sulfur-oxidizing bacteria, the H2S concentration is reduced until it no longer decreases, and a culture solution is obtained; ③ Methane oxidizing bacteria are enriched, and the CH4 concentration decreases until it no longer decreases, thereby obtaining a culture solution.

[0081] S2: Take 10 mL of culture medium from S1 and add it back into the serum bottle (500 mL), and add 100 mL of the corresponding secondary seed culture medium.

[0082] The secondary seed culture medium is prepared by adding 1.5wt.% of glucose and 0.6wt.% of yeast powder and 1.5wt.% of casein peptone to the corresponding primary seed culture medium, which are respectively referred to as the first secondary seed culture medium (for hydrogen oxidizing bacteria enrichment), the second secondary seed culture medium (for sulfur oxidizing bacteria enrichment) and the third secondary seed culture medium (for methane oxidizing bacteria enrichment), all of which are dissolved in 1L of distilled water, adjusted to pH 6.8-7.4 with phosphate buffer, and sterilized at 121°C under high pressure for 30min.

[0083] After the culture solution is added to the secondary seed culture medium, the gas is replaced with nitrogen and the first mixed gas is introduced. The culture conditions are the same as S1.

[0084] Similarly, for ① hydrogen oxidizing bacteria enrichment, when H2 consumption exceeds 50% or CO2 concentration no longer decreases; For ② sulfur-oxidizing bacteria enrichment, the H2S concentration decreased until it no longer decreased; For ③ enrichment of methane oxidizing bacteria, the CH4 concentration does not decrease after it decreases; After ①, ② and ③ are carried out until the corresponding gases no longer change, sub-culture is carried out respectively: 10mL of culture solution is added back into (100mL) of new corresponding secondary seed culture medium to obtain a culture system, and then 110mL of the culture system is cultured in a 500mL serum bottle. During this period, the secondary seed culture medium and the first mixed gas are replaced every 2 days, and the replacement is repeated 5 times in succession. After the last replacement of the first mixed gas, the corresponding gas volume remains unchanged, and hydrogen oxidizing bacteria enriched bacterial population, sulfur oxidizing bacteria enriched bacterial population and methane oxidizing bacteria enriched bacterial population are obtained respectively.

[0085] S3: Inoculate different secondary enriched bacterial communities into the same reactor containing the acclimation culture medium, i.e., inoculate the hydrogen oxidizing bacteria enriched bacterial community, sulfur oxidizing bacteria enriched bacterial community and methane oxidizing bacteria enriched bacterial community obtained in S2, add the acclimation culture medium in stages, and introduce the third mixed gas for intermittent culture: The composition of each 1L acclimation medium is: 1.00 g MgSO4·7H2O, 1.00 g KNO3, 0.27 g KH2PO4, 0.28 g Na2HPO4, 0.13 g CaCl2·2H2O, 1 mL trace elements. The composition of each 1L trace elements is: 1.00 g EDTA-2Na, 0.50 g FeSO4·7H2O, 0.07 g ZnSO4·7H2O, 0.03 g MnCl2·4H2O, 0.30 g H3BO3, 0.60 gCoCl2·6H2O, 0.02 g NiCl2·6H2O, 0.03 g Na2MoO4·2H2O and 0.01 g CuCl2·H2O. The acclimation medium also contains 1 mg / L Na2S and 100 mg / L ammonium chloride. The culture medium and trace elements were dissolved in 1 L of distilled water, the pH was adjusted to 6.8 to 7.2, and sterilized by high pressure at 121 °C for 30 min.

[0086] The bacterial cultures cultured in S2 (i.e., hydrogen oxidizing bacteria enriched flora, sulfur oxidizing bacteria enriched flora, and methane oxidizing bacteria enriched flora) were mixed in the acclimation medium at an inoculation ratio of 20% (v), and the acclimation medium in the culture bottle was 25% (v). The gas was replaced with nitrogen, the original gas in the culture bottle was emptied, and the third mixed gas was introduced to fill the headspace (i.e., fill the entire culture bottle). Place it in a constant temperature incubator at 28°C, shaker at 180rpm, and shake in intermittent mode, i.e., stand still for 15 minutes, then swing for 20 minutes. Replace the new acclimation medium and new gas (new third mixed gas) every 2 days. Each time the new acclimation medium is replaced, the acclimation medium in the culture bottle is 25% (v), and the bacteria in the original bottle are inoculated into the new bottle. The inoculation amount is 50% of the bacterial solution in the original bottle inoculated into the new acclimation medium. Continue to subculture for 3 rounds. During each subculture, measure the OD every 4 hours. 600 The third round of bacterial growth in this process is as follows Figure 1 shown.

[0087] For the first acclimation, 1 mg / L Na2S and 100 mg / L ammonium chloride were added to the culture medium. The amount of Na2S and ammonium chloride added in each subsequent passage was 1.5 times that of the previous one.

[0088] The third mixed gas contains H2, O2, CO2, CH4 and H2S, and the volume ratio of H2:O2:CO2:CH4:H2S is 1:10:25:60:0.2.

[0089] S4: introducing the third mixed gas into the container containing the acclimation culture medium (the volume of the acclimation culture medium in the container is 25%), and adding 10% of the biogas slurry in the culture container to culture the complex microbial metabolites.

[0090] The bacterial liquid (complex microbial metabolites) in the S3 medium was inoculated into the culture bottle containing the acclimatization medium of the biogas slurry, with an inoculation ratio of 40% (v). The culture time was 20 days. During the culture process, the culture temperature was 28°C, the shaker was 180 rpm, and the shaking mode was intermittent, that is, it was still for 15 minutes and then swung for 20 minutes. New gas (third mixed gas) was replaced every 2 days.

[0091] The above-mentioned biogas slurry is laboratory simulated biogas slurry, with the following composition: acetic acid (acetic acid): 1.5 g / L, propionic acid: 0.5 g / L, butyric acid: 0.2 g / L, glucose: 0.2 g / L, ammonium salt (NH4Cl): 0.3 g / L, potassium dihydrogen phosphate (KH2PO4): 0.05 g / L, potassium chloride (KCl): 0.05 g / L, sodium sulfate (Na2SO4): 0.02 g / L, ferric chloride (FeCl3): 0.002 g / L, magnesium sulfate (MgSO4·7H2O): 0.01 g / L, calcium chloride (CaCl2): 0.01 g / L, with water as solvent.

[0092] After the culture was completed, bacterial protein was isolated. During the culture process, the culture fluid was taken every 4 days to separate the bacterial cells, and the cell dry weight and protein content were tested.

[0093] The protein separation process includes the following steps: centrifuging the fermentation product at 10,000 rpm for 10 minutes and discarding the supernatant; washing the centrifuged product with phosphate buffer and centrifuging again, repeating this process three times; and baking the fermentation product after multiple centrifugation and washing at 105° C. for 24 hours to obtain microbial protein.

[0094] During the culture process, the results of cell dry weight and protein content were as follows: Figure 2 The final biomass dry weight (i.e., cell dry weight) was 495 mg / L, and the protein content in the biomass was 59.4%.

[0095] Example 2 A method for simultaneous carbon fixation and denitrification, the steps are the same as those in Example 1, the only difference being that: ① the soil corresponding to the enrichment of hydrogen oxidizing bacteria is 2 to 10 cm of soil below the surface of a water-retaining paddy field. ② the soil corresponding to the enrichment of sulfur oxidizing bacteria is 2 to 10 cm of soil below the surface of a swamp. ③ the soil corresponding to the enrichment of methane oxidizing bacteria is 2 to 10 cm of soil below the surface of a water-retaining paddy field.

[0096] The dry weight of biomass was 494 mg / L, and the protein content in biomass was 51.8%.

[0097] Example 3 A method for simultaneous carbon fixation and denitrification, the steps are the same as those in Example 1, the only difference being that: ① the soil corresponding to the enrichment of hydrogen oxidizing bacteria is the soil collected from the compost residue of livestock and poultry manure. ② the soil corresponding to the enrichment of sulfur oxidizing bacteria is the soil collected from the sludge of the oxidation pond of the sewage treatment plant. ③ the soil corresponding to the enrichment of methane oxidizing bacteria is the soil collected from the sludge of the oxidation pond of the sewage treatment plant.

[0098] The dry weight of biomass was 505 mg / L, and the protein content in biomass was 50.2%.

[0099] Example 4 A method for simultaneous carbon fixation and denitrification, the steps are the same as those in Example 1, the only difference being that: ① the soil corresponding to the enrichment of hydrogen oxidizing bacteria is the soil 2 to 10 cm below the surface of the water-retaining paddy field. ② the soil corresponding to the enrichment of sulfur oxidizing bacteria is the soil collected from the sludge in the oxidation pond of the sewage treatment plant. ③ the soil corresponding to the enrichment of methane oxidizing bacteria is the soil collected from the sludge in the oxidation pond of the sewage treatment plant.

[0100] The dry weight of biomass was 478 mg / L, and the protein content in biomass was 43.5%.

[0101] Example 5 A method for simultaneous carbon fixation and denitrification, the steps are the same as those in Example 1, the only difference being that: ① the soil corresponding to the enrichment of hydrogen oxidizing bacteria is 2 to 10 cm of soil below the surface of a water-retaining paddy field. ② the soil corresponding to the enrichment of sulfur oxidizing bacteria is 2 to 10 cm of soil below the surface of a swamp. ③ the soil corresponding to the enrichment of methane oxidizing bacteria is the soil collected from the sludge in the oxidation pond of a sewage treatment plant.

[0102] The dry weight of biomass was 483 mg / L, and the protein content in biomass was 46.8%.

[0103] Comparative Example 1 A method for simultaneous carbon fixation and nitrogen removal, the steps are the same as those in Example 1, the only difference being that the enrichment and use of hydrogen oxidizing bacteria are not performed.

[0104] The final biomass dry weight (i.e. cell dry weight) was 358 mg / L, and the protein content in the biomass was 42.7%. In addition, during the culture process, CO2 was produced due to the metabolism of methane oxidizing bacteria, which reduced the carbon fixation effect.

[0105] Comparative Example 2 A method for simultaneous carbon fixation and nitrogen removal, the steps are the same as those in Example 1, the only difference being that sulfur oxidizing bacteria enrichment and use are not performed. The culture process is inhibited by H2S in the biogas, and the growth of microorganisms is seriously affected.

[0106] The final biomass dry weight (i.e., cell dry weight) was 215 mg / L, and the protein content in the biomass was 15.2%.

[0107] Comparative Example 3 A method for simultaneous carbon fixation and nitrogen removal, the steps are the same as those in Example 1, the only difference being that methane oxidizing bacteria are not enriched and used, and methane in biogas is not fully utilized.

[0108] The final biomass dry weight (i.e., cell dry weight) was 362 mg / L, and the protein content in the biomass was 39.5%.

[0109] Example 6 A method for simultaneous carbon fixation and nitrogen removal, the steps are the same as those in Example 1, the only difference being that the composition of the first mixed gas of hydrogen oxidizing bacteria is H2:O2:CO2 in a volume ratio of 50:30:20.

[0110] The final biomass dry weight (i.e., cell dry weight) was 462 mg / L, and the protein content in the biomass was 50.6%.

[0111] Example 7 A method for simultaneous carbon fixation and nitrogen removal, the steps are the same as those in Example 1, the only difference being that the composition of the first mixed gas of hydrogen oxidizing bacteria is H2:O2:CO2 in a volume ratio of 60:25:15.

[0112] The final biomass dry weight (i.e., cell dry weight) was 471 mg / L, and the protein content in the biomass was 51.5%.

[0113] Example 8 A method for simultaneous carbon fixation and nitrogen removal, the steps are the same as those in Example 1, the only difference being that the first mixed gas composition of sulfur-oxidizing bacteria is H2S, with a volume ratio of H2:O2:CO2:H2S of 30:20:20:20.

[0114] The final biomass dry weight (i.e., cell dry weight) was 427 mg / L, and the protein content in the biomass was 51.6%.

[0115] Example 9 A method for simultaneous carbon fixation and denitrification, the steps are the same as those in Example 1, the only difference being that the first mixed gas composition of sulfur oxidizing bacteria is H2S, H2:O2:CO2:H2S volume ratio: 50:30:30:30. An excessively high initial ratio of hydrogen sulfide may have a counter-effect, and instead reduce the tolerance of sulfur oxidizing bacteria.

[0116] The final biomass dry weight (i.e., cell dry weight) was 401.5 mg / L, and the protein content in the biomass was 49.5%.

[0117] Example 10 A method for simultaneous carbon fixation and nitrogen removal, the steps are the same as those in Example 1, the only difference being that the first mixed gas composition of sulfur-oxidizing bacteria is H2S, with a volume ratio of H2:O2:CO2:H2S of 40:25:25:25.

[0118] The final biomass dry weight (i.e., cell dry weight) was 435 mg / L, and the protein content in the biomass was 50.4%.

[0119] Embodiment 11 A method for simultaneous carbon fixation and nitrogen removal, the steps are the same as those in Example 1, the only difference being that the first mixed gas of methane oxidizing bacteria is composed of CH4:O2 in a volume ratio of 50:50.

[0120] The final biomass dry weight (i.e., cell dry weight) was 436 mg / L, and the protein content in the biomass was 52.8%.

[0121] Example 12 A method for simultaneous carbon fixation and nitrogen removal, the steps are the same as those in Example 1, the only difference being that the first mixed gas of methane oxidizing bacteria is composed of CH4:O2 in a volume ratio of 60:40.

[0122] The final biomass dry weight (i.e., cell dry weight) was 485 mg / L, and the protein content in the biomass was 55.4%.

[0123] Example 13 A method for enriching methane oxidizing bacteria, the steps are the same as those in Example 1, the only difference being that the volume ratio of CH4 to O2 is 80:20.

[0124] Comparative Example 4 A method for enriching hydrogen oxidizing bacteria, the steps are the same as those in Example 1, the only difference being that the composition of the first mixed gas of hydrogen oxidizing bacteria is H2:O2:CO2 in a volume ratio of 50:20:30.

[0125] Comparative Example 5 A method for enriching hydrogen oxidizing bacteria, the steps are the same as those in Example 1, the only difference being that the composition of the first mixed gas of hydrogen oxidizing bacteria is H2:O2:CO2 in a volume ratio of 75:5:20.

[0126] Comparative Example 6 A method for enriching sulfur-oxidizing bacteria, the steps are the same as those in Example 1, the only difference being that the first mixed gas composition of the sulfur-oxidizing bacteria is H2S, with a volume ratio of H2:O2:CO2:H2S of 30:20:20:40.

[0127] Comparative Example 7 A method for enriching sulfur-oxidizing bacteria, the steps are the same as those in Example 1, the only difference being that the first mixed gas composition of the sulfur-oxidizing bacteria is H2S, with a volume ratio of H2:O2:CO2:H2S of 50:30:30:10.

[0128] Comparative Example 8 A method for enriching methane oxidizing bacteria, the steps are the same as those in Example 1, the only difference being that the volume ratio of CH4 to O2 is 40:60.

[0129] Application Example 1 The time for enriching functional bacteria in Example 1 and Example 13 and Comparative Examples 4 to 8 is shown in Table 1. The time here is the time for obtaining the corresponding functional bacteria enriched bacterial population after the subculture is completed.

[0130] Table 1 Time for enriching functional bacteria in Example 1 and Example 13 and Comparative Examples 4 to 8

[0131] In summary, the method for simultaneous carbon fixation and denitrification provided by the present invention enriches and screens a composite microbial metabolite group in which multiple microbial populations mainly composed of methane-oxidizing bacteria coexist to form a synergistic metabolic system. The synergistic metabolic system has H2S tolerance and can metabolize methane and carbon dioxide in biogas and N and P nutrients in biogas slurry to achieve the purpose of simultaneous carbon fixation and denitrification.

[0132] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for simultaneous carbon fixation and nitrogen removal, characterized in that: The following steps are involved: The hydrogen-oxidizing bacteria mixed gas is used to enrich the microbial population dominated by hydrogen-oxidizing bacteria in the soil to obtain a hydrogen-oxidizing bacteria-enriched bacterial population; The sulfur-oxidizing bacteria mixed gas is used to enrich the microbial population dominated by sulfur-oxidizing bacteria in the soil to obtain a sulfur-oxidizing bacteria enriched bacterial population; The methane-oxidizing bacteria mixed gas is used to enrich the microbial population dominated by methane-oxidizing bacteria in the soil to obtain a methane-oxidizing bacteria-enriched bacterial population; The enriched bacterial groups of methane oxidizing bacteria, the enriched bacterial groups of sulfur oxidizing bacteria and the enriched bacterial groups of hydrogen oxidizing bacteria are mixed and inoculated into an acclimation medium for acclimation and culture to obtain a composite microbial metabolic group; The composite microbial metabolites are inoculated into an acclimated culture medium containing biogas and biogas is introduced to perform simultaneous carbon fixation and nitrogen removal; The hydrogen oxidizing bacteria mixed gas comprises H2, O2 and CO2, and the volume ratio of H2, O2 and CO2 is (50-70): (30-20): (20-10); The sulfur oxidizing bacteria mixed gas includes H2, O2, CO2 and H2S, and the volume ratio of H2, O2, CO2 and H2S is (30-50): (20-30): (20-30): (20-30); The methane oxidizing bacteria mixed gas includes CH4 and O2, and the volume ratio of CH4 to O2 is (50-80): (50-20).

2. The method according to claim 1, characterized in that: Soils rich in hydrogen oxidizing bacteria include soil 2 to 10 cm below the surface of waterlogged rice fields and / or compost residues of livestock and poultry manure; Soils rich in sulfur-oxidizing bacteria include 2-10 cm soil below the surface of marshes and / or sludge from oxidation ponds in sewage treatment plants; Soils enriched with methane-oxidizing bacteria include soil 2 to 10 cm below the surface of impounded rice fields and / or sludge from oxidation ponds in sewage treatment plants.

3. The method according to claim 1, characterized in that: Methods for enriching soil microbial populations dominated by hydrogen oxidizing bacteria include: Inoculate the microorganisms in the soil into the primary seed culture medium of hydrogen oxidizing bacteria and introduce the hydrogen oxidizing bacteria mixed gas for cultivation until the H2 consumption is more than 50% or the CO2 concentration no longer decreases, thereby obtaining the primary seed solution of hydrogen oxidizing bacteria; The hydrogen oxidizing bacteria primary seed liquid is inoculated into the hydrogen oxidizing bacteria secondary seed culture medium, and a hydrogen oxidizing bacteria mixed gas is introduced for culturing to obtain a hydrogen oxidizing bacteria enriched bacterial population; The composition of the primary seed culture medium for hydrogen oxidizing bacteria includes: Na2HPO4: 2200~2800 mg / L, KH2PO4: 500~1500mg / L, (NH4)2SO4: 450~500 mg / L, MgCl2: 45~60mg / L, CaCl2·2H2O: 40~50 mg / L, disodium EDTA: 0.2~0.7 mg / L, FeSO4·7H2O: 0.1~0.5 mg / L, ZnSO4·7H2O: 0.01~0.02 mg / L, MnCl2·4H2O: 0.002~0.003 mg / L, H3BO3: 0.01~0.04 mg / L, CoCl2·6H2O: 0.01~0.02 mg / L, CuCl2·2H2O: 0.001~0.01 mg / L, NiCl2·6H2O: 0.002~0.004 mg / L, Na2MoO4·2H2O: 0.001~0.003 mg / L; The composition of the hydrogen oxidizing bacteria secondary seed culture medium is based on the hydrogen oxidizing bacteria primary seed culture medium and further includes: 1%~3% glucose, 0.6%~2% yeast powder and 1.5%~3.2% casein peptone.

4. The method according to claim 1, characterized in that: Methods for enriching the soil microbial population dominated by sulfur-oxidizing bacteria include: Inoculating soil microorganisms into a primary seed culture medium for sulfur-oxidizing bacteria and introducing a sulfur-oxidizing bacteria mixed gas for culturing until the H2S concentration no longer decreases, thereby obtaining a primary seed solution for sulfur-oxidizing bacteria; The primary seed liquid of sulfur-oxidizing bacteria is inoculated into the secondary seed culture medium of sulfur-oxidizing bacteria, and a sulfur-oxidizing bacteria mixed gas is introduced for culturing to obtain a sulfur-oxidizing bacteria enriched bacterial population; The composition of the primary seed culture medium for sulfur oxidizing bacteria includes: phenol red: 0.001-0.003 g / L, 1,4-piperazine diethanesulfonic acid: 4.5-8.5 g / L, sodium chloride: 10-25.000 g / L, magnesium sulfate heptahydrate: 1.1-2.9 g / L, potassium chloride: 0.1-1 g / L, ammonium chloride: 0.015-0.3 g / L, sodium thiosulfate pentahydrate: 1.5-3.2 g / L, calcium chloride dihydrate: 0.05-0.2 g / L, magnesium chloride hexahydrate: 2.5-4.5 g / L, potassium hydrogen phosphate: 0.05-0.15 g / L, ammonium ferrous sulfate hexahydrate: 0.001-0.002 g / L, trace element solution: 1 mL / L and vitamin solution: 10 mL / L; The composition of the secondary seed culture medium for sulfur oxidizing bacteria is based on the primary seed culture medium for sulfur oxidizing bacteria and further includes: 1% to 3% glucose, 0.6% to 2% yeast powder and 1.5% to 3.2% casein peptone.

5. The method according to claim 1, characterized in that: Methods for enriching soil microbial populations dominated by methanotrophs include: Inoculating soil microorganisms into a primary seed culture medium for methane oxidizing bacteria and introducing a mixed gas of methane oxidizing bacteria for cultivation until the CH4 concentration no longer decreases, thereby obtaining a primary seed solution for methane oxidizing bacteria; The primary seed liquid of methanotrophic bacteria is inoculated into the secondary seed culture medium of methanotrophic bacteria, and a mixed gas of methanotrophic bacteria is introduced for cultivation to obtain an enriched bacterial population of methanotrophic bacteria; The composition of the primary seed culture medium for methanotrophic bacteria includes: KNO3: 100~200 mg / L, KH2PO4: 100~200 mg / L, MgSO4·7H2O: 25~55 mg / L, CaCl2·2H2O: 5~20 mg / L, EDTA: 2~5 mg / L, CuCl2·5H2O: 0.05~0.1 mg / L, FeSO4·7H2O: 1~2 mg / L, ZnSO4·7H2O: 0.1~0.2 mg / L, NiCl2·6H2O: 0.008~0.02 mg / L, CoCl2·6H2O: 0.1~0.2 mg / L and Na2MoO4: 0.01~0.03 mg / L; The composition of the secondary seed culture medium for methanotrophic bacteria is based on the primary seed culture medium for methanotrophic bacteria and further includes: 1% to 3% glucose, 0.6% to 2% yeast powder and 1.5% to 3.2% casein peptone.

6. The method according to claim 1, characterized in that: A mixed gas is introduced during the acclimation culture; the mixed gas consists of H2, O2, CO2, CH4 and H2S; the volume ratio of H2, O2, CO2, CH4 and H2S is (0.1-1): (10-20): (15-25): (50-60): (0.1-0.3).

7. The method according to claim 1, characterized in that: The composition of each 1L acclimatization medium includes: 0.5~1.00 g MgSO4·7H2O, 1.00~2.5 g KNO3, 0.15~0.3 g KH2PO4, 0.2~0.45 g Na2HPO4, 0.1~0.15 g CaCl2·2H2O and 1 mL trace element solution.

8. The method according to claim 1, characterized in that: The acclimation culture is continuously subcultured 3 to 4 times; the acclimation culture medium also includes Na2S and ammonium chloride; during the first acclimation culture, the mass concentration of Na2S in the acclimation culture medium is 1 to 2 mg / L, and the mass concentration of ammonium chloride is 100 to 300 mg / L; during each subculture, the mass concentration of Na2S and ammonium chloride in the acclimation culture medium is 1.5 to 2 times the mass concentration of Na2S and ammonium chloride in the previous culture.

9. Application of the method according to any one of claims 1 to 8 in the resource utilization of biogas slurry and / or biogas.

10. Use of the method according to any one of claims 1 to 8 in preparing protein.

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