Bacterial agent for producing methane by fermenting blue-green algae bottom mud

Abstract

The invention discloses a cyanobacteria bottom mud fermentation methanogenic bacterial agent, which comprises clostridium butyricum, bacillus amyloliquefaciens and methanosarcina martensii, and is characterized in that the clostridium butyricum accounts for 30-60% of the total number of bacterial cells, the bacillus amyloliquefaciens accounts for 25-50% of the total number of the bacterial cells, and the methanosarcina martensii accounts for 30-60% of the total number of the bacterial cells. The methanosarcina marzeberi accounts for 10%-25% of the total number of bacterial cells. The advanced technology of adding microbial compound bacteria and combining high-temperature and high-pressure hydrolysis and anaerobic digestion technologies is utilized, the biodegradability is improved by destroying the cell structure of organic matter, the efficiency and stability of traditional anaerobic fermentation are remarkably improved, the VS removal rate after pyrohydrolysis fermentation reaches 67.5%-70.4%, the gas production rate in the anaerobic fermentation process after pyrohydrolysis is effectively increased, and the production cost is reduced. The yield of methane can be increased, the water body environment can be improved, recycling of bottom mud resources can be promoted, and green and sustainable ecological restoration is achieved.

Description

Technical Field

[0001] This invention relates to the field of anaerobic fermentation technology, and in particular to a methanogenic agent for fermenting cyanobacteria sediment. Background Technology

[0002] With industrial development, eutrophication of freshwater bodies is becoming increasingly serious. Lakes, reservoirs, and rivers frequently experience severe cyanobacterial blooms. Cyanobacteria proliferate rapidly in eutrophic waters, reducing water transparency and damaging water function. Long-term accumulation of cyanobacteria leads to decay and foul odors, releasing large amounts of nitrogen, phosphorus, and other nutrients, as well as harmful substances such as hydrogen sulfide and microcystin, causing secondary pollution. Currently, the primary method for dealing with cyanobacteria is harvesting. Timely harvesting of cyanobacteria can purify water quality. However, cyanobacteria cannot be directly consumed by poultry and livestock. The large quantities of harvested cyanobacteria are not being processed effectively and promptly. How to dispose of or utilize the enormous biomass of harvested cyanobacteria remains a challenging problem.

[0003] In the process of river and lake pollution control, sediment pollution remediation is one of the main challenges. To address the problem of sediment accumulation and improve water quality in lake areas and urban rivers, a large amount of sediment is dredged from Taihu Lake, Liangxi River, and its tributaries every year. The sediment in eutrophic water bodies contains abundant anaerobic microorganisms, which can serve as inoculum for the anaerobic fermentation of cyanobacteria. Therefore, simultaneous anaerobic fermentation of sediment and cyanobacteria is a feasible method for the disposal and resource recovery of sediment and cyanobacteria pollution.

[0004] In current technologies that use anaerobic fermentation of mixed cyanobacteria and sludge to produce methane for biogas, biomass waste is directly used for anaerobic fermentation under normal temperature conditions. However, the microbial community in this fermentation is singular, resulting in very low or no gas production efficiency. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a methanogenic agent for fermentation of cyanobacteria sediment.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A methanogenic agent for fermentation of cyanobacteria sediments, comprising Clostridium butyricum, Bacillus amyloliquefaciens, and Methanococcus marzeri, characterized in that the percentage of Clostridium butyricum in the total bacterial cell count is 30%-60%, the percentage of Bacillus amyloliquefaciens in the total bacterial cell count is 25%-50%, and the percentage of Methanococcus marzeri in the total bacterial cell count is 10%-25%.

[0008] Preferably, the *Clostridium butyricum*, *Bacillus amyloliquefaciens*, and *Methanococcus maszei* are cultured in a growth medium to expand the bacterial count to 10.10 The concentration of cells / mL was determined by mixing Clostridium butyricum, Bacillus amyloliquefaciens, and Methanococcus maserii according to the percentage of cells to obtain a mixed bacterial agent.

[0009] Preferably, the growth medium comprises: CH3COONa (0.3 g / L), NH4Cl (0.1 g / L), K2HPO4 (0.02 g / L), NaHCO3 (0.2 g / L), NaCl (0.2 g / L), MgSO4·7H2O (0.02 g / L), and yeast extract (0.1 g / L), and the medium is sterilized at 0.1 MPa for 20 min, with an inoculum volume of 10% of the medium.

[0010] Preferably, when using the growth medium, the Clostridium butyricum is further supplemented with 10 g / L glucose, 0.1 mg / L biotin and 0.5 mg / L folic acid, and the pH is adjusted to 6.8-7.2 using hydrochloric acid and sodium hydroxide. The culture is carried out in an anaerobic tank using a shaker at 100-200 rpm and a temperature of 30℃-37℃.

[0011] Preferably, when using the growth medium, the Bacillus amyloliquefaciens is further supplemented with 10 g / L starch and 5 g / L yeast extract, placed on a constant temperature shaker, and cultured at 37°C with the shaker speed set to 150-200 rpm for constant temperature aerobic culture.

[0012] Preferably, when using the growth medium for *Methanococcus maszei*, 0.2 g / L of acidified amino acid solution and 10 ml / L of vitamin solution are added. The acidified amino acid solution is lysine and glycine, and the vitamin solution is thiamine and vitamin B12. The pH is adjusted to 7.0-7.5 using HCl or NaOH. The inoculated culture medium is placed in a temperature-controlled incubator at 37°C, and a mixed gas of 5% CO2, 5% H2 and 90% N2 is introduced to remove oxygen for anaerobic culture.

[0013] Preferably, when using mixed bacterial agents for methanogenic fermentation of cyanobacteria in sediment, the cyanobacteria are first crushed and pulped, with a particle size of <5mm after crushing. The cyanobacteria and sediment are mixed at a V / S ratio of 60:40, and the total solids concentration is controlled at about 12%. The cyanobacteria-sludge mixture is then placed in an autoclave for hot water hydrolysis pretreatment at a temperature of 115℃-175℃ for a reaction time of 25min.

[0014] Preferably, fermentation is carried out using a mixture of mixed bacterial agent and hot hydrolyzed cyanobacterial sludge. A 10L sealed glass fermenter is selected. The initial inoculum in the reactor is 2L, which includes 1.8L of hot hydrolyzed product and 200mL of bacterial agent. Subsequently, 200mL of feed is added daily, along with a sludge mixture that has not undergone hot hydrolysis pretreatment. The sludge fermentation process takes 15 days. The top space of the fermenter is filled with nitrogen, and the reactor is sealed with a rubber stopper and paraffin wax.

[0015] Preferably, during anaerobic fermentation, the pH will change as acidic substances accumulate. The pH should be checked and adjusted regularly using sodium hydroxide to control the pH value at 6.8-7.2. The carbon-nitrogen ratio should be maintained at 20:1 to 30:1, while ensuring that the organic loading rate is between 1.66-4.19 gVS / (L·d). The temperature for anaerobic fermentation is 25±1℃, and stirring is performed at a speed of 60 r / min.

[0016] Preferably, during the fermentation process, the content of gases such as methane and carbon dioxide is monitored by a gas analyzer to determine the methane production efficiency. After fermentation, the methane gas in the cyanobacteria sediment is collected and can be used for energy utilization. The remaining solid matter can be further analyzed or processed. The fermentation liquid and gas are analyzed to detect key indicators such as methane yield and organic matter degradation rate in the sediment.

[0017] The methanogenic bacteria agent for fermentation of cyanobacteria sediment proposed in this invention has the following beneficial effects:

[0018] 1. Fermentation of cyanobacterial sediment mixture by combining compound bacterial agents with high-temperature hydrolysis: By applying Clostridium butyricum, Bacillus amyloliquefaciens and Methanococcus maserii in combination, a complete microbial community can be formed during the fermentation of cyanobacterial sediment. These communities play roles in decomposing organic matter, producing fermentation intermediates and ultimately generating methane, thereby significantly increasing methane production.

[0019] 2. Using these microbial combinations can effectively accelerate the degradation process of cyanobacteria sediment, reduce organic pollutants in the sediment, and thus achieve environmental remediation. This biological treatment method has a lower environmental risk and is greener and more sustainable than physical and chemical methods.

[0020] 3. Methane is a clean energy source. Utilizing the fermentation of blue-green algae sediment to produce methane can not only treat water sediment but also convert waste into valuable energy. This resource utilization helps promote the development of a circular economy.

[0021] 4. The organic matter and nutrients (such as nitrogen and phosphorus) in the cyanobacterial sediment can be reduced by the microbial fermentation and degradation of the compound bacterial agent of the present invention, thereby reducing the risk of eutrophication of water bodies and playing a positive role in improving the quality of the water environment. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the bacterial strain mixing ratio for a methanogenic agent produced by fermentation of cyanobacteria in sediment proposed in this invention.

[0023] Figure 2 This is a schematic diagram showing the anaerobic fermentation gas production results of a methanogenic bacteria agent for cyanobacteria sediment fermentation proposed in this invention. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0025] In the description of this invention, it should be understood that the terms "upper", "lower", "front", "rear", "left", "right", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0026] Example 1: This application provides a methanogenic agent for fermentation of cyanobacteria sediment, comprising Clostridium butyricum, Bacillus amyloliquefaciens, and Methanococcus marzere. Clostridium butyricum accounts for 40% of the total bacterial cell count, Bacillus amyloliquefaciens accounts for 50% of the total bacterial cell count, and Methanococcus marzere accounts for 10% of the total bacterial cell count.

[0027] Furthermore, the *Clostridium butyricum* strain is identified as *Clostridium butyricum* CGMCC 1.5205; the *Bacillus amyloliquefaciens* strain is identified as *Bacillus amyloliquefaciens* CGMCC 1.857; and the *Methanosarcina mazei* strain is identified as *Methanosarcina mazei* CGMCC 1.5193.

[0028] Furthermore, *Clostridium butyricum*, *Bacillus amyloliquefaciens*, and *Methanococcus maszei* were cultured on growth media to expand the bacterial count, reaching a total bacterial count of 10. 10 The concentration of cells / mL was determined by mixing Clostridium butyricum, Bacillus amyloliquefaciens, and Methanococcus maserii according to the percentage of cells to obtain a mixed bacterial agent.

[0029] Furthermore, the growth medium consisted of: CH3COONa (0.3 g / L), NH4Cl (0.1 g / L), K2HPO4 (0.02 g / L), NaHCO3 (0.2 g / L), NaCl (0.2 g / L), MgSO4·7H2O (0.02 g / L), and yeast extract (0.1 g / L). The medium was sterilized at 0.1 MPa for 20 min, and the inoculum volume was 10% of the medium volume.

[0030] Furthermore, when using the growth medium for Clostridium butyricum, 10 g / L glucose, 0.1 mg / L biotin, and 0.5 mg / L folic acid were added, and the pH was adjusted to 6.8-7.2 using hydrochloric acid and sodium hydroxide. The culture was carried out in an anaerobic tank using a shaker at 100-200 rpm and a temperature of 30℃-37℃.

[0031] Furthermore, when using the growth medium for Bacillus amyloliquefaciens, 10 g / L of starch and 5 g / L of yeast extract were added, and the mixture was placed on a constant temperature shaker and cultured at 37°C with the shaker speed set to 150-200 rpm for constant temperature aerobic culture.

[0032] Furthermore, when using the growth medium for *Methanococcus maszei*, 0.2 g / L of acidified amino acid solution and 10 ml / L of vitamin solution were added. The acidified amino acid solution consisted of lysine and glycine, and the vitamin solution consisted of thiamine and vitamin B12. The pH was adjusted to 7.0-7.5 using HCl or NaOH. The inoculated medium was placed in a temperature-controlled incubator at 37°C, and a mixed gas of 5% CO2, 5% H2, and 90% N2 was introduced to remove oxygen for anaerobic culture.

[0033] Furthermore, when using mixed bacterial agents for methanogenic fermentation of cyanobacteria in sediment, the cyanobacteria are first crushed and pulped to a particle size of <5mm. The cyanobacteria and sediment are mixed at a V / S ratio of 60:40, and the total solids concentration is controlled at around 12%. The cyanobacteria-sludge mixture is then placed in an autoclave for hot water hydrolysis pretreatment at a temperature of 115℃-175℃ for a reaction time of 25min.

[0034] Furthermore, fermentation was carried out using a mixture of mixed microbial agents and hot-hydrolyzed cyanobacterial sludge. A 10L sealed glass fermenter was selected, with an initial inoculum of 2L, including 1.8L of hot-hydrolyzed product and 200mL of microbial agents. Subsequently, 200mL of feed was added daily, along with a mixture of sludge without hot-hydrolyzed pretreatment. The sludge fermentation process lasted 15 days. The top space of the fermenter was filled with nitrogen, and the reactor was sealed with a rubber stopper and paraffin wax.

[0035] Furthermore, during anaerobic fermentation, the pH will change as acidic substances accumulate. The pH should be checked and adjusted regularly using sodium hydroxide to maintain a pH of 6.8-7.2. The carbon-to-nitrogen ratio should be maintained at 20:1 to 30:1, while ensuring that the organic loading rate is between 1.66-4.19 gVS / (L·d). The anaerobic fermentation temperature should be 25±1℃, and stirring should be performed at a speed of 60 r / min.

[0036] Furthermore, during the fermentation process, the content of gases such as methane and carbon dioxide is monitored using a gas analyzer to determine the methane production efficiency. After fermentation, the methane gas in the cyanobacteria sediment is collected and can be used for energy utilization, while the remaining solids can be further analyzed or processed. The fermentation broth and gases are analyzed to detect key indicators such as methane yield and the degradation rate of organic matter in the sediment.

[0037] Example 2: This application provides a methanogenic agent for fermentation of cyanobacteria sediments. The difference from Example 1 is that Clostridium butyricum accounts for 50% of the total number of bacterial cells, Bacillus amyloliquefaciens accounts for 40% of the total number of bacterial cells, and Methanococcus maszei accounts for 10% of the total number of bacterial cells.

[0038] Example 3: This application provides a methanogenic agent for fermentation of cyanobacteria sediments. The difference from Example 1 is that Clostridium butyricum accounts for 60% of the total bacterial cell count, Bacillus amyloliquefaciens accounts for 25% of the total bacterial cell count, and Methanococcus marzere accounts for 15% of the total bacterial cell count.

[0039] Example 4: This application provides a methanogenic agent for fermentation of cyanobacteria sediments. The difference from Example 1 is that Clostridium butyricum accounts for 30% of the total number of bacterial cells, Bacillus amyloliquefaciens accounts for 50% of the total number of bacterial cells, and Methanococcus marzeri accounts for 20% of the total number of bacterial cells.

[0040] Example 5: This application provides a methanogenic agent for fermentation of cyanobacteria sediments. The difference from Example 1 is that Clostridium butyricum accounts for 40% of the total bacterial cell count, Bacillus amyloliquefaciens accounts for 35% of the total bacterial cell count, and Methanococcus marzere accounts for 25% of the total bacterial cell count.

[0041] The anaerobic fermentation gas production results of multiple embodiments are shown in the table below:

[0042]

[0043] This invention utilizes an advanced process combining the addition of microbial complex bacteria with high-temperature and high-pressure hydrolysis and anaerobic digestion technology. By destroying the cell structure of organic matter and improving its biodegradability, it significantly improves the efficiency and stability of traditional anaerobic fermentation. The VS removal rate after hot hydrolysis fermentation reaches 67.5%-70.4%, effectively improving the gas production rate of the anaerobic fermentation process after hot hydrolysis.

[0044] Secondly, the microbial agent used in this invention is a compound microbial agent. Among them, Clostridium butyricum is an anaerobic bacterium that can decompose complex organic matter and mainly produces short-chain fatty acids (such as butyric acid) through fermentation, which plays an important role in the initial degradation process of bottom sediment. Bacillus amyloliquefaciens can efficiently decompose starchy substances and convert them into simple sugars that can be fermented, thus promoting the subsequent fermentation process. Methanococcus marzeri is an anaerobic microorganism that specifically participates in methane production and can convert acids, hydrogen and other substances produced during the fermentation process of other microorganisms into methane. By making full use of the synergistic effect between strains, the shortcomings of single-strain fermentation and application can be overcome, and it has stronger environmental adaptability.

[0045] This invention utilizes a combination of compound microbial agents and high-temperature hydrolysis for the fermentation of cyanobacterial sediment mixtures. By applying a mixture of Clostridium butyricum, Bacillus amyloliquefaciens, and Methanococcus maserii, a complete microbial community can be formed during the fermentation of cyanobacterial sediment. These communities play roles in decomposing organic matter, producing fermentation intermediates, and ultimately generating methane, thereby significantly increasing methane production.

[0046] Using these microbial combinations can effectively accelerate the degradation process of cyanobacteria sediment, reduce organic pollutants in the sediment, and thus achieve environmental remediation. This biological treatment method has lower environmental risks and is greener and more sustainable than physicochemical methods.

[0047] Methane is a clean energy source. Utilizing the fermentation of blue-green algae sediment to produce methane can not only treat water sediment but also convert waste into valuable energy. This resource utilization helps promote the development of a circular economy.

[0048] Organic matter and nutrients (such as nitrogen and phosphorus) in cyanobacterial sediments can be degraded by the compound bacterial agent of this invention through microbial fermentation, thereby reducing the concentration of these nutrients, decreasing the risk of eutrophication, and playing a positive role in improving water quality.

[0049] In summary, the mixed inoculum of Clostridium butyricum, Bacillus amyloliquefaciens, and Methanococcus maserensis in the fermentation of cyanobacteria in sediments for methane production can not only increase methane yield and improve the aquatic environment, but also promote the reuse of sediment resources and achieve green and sustainable ecological restoration.

[0050] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A methanogenic bacterial agent for fermentation of cyanobacteria sediment, comprising Clostridium butyricum, Bacillus amyloliquefaciens, and Methanococcus maserii, characterized in that, The percentage of *Clostridium butyricum* in the total bacterial cell count is 30%-60%, the percentage of *Bacillus amyloliquefaciens* in the total bacterial cell count is 25%-50%, and the percentage of *Methanococcus marzeri* in the total bacterial cell count is 10%-25%.

2. The methanogenic bacteria agent for cyanobacterial sediment fermentation according to claim 1, characterized in that, The *Clostridium butyricum*, *Bacillus amyloliquefaciens*, and *Methanococcus maszei* strains were cultured in a growth medium to expand the bacterial count to 10⁻⁶. 10 The concentration of cells / mL was determined by mixing Clostridium butyricum, Bacillus amyloliquefaciens, and Methanococcus maserii according to the percentage of cells to obtain a mixed bacterial agent.

3. The methanogenic bacteria agent for cyanobacterial sediment fermentation according to claim 2, characterized in that, The growth medium comprises: CH3COONa (0.3 g / L), NH4Cl (0.1 g / L), K2HPO4 (0.02 g / L), NaHCO3 (0.2 g / L), NaCl (0.2 g / L), MgSO4·7H2O (0.02 g / L), and yeast extract (0.1 g / L). The medium is sterilized at 0.1 MPa for 20 min, and the inoculum volume is 10% of the medium volume.

4. The methanogenic bacteria agent for cyanobacterial sediment fermentation according to claim 3, characterized in that, When using the growth medium, the Clostridium butyricum was also given 10 g / L glucose, 0.1 mg / L biotin and 0.5 mg / L folic acid, and the pH was adjusted to 6.8-7.2 with hydrochloric acid and sodium hydroxide. The bacteria were cultured in an anaerobic tank using a shaker at 100-200 rpm and a temperature of 30℃-37℃.

5. The methanogenic bacteria agent for cyanobacterial sediment fermentation according to claim 3, characterized in that, When using the growth medium, the Bacillus amyloliquefaciens was also cultured with 10 g / L starch and 5 g / L yeast extract, placed on a constant temperature shaker at 37°C, and the shaker speed was set to 150-200 rpm for constant temperature aerobic culture.

6. The methanogenic bacteria agent for cyanobacterial sediment fermentation according to claim 3, characterized in that, When using the growth medium for *Methanococcus maszei*, 0.2 g / L of acidified amino acid solution and 10 ml / L of vitamin solution are added. The acidified amino acid solution consists of lysine and glycine, and the vitamin solution consists of thiamine and vitamin B12. The pH is adjusted to 7.0-7.5 using HCl or NaOH. The inoculated culture medium is placed in a temperature-controlled incubator at 37°C, and a mixed gas of 5% CO2, 5% H2, and 90% N2 is introduced to remove oxygen for anaerobic culture.

7. The methanogenic bacteria agent for cyanobacterial sediment fermentation according to claim 2, characterized in that, When using mixed bacterial agents for methanogenesis from cyanobacteria sediment fermentation, the cyanobacteria are first crushed and pulped until the particle size is <5mm. The cyanobacteria and sediment are mixed at a V / S ratio of 60:40, and the total solids concentration is controlled at about 12%. The cyanobacteria-sludge mixture is then placed in an autoclave for hot water hydrolysis pretreatment at a temperature of 115℃-175℃ for 25 minutes.

8. The methanogenic bacteria agent for cyanobacterial fermentation in cyanobacteria sediment according to claim 7, characterized in that, Fermentation was carried out using a mixture of mixed microbial agents and hydrolyzed cyanobacterial sludge. A 10L sealed glass fermenter was selected. The initial inoculum in the reactor was 2L, containing 1.8L of hydrolyzed product and 200mL of microbial agents. Subsequently, 200mL of feed was added daily, along with a mixture of sludge without hydrolyzed pretreatment. The sludge fermentation process lasted 15 days. The top space of the fermenter was filled with nitrogen, and the reactor was sealed with a rubber stopper and paraffin wax.

9. The methanogenic bacteria agent for cyanobacterial fermentation in cyanobacteria sediment according to claim 8, characterized in that, During anaerobic fermentation, the pH will change as acidic substances accumulate. The pH should be checked and adjusted regularly using sodium hydroxide. The pH value should be controlled at 6.8-7.2, and the carbon-nitrogen ratio should be maintained at 20:1 to 30:

1. At the same time, the organic loading rate should be ensured to be between 1.66-4.19 gVS / (L·d). The temperature for anaerobic fermentation should be 25±1℃, and stirring should be carried out at a speed of 60 r / min.

10. The methanogenic bacteria agent for cyanobacterial sediment fermentation according to claim 8, characterized in that, During fermentation, the content of gases such as methane and carbon dioxide is monitored by a gas analyzer to determine the methane production efficiency. After fermentation, the methane gas in the cyanobacteria sediment is collected and can be used for energy utilization. The remaining solid matter can be further analyzed or processed. The fermentation broth and gases are analyzed to detect key indicators such as methane yield and organic matter degradation rate in the sediment.

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