Method for promoting methane production by using magnetic charcoal-based microbial inoculum

Abstract

The invention discloses a method for promoting methane production by using a magnetic charcoal-based microbial inoculum. The method comprises the following steps: performing magnetic modification on biochar to obtain magnetic biochar, then co-culturing the magnetic biochar and electroactive bacteria to obtain a magnetic biochar-based microbial inoculum, and finally adding the magnetic biochar-based microbial inoculum into an anaerobic digestion system containing methanogens to promote methanogenesis. The method for promoting methanogenesis by using the magnetic charcoal-based microbial inoculum has the characteristics of increasing the methanogenesis rate and increasing the abundance of methanogens, and can provide important reference for developing the technology of low-cost and efficient resource utilization of organic solid wastes.

Description

Technical Field

[0001] This invention specifically relates to a method for promoting methanogenesis using magnetic biochar-based bacterial agents, belonging to the field of organic solid waste resource utilization technology. Background Technology

[0002] my country's annual production of organic solid waste has now exceeded 4 billion tons and is on the rise. The efficient resource utilization of organic solid waste is a crucial guarantee for my country's efforts to reduce pollution and carbon emissions, develop a circular economy, and achieve its strategic goals of "carbon neutrality and carbon peaking." Anaerobic digestion with methanogenesis is an important technology for the efficient resource utilization of organic solid waste; however, anaerobic digestion involves multiple stages, including hydrolysis, acid production, and methanogenesis involving microorganisms, often leading to problems such as low methanogenesis yield, low methanogenesis rate, and poor operational stability in anaerobic digestion systems.

[0003] Traditional research suggests that microorganisms produce methanogens through interspecies electron transport, using formic acid or hydrogen as electron carriers. Recent studies have shown that conductive materials such as biochar can mediate interspecies electron transport in microorganisms, thereby promoting methanogenesis. However, traditional biochar materials suffer from drawbacks such as low electron transport efficiency and insufficient functional groups, resulting in limited methanogenesis-promoting effects. Electroactive bacteria possess extracellular electron transport capabilities and can significantly enhance electron transport efficiency in the methanogenesis process by mediating electron transport. However, existing methods for enriching electroactive bacteria using microbial electrochemical devices in anaerobic digestion systems suffer from drawbacks such as complex equipment, expensive electrode materials, and the need for replacement, increasing the cost of organic solid waste treatment. Therefore, researching and developing a biochar-electroactive bacteria composite enhanced methanogenesis technology is expected to combine the advantages of both, providing new ideas and references for developing low-cost, high-efficiency resource utilization technologies for organic solid waste, and has significant application value. Summary of the Invention

[0004] To address the shortcomings of existing anaerobic digestion methanogenesis technologies, the present invention aims to provide a method for promoting methanogenesis using magnetic biochar-based bacterial agents. Compared with existing methanogenesis technologies, the method provided by the present invention has the advantages of increasing the methanogenesis rate and the number of methanogenic bacteria.

[0005] The objective of this invention is achieved through the following technical solution: A method for promoting methanogenesis using magnetic biochar-based bacterial agents involves magnetically modifying biochar to obtain magnetic biochar, then co-culturing the magnetic biochar with electroactive bacteria to obtain a magnetic biochar-based bacterial agent, and finally adding the magnetic biochar-based bacterial agent to an anaerobic digestion system containing methanogenic bacteria to promote methanogenesis.

[0006] Preferably, the electroactive bacteria can be various electroactive strains, such as Shewanella (…). Shewanella ), Geyserum ( Geobacter ), Pseudomonas (Pseudomonas ), Lysine Bacillus ( Lysinibacillus Specifically, it could be... Shewanella oneidensis , Lysinibacillus sphaericus wait.

[0007] Preferably, the magnetic biochar is prepared by grinding and sieving biomass raw materials, then firing them at high temperature under a nitrogen atmosphere to obtain biochar. Ferrous trivalent salts and ferrous divalent salts are added to oxygen-free water, and ammonia water is added dropwise under magnetic stirring to form Fe3O4. Then, the biochar obtained by firing is added and stirring is continued. The mixture is filtered and the precipitate is collected. The precipitate is washed with water until neutral and then dried to obtain magnetic biochar.

[0008] Preferably, the magnetic biochar-based bacterial agent is prepared by adding magnetic biochar to the culture medium of electroactive bacteria, inoculating the electroactive bacteria, culturing the bacterial solution, collecting the biochar precipitate by centrifugation, and rinsing and resuspending the precipitate using the culture medium of methanogenic bacteria to obtain the magnetic biochar-based bacterial agent.

[0009] Preferably, the biomass raw materials are lignocellulosic and organic waste biomass, including but not limited to straw, sawdust, and sewage sludge.

[0010] Preferably, the biochar obtained by high-temperature firing under a nitrogen atmosphere is obtained by firing biomass raw materials at 700°C-900°C for 0.5-1 h under nitrogen protection with a heating rate of 5°C-10°C / min.

[0011] Preferably, the amounts of the ferric salt and ferrous salt are such that the iron molar ratio is 2:1 and the mass ratio of iron to biochar is 1:2-1:10.

[0012] Preferably, the ammonia water is added to maintain the pH of the solution between 10 and 11, and the reaction time to form Fe3O4 is 0.5-2 h.

[0013] Preferably, the magnetic biochar is added to the culture medium of the electroactive bacteria at a concentration of 0.1-0.5 g / L.

[0014] Preferably, the electroactive bacteria include, but are not limited to, *Bacillus spheroidans*. Lysinibacillus sphaericus Oneda Shewanella Shewanella oneidensis wait.

[0015] Preferably, the magnetic biochar-based bacterial agent is added to the anaerobic digestion system containing methanogens at a dosage of 1-5 g / L.

[0016] The present invention has the following beneficial effects: The method for promoting methanogenesis using magnetic biochar-based bacterial agents provided by this invention has the characteristics of increasing the methanogenesis rate and the number of methanogenic bacteria, and can provide an important reference for developing low-cost and efficient technologies for the resource utilization of organic solid waste. Attached Figure Description

[0017] Figure 1 This is a photograph of the magnetic biochar in Example 1 of the present invention;

[0018] Figure 2 This refers to the biomass on the materials in Embodiment 1 and Comparative Examples 1-2 of this invention;

[0019] Figure 2 This refers to the biomass on the materials in Embodiment 1 and Comparative Examples 1-2 of this invention;

[0020] Figure 3 This refers to the headspace methane concentration in the reactors of Examples 2 and Comparative Examples 3-6 of this invention;

[0021] Figure 4 These are the samples from Embodiment 2 and Comparative Examples 3-6 of the present invention. mcrA Gene copy number. Detailed Implementation

[0022] The following examples further illustrate a method for promoting methanogenesis using a magnetic biochar-based bacterial agent provided by the present invention. These examples are intended to provide a detailed description of the present invention and are not intended to limit the scope of the claims of the present invention in any way.

[0023] Example 1:

[0024] A suitable amount of sewage sludge from a wastewater treatment plant is ground and crushed, then sieved through a 200-mesh sieve and loaded into an alumina boat. The sludge is then heated to 900°C in a tubular furnace under nitrogen protection at a heating rate of 7°C / min and pyrolyzed for 0.5 h to obtain biochar, which is then cooled and set aside for later use.

[0025] Measure 400 mL of deionized water, boil it, and then aerate it with high-purity nitrogen for 0.5 h. Add 9.44 g of FeCl3·6H2O and 3.44 g of FeCl2·4H2O to the mixture. Under magnetic stirring, slowly add 20 mL of NH3·6H2O dropwise to the mixture and stir for 1 hour to precipitate Fe3O4. Then add 10 g of the biochar prepared by the aforementioned pyrolysis and continue stirring for 0.5 h.

[0026] The above mixture was filtered, and the filter residue was washed with deionized water until neutral, and then dried to obtain magnetic biochar. Figure 1 ).

[0027] Add 0.2 g / L magnetic biochar to Luria-Bertani medium (LB medium), autoclave the medium (121°C, 20 min), and then incubate aerobically in a constant temperature shaker at 180 rpm and 30°C. Shewanella oneidensis ( S. oneidensis Published in reference: Venkateswaran K., et al. Int. J. Syst. Evol. Microbiol, 1999, 49: 705-724. The applicant also holds and guarantees to provide it to the public for 20 years from the date of application) 12 h (vaccination) S. oneidensis Initial OD of post-culture medium 600 The value was 0.01-0.02. The cultured bacterial solution was centrifuged at 6500 rpm for 5 min to collect the precipitate. The precipitate was washed three times with methanogenic culture medium to obtain a magnetic biochar-based bacterial agent. Samples of the precipitate were taken to determine the protein content on the magnetic biochar-based bacterial agent. The composition of the LB culture medium was as follows: 5 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, and deionized water. The composition of the methanogenic culture medium was as follows: 4.76 g / L C8H... 18 N2O4S (HEPES), 0.5 g / L KCl, 0.1 g / L NH4Cl, 0.1 g / L CaCl2·2H2O, 0.4 g / L MgCl2·6H2O, 12.5 mL / L Wolfe trace element solution, 5 mL / L Wolfe vitamin solution, and deionized water.

[0028] Comparative Example 1:

[0029] Compared with Example 1, no magnetic modification was performed after the biochar was prepared. Instead of adding 0.2 g / L magnetic biochar to the LB medium, biochar was added. The other steps were the same as in Example 1.

[0030] Comparative Example 2:

[0031] Compared to Example 1, no biochar was prepared. FeCl3·6H2O and FeCl2·4H2O were co-precipitated to form Fe3O4, and no biochar was added. The other steps were the same as in Example 1.

[0032] The results showed that the modified biochar in Example 1 could be adsorbed by a magnet outside the beaker in the solution. Figure 1 This indicates that it possesses magnetic properties. For example... Figure 2 As shown, S. oneidensisAfter 12 hours of cultivation, the biomass (calculated as protein content) per unit mass of biochar and iron oxide was comparable, both approximately 280 mg / g. However, the biomass per unit mass of magnetic biochar reached 323.01 ± 14.98 mg / g, which was 1589% and 17.30% higher than that of biochar and iron oxide, respectively, indicating that magnetic modification of biochar can effectively improve its loading capacity for electroactive bacteria. Example 2:

[0033] 0.2 g / L of the magnetic biochar prepared in Example 1 was added to LB medium. The medium was then autoclaved (121°C, 20 min) and aerobically cultured in a constant temperature shaker at 180 rpm and 30°C. Lysinibacillus sphaericus ( L. sphaericus Published in reference: Zhu CJ, et al. Int.J. Syst. Evol. Microbiol, 2014, 64: 3644-3649. The applicant also holds and guarantees to provide it to the public for 20 years from the date of application) 14 h (vaccination) L. sphaericus Initial OD of post-culture medium 600 The value is 0.01-0.02). After cultivation, the bacterial solution was centrifuged at 6500 rpm for 5 min to collect the precipitate. The precipitate was washed three times with methanogenic culture medium (same as in Example 1) to obtain magnetic biochar-based bacterial agent.

[0034] 100 mL of methanogenic culture medium (same as in Example 1) and 1.5 g / L sodium propionate were added to the reactor. After aeration with high-purity nitrogen for 0.5 h to remove oxygen, 10 mL of methanogenic enrichment culture was inoculated and 2 g / L of magnetic biochar-based inoculant was added. The reactor was then sealed with a butyl rubber stopper and an aluminum cap. The reactor was incubated statically at 25°C-35°C and designated as the magnetic biochar-based inoculant group. The methane concentration in the reactor headspace was determined using gas chromatography, and the sodium propionate concentration in the culture medium was determined using high-performance liquid chromatography. Quantitative real-time PCR was used to determine... mcrA Gene copy number was used to quantify the number of methanogens. The methanogen enrichment culture used was obtained from a methanogenic reactor constructed in the laboratory using river sediments as inoculum. Specifically, it was obtained as follows: methanogen culture medium and an appropriate amount of sodium propionate were added to the anaerobic reactor, and after aeration with high-purity nitrogen for 0.5 h, river sediments from Guangdong Province were inoculated into the reactor. The reactor was then sealed with a butyl rubber stopper and an aluminum cap. The reactor was incubated statically at 25°C-35°C. The methane concentration in the reactor headspace was measured periodically. The methanogenic cycle was considered complete when methane production stabilized. The culture medium was then replaced with fresh methanogen culture medium and sodium propionate was added. This process was repeated until the methane production in each cycle stabilized, thus obtaining the methanogen enrichment culture.

[0035] Comparative Example 3:

[0036] Compared with Example 2, no magnetic biochar-based bacterial agent was prepared, no magnetic biochar-based bacterial agent was added to the reactor, and the other steps were the same as in Example 2. This was designated as the control group.

[0037] Comparative Example 4:

[0038] Compared with Example 2, instead of preparing magnetic biochar-based bacterial agents, the magnetic biochar prepared in Example 1 was added to the reactor instead of the magnetic biochar-based bacterial agents. The other steps were the same as in Example 2, and this group was referred to as the magnetic biochar group.

[0039] Comparative Example 5:

[0040] Compared with Example 2, instead of preparing magnetic biochar-based bacterial agents, the addition of magnetic biochar-based bacterial agents to the reactor was replaced with the addition of biochar prepared in Example 1. The other steps were the same as in Example 2, and this group was referred to as the biochar group.

[0041] Comparative Example 6:

[0042] Sterile LB medium was used for aerobic culture in a constant temperature shaker at 180 rpm and 30°C. L. sphaericus 14 hours. L. sphaericus The bacterial culture was centrifuged at 6500 rpm for 5 min to collect the bacterial cells. The bacterial cells were washed three times with methanogenic culture medium and then resuspended in methanogenic culture medium.

[0043] Add 100 mL of methanogenic culture medium (same as in Example 1) and 1.5 g / L sodium propionate to the reactor. After aerating with high-purity nitrogen for 0.5 h to remove oxygen, inoculate with 10 mL of methanogenic enrichment culture and add an equal amount of biomass of magnetic biochar agent as added in Example 2. L. sphaericus The bacterial culture was then sealed with butyl rubber stoppers and aluminum caps. The reactor was incubated statically at 25°C-35°C, and this group was designated as the bacterial culture group.

[0044] The results showed that methane production in the control group stabilized around day 9, while the other groups only needed a few days to stabilize. Figure 3The maximum cumulative headspace methane concentrations were as follows: magnetic biochar-based bacterial agent group (82.28±5.45 mg / L), bacterial agent group (75.65±3.48 mg / L), magnetic biochar group (71.67±1.77 mg / L), biochar group (65.58±2.72 mg / L), and control group (58.80±2.48 mg / L). This indicates that biochar, magnetic biochar, and electroactive bacteria can all effectively promote methanogenesis. Magnetic modification of biochar significantly enhances the methanogenesis-promoting effect, and loading electroactive bacteria onto magnetic biochar is more effective than either electroactive bacteria alone or magnetic biochar alone. Quantitative real-time PCR results showed that on day 12 post-inoculation, the concentrations of methane per unit volume (mL) in the magnetic biochar-based bacterial agent group, bacterial agent group, magnetic biochar group, and biochar group were significantly higher than those in the control group. mcrA The gene copy numbers were significantly higher than those of the control group, indicating that biochar, magnetic biochar, and electroactive bacteria are all beneficial to the growth and accumulation of methanogens, with magnetic biochar-based bacterial agents showing the best effect.

Claims

1. A method for promoting methanogenesis using magnetic biochar-based inoculum, characterized by, The method comprises the following steps: modifying biochar to obtain magnetic biochar, then co-culturing the magnetic biochar with electroactive bacteria to obtain a magnetic biochar-based microbial inoculum, and finally adding the magnetic biochar-based microbial inoculum to an anaerobic digestion system containing methanogens to promote methanogenesis.

2. The method of claim 1, wherein, The electroactive bacteria can be various strains of electroactive bacteria, including but not limited to Shewanella ( Shewanella ), Geobacter ( Geobacter ), Pseudomonas ( Pseudomonas ), and Lysinibacillus ( Lysinibacillus ), etc., preferably, the Shewanella is Shewanella oneidensis , and the Lysinibacillus is Lysinibacillus sphaericus .

3. The method of claim 1, wherein, The magnetic biochar is obtained by grinding and screening biomass raw materials, high-temperature firing under a nitrogen atmosphere to obtain biochar, adding ferric and ferrous salts to anaerobic water, and dropwise adding ammonia water under magnetic stirring to form Fe3O4 through a coprecipitation reaction, then adding the aforementioned biochar to continue stirring, collecting the precipitate by suction filtration, drying the precipitate after washing to neutral to obtain the magnetic biochar.

4. The method of claim 1, wherein, The magnetic biochar-based microbial inoculum is obtained by adding magnetic biochar to the culture medium of electroactive bacteria, inoculating electroactive bacteria culture, centrifuging the bacterial solution to collect the biochar precipitate, and resuspending the precipitate using a methanogen culture medium to obtain the magnetic biochar-based microbial inoculum.

5. The method of claim 3, wherein, The biomass raw material is lignocellulose and organic waste biomass, preferably including but not limited to straw, wood chips, and sewage sludge.

6. The method of claim 3, wherein, The biochar is obtained by high-temperature firing under a nitrogen atmosphere, i.e., firing the biomass raw material under nitrogen protection at a temperature increasing rate of 5-10°C / min for 0.5-1 h at 700-900°C.

7. The method of claim 3, wherein, The ferric and ferrous salts are used in a molar ratio of 2:1, and the mass ratio of iron to biochar is 1:2-1:

10.

8. The method of claim 3, wherein, The ammonia water is added to maintain the pH of the solution at 10-11, and the reaction time for forming Fe3O4 is 0.5-2 h.

9. The method of claim 4, wherein, The magnetic biochar is added to the culture medium of electroactive bacteria at a concentration of 0.1-0.5 g / L.

10. The method of claim 1, wherein, The magnetic biochar-based microbial inoculum is added to the anaerobic digestion system containing methanogens at a dosage of 1-5 g / L.

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