CN111100880A - Magnetic corpuscle promotes microbial electric fermentation to reduce CO2Process for producing methane - Google Patents
Magnetic corpuscle promotes microbial electric fermentation to reduce CO2Process for producing methane Download PDFInfo
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Abstract
The invention relates to a biomass energy utilization technology, and aims to provide a magnetosome for promoting microbial electric fermentation and reduction of CO2A process for producing methane. The method comprises the following steps: (1) extracting purified magnetosomes from Magnetospirillum in logarithmic growth phase; (2) the microbial electric fermentation system adopts a three-electrode system; taking small molecular organic acid salt as a carbon source, adding the small molecular organic acid salt and anaerobic activated sludge into a system, and taking magnetosome as an exogenous conductive additive; introducing pure nitrogen into the system in a sealed manner to remove air, connecting the system with an electrochemical workstation, and electrolyzing the micromolecular organic acid salt to generate CO at the anode of the graphite rod after the system is started to operate2，CO2Reduced at the cathode of the graphite rod to produce methane. The invention can effectively improve the concentration of the biological methane and reduce the subsequent purification cost of the methane; can effectively optimize the electron transfer performance of the system, improve the content of extracellular polymer with redox activity, and effectively enrich and be responsible for reducing CO2A methanogenic microorganism.
Description
Technical Field
The invention relates to a biomass energy utilization technology, in particular to a magnetosome-promoted microbial electric fermentation reduction CO2A process for producing methane.
Background
Under the conditions of energy crisis and continuously aggravated environmental pollution, people are increasingly concerned about producing advanced biological energy by using alternative raw materials. Anaerobic fermentation is a widely used organic waste treatment and energy utilization method for producing biological methane, and is a multi-stage and multi-phase biochemical process under the synergistic action of various microorganisms. Complex macromolecular organic substances (such as polysaccharide, protein, lipid and the like) are firstly degraded into micromolecular organic substances such as monosaccharide, amino acid and the like by hydrolytic bacteria and then are continuously degraded into micromolecular volatile fatty acid and alcohol under the action of acid-producing bacteria. Volatile fatty acid and alcohol generated in the acidification stage can be converted into acetic acid and CO under the action of acetogenic bacteria2、H2And the methane is further utilized and converted into methane by methanogens. In the stage of the inter-nutrition metabolism of acetogenic bacteria and methanogenic bacteria, a transfer mechanism which does not need an electron transfer carrier (such as hydrogen, formic acid and the like) -direct electrons between species are discovered in recent yearsTransfer (Direct Interspecies Electron Transfer, DIET). The electron transfer mode is more beneficial to saving energy, and the Gibbs free energy is lower, so that the thermodynamic reaction is more beneficial.
In order to strengthen the methane bacteria to obtain electron reduction CO in a direct electron transfer mode2Methane is produced and electrodes may be inserted in the fermentation system and an electrical potential applied to provide electrons. It has been found by the scholars that the applied current is effective in increasing the yield of different target products, because the microbial metabolic pathway and thus the product production can be influenced by adjusting the extracellular oxidation-reduction potential (ORP) in the bioelectrochemical system. In order to enhance the mutual-feeding metabolism and synergistic effect between anaerobic fermentation microorganisms, conductive materials such as metal nanoparticles and carbon-based conductive materials (including carbon-based conductive materials such as granular activated carbon, biochar and carbon cloth, hematite and magnetite) can be added into an anaerobic fermentation system, so that the delay time of methane production starting is shortened, and the methane production rate and yield and the resistance to inhibition conditions are improved. Research reports that the abundance of electrogenic microorganisms (such as Geobacter) in the reactor is remarkably improved after the conductive material is added, and the electrogenic bacteria and the receiving electrons reduce CO2The methanogenic archaea can be metabolized to produce methane by DIET, and the methane production rate in this mode is not limited by the hydrogen partial pressure in the reactor.
Although the above technology of inserting an electrode in a fermentation system to apply an external current can improve the electron transfer between microorganisms and the electrode, the direct electron transfer between species of microorganisms still needs to be enhanced by adding an exogenous conductive substance; the above-mentioned technology of adding conductive material into fermentation system also has some disadvantages, such as poor dispersibility of metal nanoparticles, poor biocompatibility, relatively small specific surface area of traditional carbon-based conductive material, difficult recycling, etc., which limits its application in anaerobic fermentation. Generally, the technology of independently applying a certain potential or adding a conductive material has a limited effect on improving the performance of a fermentation system, the concentration of the biological methane generated by the system is not high enough, further methane purification is needed, and the process is complicated; the electron transfer performance of the microorganisms in the system is poor, and the reduction of CO is difficult to meet2OfTransferring the demand; the content of redox active extracellular polymers playing an important role in microbial extracellular electron transfer is not high; responsible for reducing CO in the system2Methanogenic microorganisms are to be further enriched, etc. Therefore, the invention provides a technical means that the magnetosome which has good dispersibility, good biocompatibility and large specific surface area and can be recycled and recycled by magnetic separation and other modes is selected as the exogenous conductive additive, and the microbial electron transfer is enhanced by adopting the synergy of the external potential and the added magnetosome, so that the technical problems are hopefully solved.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects of the prior art and provides a magnetosome for promoting microbial electrical fermentation to reduce CO2A process for producing methane.
In order to solve the technical problem, the solution of the invention is as follows:
provides a magnetic corpuscle for promoting microbial electric fermentation and reduction of CO2The method for preparing methane specifically comprises the following steps:
(1) extracting purified magnetosomes from Magnetospirillum gryphisaldense (Magnetospirillum gryphisaldense) in logarithmic growth phase;
(2) the microbial electric fermentation system adopts a three-electrode system, and the working volume of the system is 50 mL; two graphite rod electrodes are used as a cathode and an anode, and an Ag/AgCl electrode is used as a reference electrode; taking 1g of micromolecular organic acid salt as a carbon source of an electric fermentation system, and adding the micromolecular organic acid salt and anaerobic activated sludge serving as inoculated thalli into the system, wherein the mass ratio of the Total Volatile Solids (TVS) of the anaerobic activated sludge to the micromolecular organic acid salt is 2: 1; adjusting the pH value of the mixture in the system to 7.5, then adding magnetosomes as exogenous conductive additives into the system, and controlling the addition amount to ensure that the concentration of the magnetosomes in the system is 100 mg/L;
(3) sealing the microbial electric fermentation system, and introducing pure nitrogen for 10 minutes to remove air; then the system is connected with an electrochemical workstation, the potential is applied to-0.6V to-0.8V at the temperature of 37 ℃, the operation is started, and the small molecular organic acid salt is electrolyzed at the anode of the graphite rod to generate CO2,CO2Is reduced to generate methane at the cathode of the graphite rod and utilizesThe gas chromatograph measures the methane product concentration.
In the invention, in the step (1), the magnetic corpuscle is extracted and purified from the magnetospirillum by an ultrasonic crushing extraction method, which specifically comprises the following steps: culturing the Magnaporthe magnus to logarithmic growth phase, centrifugally collecting the thalli and resuspending the thalli in a phosphate buffer; ultrasonically crushing cells in an ice bath environment, controlling the power to be 100W, crushing for 10s and spacing for 10s, and repeating for 50 times; separating magnetosome with magnet, washing with phosphate buffer solution for 5 times to obtain purified magnetosome.
In the invention, in the step (2), the pH value of the mixture is adjusted by NaOH and HCl solution with the molar concentration of 6 mol/L.
In the invention, in the step (2), the small molecule organic acid salt is any one of acetate, propionate or butyrate.
In the invention, in the step (2), the anaerobic activated sludge is taken from an electric fermentation methanogenesis reactor which runs stably and is rich in electric active bacteria and methanogenic archaea.
In the invention, in the step (2), the diameter of the graphite rod electrode is 0.5cm, and the length of the graphite rod electrode is 7.5 cm.
Description of the inventive principles:
magnetosomes are magnetic nanoparticles synthesized in magnetotactic bacteria cells, the size of the magnetosome is about 20-120nm, the components are ferroferric oxide or ferroferric sulfide, the size form and the component composition of the magnetosome depend on the type of magnetotactic bacteria, and the size form of the magnetosome synthesized by each magnetotactic bacteria is uniform. The Magnetospirilluloscompressive strain MSR-1 used in the invention is a typical magnetotactic bacterium, the spiral cells contain 40-45nm cubic magnetite with different quantity, and the magnetosomes are arranged in a chain shape, thus being a very good biological magnetic nano material. Compared with the common artificial magnetite, the magnetosome is not easy to aggregate, has good dispersibility, has a large number of bioactive groups, can be used for covalent connection with other molecules, and has good biocompatibility and safety. In addition, the magnetosome can be effectively recycled from the electric fermentation system by means of magnetic separation and the like. As an additive of an anaerobic fermentation bioreactor, the conductive magnetosome has the potential of promoting extracellular electron transfer of bacteria and can strengthen direct electron transfer between electrically active bacteria and methanogenic archaea.
Previous studies show that the system performance can be improved by applying current or adding conductive materials in a fermentation system, and the generation amount of target products can be increased. However, the microbial electric fermentation system adds magnetosome to strengthen the direct electron transfer process to promote CO2The reduction for preparing methane is not reported in the literature. There is a direct electron transfer mode in the microbial electric fermentation system with added magnetosomes: (1) a direct contact transfer mechanism, wherein the microorganisms attached to the surface of the electrode receive or release electrons through electron transfer proteins on a biological membrane; (2) a nanowire transfer mechanism in which electroactive bacteria (e.g., geobacter) transfer electrons by contacting with other microorganisms or electrodes through self-growing pili (pili) or nanowires (conductive nanowires); (3) an electron shuttle transfer mechanism in which a microorganism transfers electrons by secreting Extracellular Polymers (EPS) having redox activity as electron shuttles; (4) and (3) a conductive material transfer mechanism, wherein the electron transfer is mediated between microorganisms through an additional conductive material. The evolution mechanism of the extracellular electron transfer mode, the flora structure and the methanogenesis metabolic pathway under the synergistic effect of magnetosomes and impressed current is worthy of being deeply researched.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention can effectively improve the concentration of the biological methane: the purification rate is increased from 46% to 73% -87%, which is beneficial to reducing the subsequent biogas purification cost;
2. the invention can effectively optimize the electronic transmission performance of the system: the apparent electron transfer constant based on the cyclic voltammetry characteristic curve is improved from 0.030 to 0.092-0.108 s-1The conductivity of the anaerobic activated sludge in the system is improved from 6.9 mu s/cm to 18.6-21.9 mu s/cm, so as to meet the requirement of reducing CO2The electronic requirements of (1);
3. the invention can improve the content of extracellular polymeric substances with redox activity: the redox active extracellular polymer plays an important role in microbial extracellular electron transfer, and the invention can improve the content of the redox active extracellular polymer, namely fulvic acid, from 11.2% to 19.1-20.0% and the content of the humic acid from 11.7% to 12.2-12.8%;
4. the invention can effectively enrich and be responsible for reducing CO2Methanogenic microorganisms: the electroactive bacteria are mainly geobacillus, and the relative abundance of the electroactive bacteria is improved from 15.1% to 32.9-36.8%; the methanogenic archaea is mainly methanosarcina sarcina, and the relative abundance of the methanogenic archaea is improved from 15.8% to 33.9-34.8%.
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FIG. 1 is a flow chart of an implementation of the present invention.
Detailed Description
It should be noted that all the devices (such as microbial electric fermentation system and electrochemical workstation) used in the present invention are the prior art, and those skilled in the art can purchase or build themselves through public ways. The various strains (such as Magnetospirillum magnetospirillum, electroactive bacteria, methanogenic archaea and the like) used in the invention are all the prior art, and can be purchased or obtained by self-culture through a public way by a person skilled in the art, and the culture method of the strains is not described again.
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. The examples may provide those skilled in the art with a more complete understanding of the present invention, and are not intended to limit the invention in any way.
Example 1
Culturing the Magnetospirillum to logarithmic growth phase, centrifugally collecting thalli, resuspending in phosphate buffer, ultrasonically breaking cells in an ice bath environment (power is 100W, breaking is 10s, interval is 10s, repeating is 50 times), adsorbing and separating magnetosome by using a magnet, washing for 5 times by using the phosphate buffer to obtain a purified magnetosome sample, and taking the magnetosome as an exogenous conductive additive to strengthen the electron transfer performance in a microbial electric fermentation system. The method is characterized in that small molecular organic acid salt is used as a carbon source of an electric fermentation system, and anaerobic activated sludge (obtained from an electric fermentation methane-producing reactor which runs stably) rich in electric active bacteria and methane-producing archaea is selected as inoculated thalli. The microbial electric fermentation system adopts a three-electrode system, two graphite rod electrodes (phi 0.5cm multiplied by 7.5cm) are selected as a cathode and an anode, and an Ag/AgCl electrode is used as a referenceAnd an electrode. 1g of micromolecular organic acid salt and anaerobic activated sludge are added into a system with a working volume of 50mL, and the mass ratio of Total Volatile Solids (TVS) of the activated sludge and the micromolecular organic acid salt is 2: 1. The pH of the mixture was adjusted to 7.5 with 6mol/L NaOH and HCl solutions, and then magnetosomes were added at a concentration of 100 mg/L. And (3) sealing the system, introducing high-purity nitrogen for 10 minutes to remove air, connecting the system with an electrochemical workstation, and applying a potential of-0.6V to start operation at the temperature of 37 ℃. The electroactive bacteria (mainly geobacter) form a biofilm on the surface of the graphite rod anode, and decompose-acetate to generate electrons: CH (CH)3COO-+2H2O→2CO2+7H++8e-(ii) a Methane-producing archaea (mainly methane sarcina) forms a biological film on the surface of the cathode of the graphite rod, and CO is reduced by electrons generated by the electrode2Conversion to methane (by anodic biofilm electrolysis of acetate): CO 22+8H++8e-→CH4+2H2And O, testing the concentration of the methane product by using a gas chromatograph. Under the synergistic effect of magnetosome and impressed current, the methane concentration is increased from 46% to 73%; the electrochemical performance of the microbial electric fermentation system is effectively improved: the apparent electron transfer constant based on cyclic voltammetry curve is improved from 0.030 to 0.092s-1The conductive capacity of the activated sludge in the system is improved from 6.9 mu s/cm to 18.6 mu s/cm; the content of extracellular polymeric fulvic acid is increased from 11.2% to 19.1%, and the content of humic acid is increased from 11.7% to 12.2%; analysis of microbial community and methanogenesis metabolism indicated that: the relative abundance of the electroactive bacteria is mainly geobacter which is increased from 15.1% to 32.9%, and the relative abundance of the methanogenic archaea is mainly methanosarcina which is increased from 15.8% to 33.9%.
Example 2
Culturing Magnetospirillum to logarithmic growth phase, centrifuging to collect thallus and resuspending in phosphate buffer, ultrasonically breaking cells in ice bath environment (power 100W, breaking for 10s, spacing for 10s, repeating for 50 times), adsorbing and separating magnetosome with magnet, washing with phosphate buffer for 5 times to obtain purified magnetosome sample, and using magnetosome as exogenous conductive additive to strengthen electricity in microbial electric fermentation systemThe sub-transfer performance. The method is characterized in that small molecular organic acid salt is used as a carbon source of an electric fermentation system, and anaerobic activated sludge (obtained from an electric fermentation methane-producing reactor which runs stably) rich in electric active bacteria and methane-producing archaea is selected as inoculated thalli. The microbial electric fermentation system adopts a three-electrode system, two graphite rod electrodes (phi 0.5cm multiplied by 7.5cm) are selected as a cathode and an anode, and an Ag/AgCl electrode is selected as a reference electrode. 1g of micromolecular organic acid salt and anaerobic activated sludge are added into a system with a working volume of 50mL, and the mass ratio of Total Volatile Solids (TVS) of the activated sludge and the micromolecular organic acid salt is 2: 1. The pH of the mixture was adjusted to 7.5 with 6mol/L NaOH and HCl solutions, and then magnetosomes were added at a concentration of 100 mg/L. And sealing the system, introducing high-purity nitrogen for 10 minutes to remove air, connecting the system with an electrochemical workstation, and applying a potential of-0.7V to start operation at the temperature of 37 ℃. The electrically active bacteria (mainly geobacter) form a biological film on the surface of the graphite rod anode, and propionate is decomposed to generate electrons: CH (CH)3CH2COO-+2H2O→CH3COO-+CO2+6H++6e-(ii) a Methane-producing archaea (mainly methane sarcina) forms a biological film on the surface of the cathode of the graphite rod, and CO is reduced by electrons generated by the electrode2Conversion (by anodic biofilm electrolysis of propionate) to methane: CO 22+8H++8e-→CH4+2H2And O, testing the concentration of the methane product by using a gas chromatograph. Under the synergistic effect of magnetosome and impressed current, the methane concentration is increased from 46% to 81%; the electrochemical performance of the microbial electric fermentation system is effectively improved: the apparent electron transfer constant based on cyclic voltammetry curve increased from 0.030 to 0.102s-1The conductive capacity of the activated sludge in the system is improved from 6.9 mu s/cm to 20.7 mu s/cm; the content of extracellular polymeric fulvic acid is increased from 11.2% to 19.7%, and the content of humic acid is increased from 11.7% to 12.6%; analysis of microbial community and methanogenesis metabolism indicated that: the electroactive bacteria are mainly geobacter, and the relative abundance of the electroactive bacteria is improved from 15.1 percent to 33.9 percent; the methanogenic archaea is mainly methanosarcina sarcina, and the relative abundance of the methanogenic archaea is improved from 15.8% to 34.1%.
Example 3
Culturing the Magnetospirillum to logarithmic growth phase, centrifugally collecting thalli, resuspending in phosphate buffer, ultrasonically breaking cells in an ice bath environment (power is 100W, breaking is 10s, interval is 10s, repeating is 50 times), adsorbing and separating magnetosome by using a magnet, washing for 5 times by using the phosphate buffer to obtain a purified magnetosome sample, and taking the magnetosome as an exogenous conductive additive to strengthen the electron transfer performance in a microbial electric fermentation system. The method is characterized in that small molecular organic acid salt is used as a carbon source of an electric fermentation system, and anaerobic activated sludge (obtained from an electric fermentation methane-producing reactor which runs stably) rich in electric active bacteria and methane-producing archaea is selected as inoculated thalli. The microbial electric fermentation system adopts a three-electrode system, two graphite rod electrodes (phi 0.5cm multiplied by 7.5cm) are selected as a cathode and an anode, and an Ag/AgCl electrode is selected as a reference electrode. 1g of micromolecular organic acid salt and anaerobic activated sludge are added into a system with a working volume of 50mL, and the mass ratio of Total Volatile Solids (TVS) of the activated sludge and the micromolecular organic acid salt is 2: 1. The pH of the mixture was adjusted to 7.5 with 6mol/L NaOH and HCl solutions, and then magnetosomes were added at a concentration of 100 mg/L. And (3) sealing the system, introducing high-purity nitrogen for 10 minutes to remove air, connecting the system with an electrochemical workstation, and applying a potential of-0.8V to start operation at the temperature of 37 ℃. The electrically active bacteria (mainly geobacter) form a biological membrane on the surface of the graphite rod anode, and the butyrate is decomposed to generate electrons: CH (CH)3(CH2)2COO-+2H2O→CH3CH2COO-+CO2+6H++6e-(ii) a Methane-producing archaea (mainly methane sarcina) forms a biological film on the surface of the cathode of the graphite rod, and CO is reduced by electrons generated by the electrode2Conversion to methane (by anodic biofilm electrolysis of butyrate): CO 22+8H++8e-→CH4+2H2And O, testing the concentration of the methane product by using a gas chromatograph. Under the synergistic effect of magnetosome and external current, the concentration of methane is increased from 46% to 87%; the electrochemical performance of the microbial electric fermentation system is effectively improved: the apparent electron transfer constant based on cyclic voltammetry curve is improved from 0.030 to 0.108s-1In the systemThe conductive capacity of the activated sludge is improved from 6.9 mu s/cm to 21.9 mu s/cm; the content of extracellular polymeric fulvic acid is increased from 11.2% to 20.0%, and the content of humic acid is increased from 11.7% to 12.8%; analysis of microbial community and methanogenesis metabolism indicated that: the electro-active bacteria are mainly geobacillus, and the relative abundance of the electro-active bacteria is improved from 15.1 percent to 36.8 percent; the methanogenic archaea is mainly methanosarcina sarcina, and the relative abundance of the methanogenic archaea is improved from 15.8% to 34.8%.
Finally, it should be noted that the above-mentioned list is only a specific embodiment of the present invention. It is obvious that the present invention is not limited to the above embodiments, but many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (6)
1. Magnetic corpuscle promotes microbial electric fermentation reduction CO2The method for preparing methane is characterized by comprising the following steps:
(1) extracting purified magnetosomes from Magnetospirillum in logarithmic growth phase;
(2) the microbial electric fermentation system adopts a three-electrode system, and the working volume of the system is 50 mL; two graphite rod electrodes are used as a cathode and an anode, and an Ag/AgCl electrode is used as a reference electrode; taking 1g of small molecular organic acid salt as a carbon source of an electric fermentation system, and adding the small molecular organic acid salt and anaerobic activated sludge serving as inoculated bacteria into the system, wherein the mass ratio of total volatile solid of the anaerobic activated sludge to the small molecular organic acid salt is 2: 1; adjusting the pH value of the mixture in the system to 7.5, then adding magnetosomes as exogenous conductive additives into the system, and controlling the addition amount to ensure that the concentration of the magnetosomes in the system is 100 mg/L;
(3) sealing the microbial electric fermentation system, and introducing pure nitrogen for 10 minutes to remove air; then the system is connected with an electrochemical workstation, the potential is applied to-0.6V to-0.8V at the temperature of 37 ℃, the operation is started, and the small molecular organic acid salt is electrolyzed at the anode of the graphite rod to generate CO2，CO2The carbon dioxide is reduced at the cathode of the graphite rod to generate methane; the methane product concentration was measured using a gas chromatograph.
2. The method according to claim 1, wherein in step (1), the magnetosome is extracted and purified from the Magnetospirillum magnetospirillum by ultrasonication and extraction, and the method specifically comprises the following steps: culturing the Magnaporthe magnus to logarithmic growth phase, centrifugally collecting the thalli and resuspending the thalli in a phosphate buffer; ultrasonically crushing cells in an ice bath environment, controlling the power to be 100W, crushing for 10s and spacing for 10s, and repeating for 50 times; separating magnetosome with magnet, washing with phosphate buffer solution for 5 times to obtain purified magnetosome.
3. The method of claim 1, wherein in step (2), the pH of the mixture is adjusted with a 6mol/L NaOH and HCl solution.
4. The method according to claim 1, wherein in step (2), the small molecule organic acid salt is any one of acetate, propionate or butyrate.
5. The method according to claim 1, wherein in step (2), the anaerobic activated sludge is taken from a stably-operating electric fermentation methanogenic reactor, and is rich in electric active bacteria and methanogenic archaea.
6. The method of claim 1, wherein in step (2), the graphite rod electrode has a diameter of 0.5cm and a length of 7.5 cm.
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| CN112162013A (en) * | 2020-09-28 | 2021-01-01 | 福建农林大学 | Microbial fuel cell for monitoring electron transfer between direct inoculation and application method |
| CN112390359A (en) * | 2020-11-16 | 2021-02-23 | 浙江工商大学 | Preparation and application method of filler with enhanced methane production function |
| CN112390359B (en) * | 2020-11-16 | 2022-08-09 | 浙江工商大学 | Preparation and application method of filler with enhanced methane production function |
| CN112960880A (en) * | 2021-03-12 | 2021-06-15 | 桂林理工大学 | Method for improving methane production of anaerobic co-digestion waste oil and sludge based on carbon cloth addition |
| CN116282522A (en) * | 2023-03-28 | 2023-06-23 | 中建三局绿色产业投资有限公司 | Method for treating garbage leachate by coupling magnetite with microbial electrolytic cell |
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