Electric-driven biological nitrogen fixation system and method

Abstract

The invention relates to an electric energy driven biological nitrogen fixation system and method, according to the system and method, an electro-catalysis system and brown nitrogen-fixing bacteria are combined for use, the brown nitrogen-fixing bacteria are brown nitrogen-fixing bacteria with the preservation number of GDMCC No.11487, in the system, nitrogen in air can be converted into ammonia under the aerobic condition, nitrogen in the air can be converted into nitrogen, nitrogen in the air can be converted into nitrogen, nitrogen in the air can be converted into nitrogen, nitrogen in the air can be converted into nitrogen, nitrogen in the air can be converted into nitrogen, nitrogen in the air can be converted into nitrogen, nitrogen in the air can be converted into nitrogen, nitrogen in the air can be converted into nitrogen, nitrogen in the air can be converted into nitrogen The Azotobacter vinellatus provides a nitrogen source for the growth of the Azotobacter vinellatus and a surrounding ecological system, can meet the requirement of large-scale production of ammonia, is obviously improved in protein content in an electro-catalysis system, and can also provide a good thought for producing feed protein.

Description

Technical Field

[0001] The present disclosure relates to the field of microbial technology, and in particular, to an electric energy driven biological nitrogen fixation system and method. Background Art

[0002] Ammonia is an important raw material in agricultural production. More than 90% of the world's ammonia is produced based on the Haber-Bosch process developed more than 100 years ago. However, the Haber-Bosch process is extremely energy-intensive and causes environmental pollution. It is necessary to develop a more energy-saving and environmentally friendly method of producing ammonia.

[0003] Biological nitrogen fixation has the advantages of being environmentally friendly, green and low-carbon, and can be used as an alternative way to produce ammonia on a large scale. However, the main energy source of biological nitrogen fixation comes from ATP hydrolysis, and the slow rate of ATP hydrolysis is a limiting factor. Microbial electrosynthesis technology is a new interdisciplinary technology that is green, environmentally friendly and sustainable. It can use renewable electricity to provide reducing power for microorganisms, promote cell growth and material synthesis, and enable nitrogen-fixing bacteria to use nitrogen as a substrate to synthesize ammonia, which has good application prospects.

[0004] However, the current electric energy driven biological nitrogen fixation system still has some shortcomings. For example, the application environment is limited and it can only be carried out in anaerobic and microaerobic environments. There are no reports of aerobic systems. The ammonia production is low and it is difficult to meet large-scale production needs.

[0005] Therefore, there is an urgent need to provide a method to solve the problem that the electrically driven nitrogen fixation system only fixes nitrogen in anaerobic and microaerobic environments, while achieving electrically driven efficient ammonia production. Summary of the invention

[0006] The purpose of the present disclosure is to provide a system and method for electrically driven biological nitrogen fixation, which combines an electrocatalytic system with brown nitrogen-fixing bacteria to achieve efficient ammonia production in an aerobic environment.

[0007] In order to achieve the above-mentioned object, the present disclosure provides a system for electrically driven biological nitrogen fixation, the system comprising an electrochemical workstation and a reactor body; the reactor body comprises an H-type double-chamber electrocatalytic reactor, a proton exchange membrane, electrodes, and a gas input device; The H-type dual-chamber electrocatalytic reactor consists of a cathode chamber and an anode chamber, each of which is independent of the other. 110-130 mL of a microbial cell containing OD 600 It is a liquid culture medium of 0.2-0.4 brown nitrogen-fixing bacteria; the anode chamber is added with PBS buffer; The electrodes include a working electrode, a counter electrode and a reference electrode; the gas input device continuously introduces air into the H-type double-chamber electrocatalytic reactor; Connect the reactor body to the electrochemical workstation, set a constant voltage of -0.6 to -0.8 V, and culture the system at 28-32° C.; The classification of the brown nitrogen-fixing bacteria is named as brown nitrogen-fixing bacteria ( Azotobacter vinelandii ), the deposit number is GDMCC No.11487.

[0008] Optionally, a sterilization device is further provided inside the reactor body to sterilize the reactor body at 120-130° C. and 101-105 kPa for 25-35 min.

[0009] Optionally, the working electrode and the counter electrode are carbon cloth connected to titanium wire; and the reference electrode is a saturated calomel electrode.

[0010] Optionally, the gas input device is an air pump; the gas input device is also connected to an air filter and an air flow meter, the air filter is used to filter bacteria contamination in the air; the air flow meter controls the air ventilation volume to 120-130mL / min.

[0011] Optionally, the liquid culture medium is a Burk liquid culture medium; the Burk liquid culture medium contains 0.1-0.3 g / L MgCl2, 0.08-0.10 g / L CaCl2, 0.7-0.9 g / L KH2PO4, 0.1-0.3 g / L K2HPO4, 0.013-0.015 g / L Na2SO4, 0.10-0.14 g / L Fe2(SO4)3, 0.0020-0.0026 g / L Na2MoO4, and 18-24 g / L sucrose; the pH is 6.8-7.0, and the culture medium is sterilized by steam at 120-122°C and 101-105 kPa for 18-22 min; The PBS buffer comprises 8.5-9.0 g / L Na2HPO4, 4.4-4.6 g / L NaH2PO4, and 0.05-0.15 g / L KCl; the pH value is 6.8-7.0, and the PBS buffer is sterilized by steam at 121-125°C and 101-105 kPa for 18-22 min.

[0012] The present invention cultivates the brown nitrogen-fixing bacteria in Burk liquid culture medium, so that the brown nitrogen-fixing bacteria grows and reproduces in a suitable environment to reach a certain bacterial volume; and the present invention further controls the culture conditions to ensure that the brown nitrogen-fixing bacteria grows well in the culture medium.

[0013] In another aspect, the present disclosure provides a method for electrically driving biological nitrogen fixation using the above system, the method comprising the following steps: S1. Inoculate brown nitrogen-fixing bacteria into liquid culture medium and culture to obtain the bacterial mass OD 600 The bacterial solution is 0.2-0.4; S2, add 110-130mL of the bacterial cell volume OD in the cathode chamber 600 The bacterial solution is 0.2-0.4; PBS buffer is added to the anode chamber; S3, continuously inputting air into the H-type dual-chamber electrocatalytic reactor using a gas input device; S4, connecting the reactor body to an electrochemical workstation, setting a constant voltage of -0.6 to -0.8 V, running the system, and culturing the system at 28-32° C.; The classification of the brown nitrogen-fixing bacteria is named as brown nitrogen-fixing bacteria ( Azotobacter vinelandii ), the deposit number is GDMCC No.11487.

[0014] Optionally, the liquid culture medium is a Burk liquid culture medium; the Burk liquid culture medium contains 0.1-0.3 g / L MgCl2, 0.08-0.10 g / L CaCl2, 0.7-0.9 g / L KH2PO4, 0.1-0.3 g / L K2HPO4, 0.013-0.015 g / L Na2SO4, 0.10-0.14 g / L Fe2(SO4)3, 0.0020-0.0026 g / L Na2MoO4, and 18-24 g / L sucrose; the pH is 6.8-7.0, and the liquid culture medium is sterilized at 120-122°C, 101-105 kPa steam for 18-22 min; The PBS buffer comprises 8.5-9.0 g / L Na2HPO4, 4.4-4.6 g / L NaH2PO4, and 0.05-0.15 g / L KCl; the pH value is 6.8-7.0, and the PBS buffer is sterilized by steam at 121-125° C. and 101-105 kPa for 18-22 min.

[0015] Optionally, in step S1, the culturing process includes: The brown nitrogen-fixing bacteria were inoculated into Burk liquid medium and cultured at 200-240 rpm and 28-32°C until the bacterial cell volume OD 600 is 1.0-1.2, and the first bacterial liquid is obtained; The first bacterial solution was centrifuged at 4000-6000 rpm and 2-6°C for 10 min; the supernatant was removed, and the solution was resuspended and washed 2-4 times with 0.8-0.9% saline; after washing, the solution was centrifuged again for 10-12 min under the same conditions to remove the residual culture medium and bacterial metabolites, thereby obtaining the second bacterial solution; The second bacterial solution was added to the Burk liquid medium to obtain the bacterial cell volume OD 600 The bacterial solution is 0.2-0.4.

[0016] Optionally, step S2 further comprises: sterilizing the reactor body at 121-130°C and 101-105 kPa for 25-35 min using a sterilizing device; then adding 110-130 mL of the bacterial cell mass OD 600 The bacterial solution is 0.2-0.4.

[0017] Optionally, step S3 also includes: filtering bacteria contamination in the air through an air filter, and then using the gas input device to continuously input air into the H-type dual-chamber electrocatalytic reactor, with a ventilation volume of 120-130 mL / min.

[0018] Through the above technical scheme, the present disclosure provides a system and method for electrically driven biological nitrogen fixation, which combines an electrocatalytic system with brown nitrogen-fixing bacteria, can convert nitrogen in the air into ammonia under aerobic conditions, provide a nitrogen source for its own growth and the surrounding ecosystem, and can meet the large-scale production of ammonia.

[0019] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings: Figure 1 A standard curve plotted for the concentration of ammonium ions secreted outside the cell by Azotobacter vinelandii.

[0021] Figure 2 is the ammonium ion concentration under the conditions of applying and not applying voltage to the brown nitrogen-fixing bacteria.

[0022] Figure 3 The results of the detection of nitrogen-fixing bacteria under the conditions of applying and not applying voltage 15 N chromatographic peak.

[0023] Figure 4 The ammonium ion production of four strains of vine nitrogen-fixing bacteria after 60 h of cultivation in the electrocatalytic system with and without applied voltage.

[0024] Figure 5 is the cell density of vine nitrogen-fixing bacteria with and without voltage applied.

[0025] Figure 6 is the dry cell weight of vine nitrogen-fixing bacteria with and without voltage applied. DETAILED DESCRIPTION

[0026] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0027] In a first aspect, the present disclosure provides a system for electrically driven biological nitrogen fixation, the system comprising an electrochemical workstation and a reactor body; the reactor body comprises an H-type double-chamber electrocatalytic reactor, a proton exchange membrane, electrodes, and a gas input device; The H-type dual-chamber electrocatalytic reactor consists of a cathode chamber and an anode chamber, each of which is independent of the other. 110-130 mL of a microbial cell containing OD 600 It is a liquid culture medium of 0.2-0.4 brown nitrogen-fixing bacteria; PBS buffer is added to the anode chamber; The electrodes include a working electrode, a counter electrode and a reference electrode; the gas input device continuously introduces air into the H-type double-chamber electrocatalytic reactor; Connect the reactor body to the electrochemical workstation, set a constant voltage of -0.6 to -0.8 V, and culture the system at 28-32° C.; The classification of the brown nitrogen-fixing bacteria is named as brown nitrogen-fixing bacteria ( Azotobacter vinelandii ), the deposit number is GDMCC No.11487.

[0028] The system provided by the present disclosure combines the electrocatalytic reaction with brown nitrogen-fixing bacteria, and converts the introduced air into ammonia through the brown nitrogen-fixing bacteria, which can effectively solve the problem that the existing electrical energy-driven biological nitrogen fixation cannot be applied in an aerobic environment.

[0029] Optionally, a sterilization device is further provided inside the reactor body to sterilize the reactor body at 121-130° C. and 101-105 kPa for 25-35 min.

[0030] Optionally, the gas input device is an air pump; the gas input device is connected to an air filter and an air flow meter, the air filter is used to filter bacteria contamination in the air; the air flow meter controls the air ventilation volume to 120-130mL / min.

[0031] Optionally, the working electrode and the counter electrode are carbon cloth connected to titanium wire; and the reference electrode is a saturated calomel electrode.

[0032] The liquid culture medium is a Burk liquid culture medium; the Burk liquid culture medium contains 0.1-0.3 g / L MgCl2, 0.08-0.10 g / L CaCl2, 0.7-0.9 g / L KH2PO4, 0.1-0.3 g / L K2HPO4, 0.013-0.015 g / L Na2SO4, 0.10-0.14 g / L Fe2(SO4)3, 0.0020-0.0026 g / L Na2MoO4, and 18-24 g / L sucrose; the pH value is 6.8-7.0, and the liquid culture medium is sterilized by steam at 120-122° C. and 101-105 kPa for 18-22 min; The PBS buffer comprises 8.5-9.0 g / L Na2HPO4, 4.4-4.6 g / L NaH2PO4, and 0.05-0.15 g / L KCl; the pH value is 6.8-7.0, and the PBS buffer is sterilized by steam at 121-125° C. and 101-105 kPa for 18-22 min.

[0033] In another aspect, the present disclosure provides a method for electrically driving biological nitrogen fixation using the above system, the method comprising the following steps: S1. Inoculate brown nitrogen-fixing bacteria into liquid culture medium and culture to obtain the bacterial mass OD 600 The bacterial solution is 0.2-0.4; S2, add 110-130mL of the bacterial cell volume OD in the cathode chamber 600 The bacterial solution is 0.2-0.4; PBS buffer is added to the anode chamber; S3, continuously inputting air into the H-type dual-chamber electrocatalytic reactor using a gas input device; S4, connecting the reactor body to an electrochemical workstation, setting a constant voltage of -0.6 to -0.8 V, running the system, and culturing the system at 28-32° C.; The classification of the brown nitrogen-fixing bacteria is named as brown nitrogen-fixing bacteria ( Azotobacter vinelandii ), the deposit number is GDMCC No.11487.

[0034] In the method of electrically driven biological nitrogen fixation provided in the present disclosure, brown nitrogen-fixing bacteria with a deposit number of GDMCC No. 11487 are used to convert the air introduced into the electrocatalytic reactor into ammonia, thereby solving the problem that the current electrically driven nitrogen fixation system can only be used for nitrogen fixation in anaerobic and microaerobic environments, and at the same time, achieving efficient ammonia production.

[0035] Optionally, the liquid culture medium is a Burk liquid culture medium; the Burk liquid culture medium contains 0.1-0.3 g / L MgCl2, 0.08-0.10 g / L CaCl2, 0.7-0.9 g / L KH2PO4, 0.1-0.3 g / L K2HPO4, 0.013-0.015 g / L Na2SO4, 0.10-0.14 g / L Fe2(SO4)3, 0.0020-0.0026 g / L Na2MoO4, and 18-24 g / L sucrose; the pH is 6.8-7.0, and the liquid culture medium is sterilized at 120-122°C, 101-105 kPa steam for 18-22 min; The PBS buffer comprises 8.5-9.0 g / L Na2HPO4, 4.4-4.6 g / L NaH2PO4, and 0.05-0.15 g / L KCl; the pH value is 6.8-7.0, and the PBS buffer is sterilized by steam at 121-125° C. and 101-105 kPa for 18-22 min.

[0036] Optionally, in step S1, the culturing process includes: The brown nitrogen-fixing bacteria were inoculated into Burk liquid medium and cultured at 200-240 rpm and 28-32°C until the bacterial cell volume OD 600 is 1.0-1.2, and the first bacterial liquid is obtained; The first bacterial solution was centrifuged at 4000-6000 rpm and 2-6°C for 8-12 min; the supernatant was removed, and the solution was resuspended and washed 2-4 times with 0.8-0.9% saline; after washing, the solution was centrifuged again for 10-12 min under the same conditions to remove the residual culture medium and bacterial metabolites, thereby obtaining a second bacterial solution; The second bacterial solution was added to the Burk liquid medium to obtain the bacterial cell volume OD 600 The bacterial solution is 0.2-0.4.

[0037] Optionally, step S2 further comprises: sterilizing the reactor body at 121-130°C and 101-105 kPa for 25-35 min using a sterilizing device; then adding 110-130 mL of the bacterial cell mass OD 600 The bacterial solution is 0.2-0.4.

[0038] Step S3 also includes: filtering bacteria contamination in the air through an air filter, and then using the gas input device to continuously input air into the H-type dual-chamber electrocatalytic reactor, with a ventilation volume of 120-130 mL / min.

[0039] The present disclosure is further illustrated below by way of examples, but the present disclosure is not limited thereby.

[0040] Example 1 (1) The materials used in this embodiment are as follows: Test strain: Brown nitrogen-fixing bacteria (classification name: Azotobacter vinelandii , the deposit number is GDMCC No.11487, type: self-generating nitrogen-fixing bacteria, purchased from Guangdong Microbial Culture Collection Center); Culture medium: The formula of PBS buffer is shown in Table 1, and the formula of Burk's medium is shown in Table 2; Table 1 PBS buffer

[0041] Table 2 Burk medium

[0042] (2) Cultivation of Brown nitrogen-fixing bacteria: Cultivate the Brown nitrogen-fixing bacteria with the deposit number of GDMCC No.11487 Azotobacter vinelandii Inoculate into 1 L Burk liquid medium and culture at 220 rpm and 30 °C until the bacterial volume reaches OD 600 ≈1.0. The above bacterial solutions were centrifuged at 5000 rpm and 4 °C for 10 min, the supernatant was removed, the bacteria were resuspended and washed twice with 0.85% saline, and centrifuged for 10 min under the same conditions to remove the residual culture medium and bacterial metabolites. The washed bacterial solutions were added to Burk medium, and the OD 600 Adjust to 0.3.

[0043] (3) Construction of an electrocatalytic nitrogen fixation system: An H-type double-chamber electrocatalytic reactor was constructed. The total volume of each chamber was 220 mL, the liquid volume of each chamber was 120 mL, the top space volume was 100 mL, and the proton exchange membrane was used to separate the chambers. Carbon cloth connected to titanium wire was used as the working electrode and counter electrode, and a saturated calomel electrode (SCE) was used as the reference electrode. A sterilization device was set up in the electrocatalytic reactor. The electrocatalytic system was sterilized at 121°C and 103 kPa for 30 min. Then, 120 mL of the OD 200-containing solution obtained by culturing in step (2) was added to the cathode chamber. 600 =0.3 Burk medium of brown nitrogen-fixing bacteria; 120 mL sterile PBS buffer was added to the anode chamber. Air was continuously introduced into the working electrolytic cell using an air pump, and the air flow rate was set to 125 mL / min using an air flow meter. Air filters were used to filter out bacteria contamination in the air. The reactor was connected to an electrochemical workstation (Chenhua CHI1040C), a constant voltage of -0.8 V was set, and the reactor was placed in a constant temperature incubator at 30°C for culture reaction, and the current was recorded once every 60 seconds.

[0044] (4) NH4 +Concentration determination: a. Solution preparation: Preparation of ammonia nitrogen standard stock solution: Take a certain amount of ammonium chloride and place it in an electric blast drying oven, dry it at 100-105℃ for 2 hours, then weigh 0.3146g of dry ammonium chloride and dissolve it in the electrolyte, transfer it to a 100 mL volumetric flask, and make up the volume with the corresponding culture medium. The ammonia nitrogen standard stock solution can be stored at 2-5℃ for 1 month; Preparation of ammonia nitrogen standard working solution: Take 1.00 mL of ammonia nitrogen standard stock solution and transfer it to a 100 mL volumetric flask, and make up the volume with the corresponding culture medium; Preparation of color developer A: Weigh 5.0g of sodium salicylate and 5.0g of potassium sodium tartrate respectively, and dissolve them in 100 mL of 1.0 mol L -1 KOH solution; Preparation of oxidant B: prepare 0.05 mol L -1 NaClO solution; Preparation of catalyst C: weigh 0.18 g of sodium nitrosoferrocyanide and dissolve it in 18 mL of ultrapure water.

[0045] b. Drawing of ammonia nitrogen standard curve: Take 6 10 mL colorimetric tubes, add 0.00, 0.04, 0.08, 0.12, 0.16 and 0.20 mL of ammonia nitrogen standard working solution respectively, add electrolyte to the 4 mL scale line, add 4 mL of color developer A, 2 mL of oxidant B and 0.4 mL of catalyst C in sequence, shake well, and let stand in the dark for 1 hour. Use a 10 mm colorimetric dish as a sample pool, electrolyte as a reference solution, use a UV-visible spectrophotometer to detect its absorbance at 655 nm, and use concentration as the horizontal axis and absorbance as the vertical axis to draw an ammonia nitrogen standard curve.

[0046] c. Sample detection: Take 4 mL of the sample to be tested, add 4 mL of color developer A, 2 mL of oxidant B and 0.4 mL of catalyst C in sequence, shake well, and let stand in the dark for 1 hour. Use a 10 mm cuvette as the sample pool, electrolyte as the reference solution, use a UV-visible spectrophotometer to detect its absorbance at 655 nm, and calculate the corresponding ammonia nitrogen concentration through the ammonia nitrogen standard curve.

[0047] (5) 15 N2 verification of electrically driven biological nitrogen fixation: The headspace of the electrically driven biological nitrogen fixation system was repeatedly evacuated (0.1 kPa) and filled with high-purity helium (99.999%, 120 kPa) 5 times, and finally the pressure was adjusted to 101.3 kPa, 20 ml 15 N2 gas labeled with N (purity > 98.5%) was injected into the system. After the run, the electrolyte containing bacteria was collected using a freeze-drying instrument and measured using an isotope mass spectrometer. 15 N abundance.

[0048] (6) Result determination: Electricity-driven nitrogen-fixing bacteria Azotobacter vinelandii GDMCC No.11487 Ammonia secretion: Determination of NH4 secreted by brown nitrogen-fixing bacteria using indophenol blue spectrophotometry + Concentration, plotted NH4 + The concentration standard curve is as follows Figure 1 As shown, y=47.794x-7.0457, R²=0.9993, the R of the standard curve 2 >0.999, which proves that this standard curve is rigorous and can be used for further determination.

[0049] -0.8 V voltage condition drives the nitrogen fixation of vine nitrogen-fixing bacteria GDMCC No.11487 in Burk's medium to secrete NH4 + Concentration Figure 2 As shown, after culturing 48 h with -0.8 V voltage, NH4 was secreted into the cell. + The concentration was 14.00 µg / mL (0.78 mM) and the NH4 secreted into the cell by the vine nitrogen-fixing bacteria without voltage applied. + The concentration was 1.83 µg / mL (0.102 mM). After voltage was applied, the NH4 + The concentration increased by 7.65 times, which shows that applying voltage can significantly improve the nitrogen fixation ability of brown nitrogen-fixing bacteria.

[0050] Furthermore, elemental analyzer-isotope mass spectrometry was used to detect the activity of brown nitrogen-fixing bacteria when cultured for 60 h under -0.8 V voltage and without voltage. 15 N abundance, the results are as follows Figure 3 As shown (15N abundance after 60 h of culture with -0.8 V voltage applied and without voltage applied, * corresponds to P Value, ** P≤ 0.01; *** P≤ 0.005; **** P≤ 0.001; ns means no significant difference), and it was calculated that the brown nitrogen-fixing bacteria had a voltage of -0.8 V after 60 h of cultivation. 15 N abundance is 1.14±0.08% without voltage application 15 The N abundance is 0.83±0.01%, which shows that the electro-driven brown nitrogen-fixing bacteria provided by the present disclosure can fix more 15 N; When the strain was cultured in the electrocatalytic system for 60 h, NH4 + Output Figure 4 shown.

[0051] In the above electrocatalytic system, the growth of brown nitrogen-fixing bacteria GDMCC No.11487 reached a plateau at 36 h. At 60 h, the cell density (OD 600 ) is 2.26 at most, and the cell dry weight is 88.10 mg. Under the condition of no voltage application, the cell density of brown nitrogen-fixing bacteria is 1.71, and the cell dry weight is 56.50 mg (e.g. Figure 5-6 As shown); after applying voltage, the cell density increased by 1.32 times, and the cell dry weight increased by 1.56 times. It can be seen that applying voltage in the electrocatalytic system can promote the bacterial cell amount of brown nitrogen-fixing bacteria GDMCC No.11487, and the bacterial protein content of brown nitrogen-fixing bacteria GDMCC No.11487 is as high as 71.18%. Due to its nitrogen-fixing characteristics, nitrogen-fixing bacteria express a large amount of nitrogenase (including molybdenum-iron nitrogenase, vanadium-iron nitrogenase and iron-iron nitrogenase), which is an ideal single-cell protein rich in iron. Single-cell protein refers to the bacterial protein of microorganisms such as bacteria and yeast. Microbial cells are rich in protein, and single-cell protein contains 8 kinds of amino acids that cannot be synthesized by animal bodies, especially lysine, methionine and tryptophan that are lacking in plant feed. The biological value is far better than plant protein, and the digestibility is as high as 85-90%. The highest protein content in crops is soybean, and the protein content is only 35-40%. Therefore, brown nitrogen-fixing bacteria single-cell protein is an ideal protein feed. The system and method can significantly increase the bacterial protein of brown nitrogen-fixing bacteria.

[0052] Comparative Example 1 In order to verify whether the electric energy promoted nitrogen fixation by brown nitrogen-fixing bacteria is universal, this comparative example measured the other three brown nitrogen-fixing bacteria ATCC 13705, ATCC 17962 and ATCC 478 (all of the above strains were purchased from Guangdong Microbial Culture Collection Center) in the same way as in Example 1. The extracellular secretion of NH4 + The results showed that applying a voltage of -0.8 V could only promote the GDMCC No.1148 strain provided in Example 1 to secrete more NH4 + , unable to promote the secretion of NH4 by the other three strains of brown nitrogen-fixing bacteria +， like Figure 4 shown.

[0053] It can be seen from the above scheme that the electric energy driven biological nitrogen fixation method and system provided by the present disclosure effectively improves the ammonia production of electric driven nitrogen fixation, and can increase the ammonia secretion of nitrogen fixation by 7.65 times, with the ammonia secretion reaching up to 14.00 µg / mL (0.78 mM); when a voltage of -0.8 V is applied, air is continuously introduced into the working electrolytic cell by an air pump, thereby realizing electric driven nitrogen fixation in an aerobic environment, and the protein content of the brown nitrogen-fixing bacteria in the electrocatalytic system is significantly improved, which can also provide a good idea for the production of feed protein.

[0054] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0055] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0056] In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. An electrically driven biological nitrogen fixation system, characterized in that: The system comprises an electrochemical workstation and a reactor body; the reactor body comprises an H-type double-chamber electrocatalytic reactor, a proton exchange membrane, electrodes, and a gas input device; The H-type dual-chamber electrocatalytic reactor consists of a cathode chamber and an anode chamber, each of which is independent of the other. 110-130 mL of a microbial cell containing OD 600 It is a liquid culture medium of 0.2-0.4 brown nitrogen-fixing bacteria; PBS buffer is added to the anode chamber; The electrodes include a working electrode, a counter electrode and a reference electrode; the gas input device continuously introduces air into the H-type double-chamber electrocatalytic reactor; Connect the reactor body to the electrochemical workstation, set a constant voltage of -0.6 to -0.8 V, and culture the system at 28-32° C.; The classification of the brown nitrogen-fixing bacteria is named as brown nitrogen-fixing bacteria ( Azotobacter vinelandii ), the deposit number is GDMCC No.11487.

2. The system according to claim 1, wherein: A sterilizing device is also provided inside the reactor body, and the reactor body is sterilized at 121-130° C. and 101-105 kPa for 25-35 min.

3. The system according to claim 1, wherein: The working electrode and the counter electrode are carbon cloth connected with titanium wire; The reference electrode is a saturated calomel electrode.

4. The system according to claim 1, wherein: The gas input device is an air pump; The gas input device is connected to an air filter and an air flow meter, and the air filter is used to filter bacteria contamination in the air; The air flow meter controls the air ventilation volume to be 120-130 mL / min.

5. The system according to claim 1, wherein: The liquid culture medium is a Burk liquid culture medium; the Burk liquid culture medium contains 0.1-0.3 g / L MgCl2, 0.08-0.10 g / L CaCl2, 0.7-0.9 g / L KH2PO4, 0.1-0.3 g / L K2HPO4, 0.013-0.015 g / L Na2SO4, 0.10-0.14 g / L Fe2(SO4)3, 0.0020-0.0026 g / L Na2MoO4, and 18-24 g / L sucrose; pH is 6.8-7.0, steam sterilize at 120-122℃, 101-105 kPa for 18-22min; The PBS buffer comprises 8.5-9.0 g / L Na2HPO4, 4.4-4.6 g / L NaH2PO4, and 0.05-0.15 g / L KCl; the pH value is 6.8-7.0, and the PBS buffer is sterilized by steam at 121-125° C. and 101-105 kPa for 18-22 min.

6. A method for biological nitrogen fixation driven by electrical energy using the system according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: S1. Inoculate brown nitrogen-fixing bacteria into liquid culture medium and culture to obtain the bacterial mass OD 600 The bacterial solution is 0.2-0.4; S2, add 110-130mL of the bacterial cell volume OD in the cathode chamber 600 The bacterial solution is 0.2-0.4; PBS buffer is added to the anode chamber; S3, continuously introducing air into the H-type dual-chamber electrocatalytic reactor using a gas input device; S4, connecting the reactor body to an electrochemical workstation, setting a constant voltage of -0.6 to -0.8 V, running the system, and culturing the system at 28-32° C.; The classification of the brown nitrogen-fixing bacteria is named as brown nitrogen-fixing bacteria ( Azotobacter vinelandii ), the deposit number is GDMCC No.11487.

7. The method according to claim 6, wherein: The liquid culture medium is a Burk liquid culture medium; the Burk liquid culture medium contains 0.1-0.3 g / L MgCl2, 0.08-0.10 g / L CaCl2, 0.7-0.9 g / L KH2PO4, 0.1-0.3 g / L K2HPO4, 0.013-0.015 g / L Na2SO4, 0.10-0.14 g / L Fe2(SO4)3, 0.0020-0.0026 g / L Na2MoO4, and 18-24 g / L sucrose; pH is 6.8-7.0, steam sterilize at 120-122℃, 101-105kPa for 18-22min; The PBS buffer comprises 8.5-9.0 g / L Na2HPO4, 4.4-4.6 g / L NaH2PO4, and 0.05-0.15 g / L KCl; the pH value is 6.8-7.0, and the PBS buffer is sterilized by steam at 121-125° C. and 101-105 kPa for 18-22 min.

8. The method according to claim 7, wherein: In step S1, the culturing process includes: The brown nitrogen-fixing bacteria were inoculated into Burk liquid medium and cultured at 200-240 rpm and 28-32°C until the bacterial cell volume OD 600 is 1.0-1.2, and the first bacterial liquid is obtained; The first bacterial solution was centrifuged at 4000-6000 rpm and 2-6°C for 8-12 min; the supernatant was removed, and the solution was resuspended and washed 2-4 times with 0.8-0.9% saline; after washing, the solution was centrifuged again for 10-12 min under the same conditions to remove the residual culture medium and bacterial metabolites, thereby obtaining a second bacterial solution; The second bacterial solution was added to the Burk liquid medium to obtain the bacterial cell volume OD 600 The bacterial solution is 0.2-0.

4.

9. The method according to claim 6, wherein: Step S2 also includes: sterilizing the reactor body at 121-130°C and 101-105 kPa for 25-35 min using a sterilizing device; then adding 110-130 mL of the bacterial cell mass OD 600 The bacterial solution is 0.2-0.

4.

10. The method according to claim 6, wherein: Step S3 also includes: filtering bacteria contamination in the air through an air filter, and then using the gas input device to continuously introduce air into the H-type dual-chamber electrocatalytic reactor, with a ventilation volume of 120-130 mL / min.

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