An electric energy driven biological nitrogen fixation system and method

Abstract

The present disclosure relates to a system and method for electrically driven biological nitrogen fixation, wherein the system and method combine an electrocatalytic system with a nitrogen-fixing bacterium of the brown variety, the nitrogen-fixing bacterium being a nitrogen-fixing bacterium of the brown variety with a deposit number of GDMCC No. 11487 ( Azotobacter vinelandii) In this system, nitrogen in the air can be converted into ammonia under aerobic conditions, providing a nitrogen source for its own growth and the surrounding ecosystem, and can meet the large-scale production of ammonia. 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.

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 more energy-saving and environmentally friendly methods to produce ammonia.

[0003] Biological nitrogen fixation offers environmentally friendly, green, and low-carbon advantages, making it a viable alternative for large-scale ammonia production. However, the primary energy source for biological nitrogen fixation is ATP hydrolysis, a limiting factor due to its slow rate of ATP hydrolysis. Microbial electrosynthesis is a new, green, sustainable, and interdisciplinary technology that utilizes renewable electricity to provide reducing power for microorganisms, promoting cell growth and biosynthesis. This technology enables nitrogen-fixing bacteria to synthesize ammonia using nitrogen as a substrate, and holds great promise for future applications.

[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 on 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 vine nitrogen-fixing bacteria to achieve efficient ammonia production even in an aerobic environment.

[0007] To achieve the above objectives, 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 comprising an H-type dual-chamber electrocatalytic reactor, a proton exchange membrane, electrodes, and a gas input device;

[0008] The H-type dual-chamber electrocatalytic reactor consists of an independent cathode chamber and an anode chamber. 110-130 mL of bacteria 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;

[0009] The electrodes include a working electrode, a counter electrode and a reference electrode; the gas input device continuously introduces air into the H-type dual-chamber electrocatalytic reactor;

[0010] 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;

[0011] The classification of the brown nitrogen-fixing bacteria is named as brown nitrogen-fixing bacteria ( Azotobacter vinelandii ), the deposit number is GDMCC No.1.1487.

[0012] 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.

[0013] Optionally, the working electrode and the counter electrode are carbon cloth connected to titanium wire; and the reference electrode is a saturated calomel electrode.

[0014] 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.

[0015] Optionally, the liquid culture medium is 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;

[0016] The PBS buffer solution includes 8.5-9.0 g / L Na2HPO4, 4.4-4.6 g / L NaH2PO4, and 0.05-0.15 g / L KCl; has a pH of 6.8-7.0, and is sterilized by steam at 121-125°C and 101-105 kPa for 18-22 minutes.

[0017] The present disclosure 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 disclosure further controls the culture conditions to ensure that the brown nitrogen-fixing bacteria grows well in the culture medium.

[0018] 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:

[0019] S1. Inoculate brown nitrogen-fixing bacteria into liquid culture medium and culture to obtain bacterial volume OD 600 The bacterial solution is 0.2-0.4;

[0020] 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;

[0021] S3, continuously inputting air into the H-type dual-chamber electrocatalytic reactor using a gas input device;

[0022] S4, connecting the reactor body to an electrochemical workstation, setting a constant voltage of -0.6 to -0.8 V to operate the system, and culturing the system at 28-32° C.;

[0023] The classification of the brown nitrogen-fixing bacteria is named as brown nitrogen-fixing bacteria ( Azotobacter vinelandii ), the deposit number is GDMCC No.1.1487.

[0024] Optionally, the liquid culture medium is 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 medium is steam sterilized at 120-122°C and 101-105 kPa for 18-22 min;

[0025] The PBS buffer solution includes 8.5-9.0 g / L Na2HPO4, 4.4-4.6 g / L NaH2PO4, and 0.05-0.15 g / L KCl; has a pH of 6.8-7.0, and is sterilized by steam at 121-125° C. and 101-105 kPa for 18-22 minutes.

[0026] Optionally, in step S1, the culturing process includes:

[0027] The brown nitrogen-fixing bacteria were inoculated into Burk liquid medium and cultured at 200-240 rpm and 28-32°C until the bacterial count OD 600 is 1.0-1.2, and the first bacterial liquid is obtained;

[0028] The first bacterial solution was centrifuged at 4000-6000 rpm and 2-6°C for 10 minutes; 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 under the same conditions for 10-12 minutes to remove residual culture medium and bacterial metabolites to obtain a second bacterial solution;

[0029] 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.

[0030] 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; and then adding 110-130 mL of the bacterial cell mass OD 600 The bacterial solution is 0.2-0.4.

[0031] Optionally, step S3 further includes: filtering bacteria contamination in the air through an air filter, and then continuously inputting air into the H-type dual-chamber electrocatalytic reactor using the gas input device, with a ventilation volume of 120-130 mL / min.

[0032] Through the above technical solution, the present disclosure provides a system and method for electrically driven biological nitrogen fixation. The system and method combine an electrocatalytic system with brown nitrogen-fixing bacteria, which can convert nitrogen in the air into ammonia under aerobic conditions, providing a nitrogen source for its own growth and the surrounding ecosystem, and can meet the large-scale production of ammonia.

[0033] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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 detailed description, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings:

[0035] Figure 1 The standard curve is drawn for the concentration of ammonium ions secreted outside the cells of vine nitrogen-fixing bacteria.

[0036] Figure 2 is the ammonium ion concentration under the conditions of applying and not applying voltage to the brown nitrogen-fixing bacteria.

[0037] Figure 3 The results of the detection of nitrogen-fixing bacteria under the conditions of applied and no voltage were 15 N chromatographic peak.

[0038] 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.

[0039] Figure 5 is the cell density of vine nitrogen-fixing bacteria with and without applied voltage.

[0040] Figure 6 is the dry cell weight of vine nitrogen-fixing bacteria with and without applied voltage. DETAILED DESCRIPTION

[0041] The following describes the specific embodiments of the present disclosure in detail with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure and are not intended to limit the present disclosure.

[0042] A first aspect of 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 comprising an H-type dual-chamber electrocatalytic reactor, a proton exchange membrane, electrodes, and a gas input device;

[0043] The H-type dual-chamber electrocatalytic reactor consists of an independent cathode chamber and an anode chamber. 110-130 mL of bacteria containing OD 600 It is a liquid culture medium containing 0.2-0.4 brown nitrogen-fixing bacteria; PBS buffer is added to the anode chamber;

[0044] The electrodes include a working electrode, a counter electrode and a reference electrode; the gas input device continuously introduces air into the H-type dual-chamber electrocatalytic reactor;

[0045] 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;

[0046] The classification of the brown nitrogen-fixing bacteria is named as brown nitrogen-fixing bacteria ( Azotobacter vinelandii ), the deposit number is GDMCC No.1.1487.

[0047] The system provided by the present disclosure combines an electrocatalytic reaction with brown nitrogen-fixing bacteria, and converts the introduced air into ammonia through brown nitrogen-fixing bacteria, which can effectively solve the problem that existing electric energy-driven biological nitrogen fixation cannot be applied in an aerobic environment.

[0048] 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 minutes.

[0049] 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.

[0050] Optionally, the working electrode and the counter electrode are carbon cloth connected to titanium wire; and the reference electrode is a saturated calomel electrode.

[0051] 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 medium is sterilized by steam at 120-122°C and 101-105 kPa for 18-22 minutes;

[0052] The PBS buffer solution includes 8.5-9.0 g / L Na2HPO4, 4.4-4.6 g / L NaH2PO4, and 0.05-0.15 g / L KCl; has a pH of 6.8-7.0, and is sterilized by steam at 121-125° C. and 101-105 kPa for 18-22 minutes.

[0053] 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:

[0054] S1. Inoculate brown nitrogen-fixing bacteria into liquid culture medium and culture to obtain bacterial volume OD 600 The bacterial solution is 0.2-0.4;

[0055] 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;

[0056] S3, continuously inputting air into the H-type dual-chamber electrocatalytic reactor using a gas input device;

[0057] S4, connecting the reactor body to an electrochemical workstation, setting a constant voltage of -0.6 to -0.8 V to operate the system, and culturing the system at 28-32° C.;

[0058] The classification of the brown nitrogen-fixing bacteria is named as brown nitrogen-fixing bacteria ( Azotobacter vinelandii), the deposit number is GDMCC No.1.1487.

[0059] In the method of electrically driven biological nitrogen fixation provided in the present disclosure, a nitrogen-fixing bacterium (Vine nitrogen-fixing bacteria) with a deposit number of GDMCC No. 1.1487 is used to convert air introduced into an electrocatalytic reactor into ammonia. This solves the problem that current electrically driven nitrogen fixation systems can only be used for nitrogen fixation in anaerobic and microaerobic environments, while achieving efficient ammonia production.

[0060] Optionally, the liquid culture medium is 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 medium is steam sterilized at 120-122°C and 101-105 kPa for 18-22 min;

[0061] The PBS buffer solution includes 8.5-9.0 g / L Na2HPO4, 4.4-4.6 g / L NaH2PO4, and 0.05-0.15 g / L KCl; has a pH of 6.8-7.0, and is sterilized by steam at 121-125° C. and 101-105 kPa for 18-22 minutes.

[0062] Optionally, in step S1, the culturing process includes:

[0063] The brown nitrogen-fixing bacteria were inoculated into Burk liquid medium and cultured at 200-240 rpm and 28-32°C until the bacterial count OD 600 is 1.0-1.2, and the first bacterial liquid is obtained;

[0064] The first bacterial solution was centrifuged at 4000-6000 rpm and 2-6°C for 8-12 minutes; 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 under the same conditions for 10-12 minutes to remove residual culture medium and bacterial metabolites to obtain a second bacterial solution;

[0065] 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.

[0066] 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; and then adding 110-130 mL of the bacterial cell mass OD 600 The bacterial solution is 0.2-0.4.

[0067] Step S3 also includes: filtering the bacteria contamination in the air through an air filter, and then continuously inputting air into the H-type dual-chamber electrocatalytic reactor using the gas input device, with a ventilation volume of 120-130 mL / min.

[0068] The present disclosure is further illustrated below by way of examples, but the present disclosure is not limited thereby.

[0069] Example 1

[0070] (1) The materials used in this embodiment are as follows:

[0071] Test strain: Brown nitrogen-fixing bacteria (classification name: Azotobacter vinelandii , deposit number is GDMCC No.1.1487, type: free-living nitrogen-fixing bacteria, purchased from Guangdong Provincial Microbial Culture Collection Center);

[0072] Culture medium: The formula of PBS buffer is shown in Table 1, and the formula of Burk's medium is shown in Table 2;

[0073] Table 1 PBS buffer

[0074]

[0075] Table 2 Burk medium

[0076]

[0077] (2) Cultivation of brown nitrogen-fixing bacteria: Cultivate the brown nitrogen-fixing bacteria with the collection number of GDMCC No. 1.1487. 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 suspension was centrifuged at 5000 rpm and 4 °C for 10 min, the supernatant was removed, and the bacteria were resuspended and washed twice with 0.85% saline. The suspension was centrifuged for 10 min under the same conditions to remove the residual culture medium and bacterial metabolites. The washed bacterial suspension was added to Burk medium, and the OD 600 Adjust to 0.3.

[0078] (3) Construction of 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 middle was separated by a proton exchange membrane. Carbon cloth connected to titanium wire served as the working electrode and counter electrode, and a saturated calomel electrode (SCE) served 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 the culture in step (2) was added to the cathode chamber. 600 =0.3 Burk's medium containing Azotobacter vinifera; 120 mL of sterile PBS buffer was added to the anode chamber. Air was continuously introduced into the working electrolytic cell using an air pump, with the air flow meter setting the flow rate at 125 mL / min. An air filter was used to filter out any bacterial contamination. The reactor was connected to an electrochemical workstation (Chenhua CHI1040C) and set to a constant voltage of -0.8 V. The reaction was incubated in a constant-temperature incubator at 30°C, with the current recorded every 60 seconds.

[0079] (4) NH4 + Concentration determination:

[0080] a. Solution preparation:

[0081] To prepare the ammonia nitrogen standard stock solution: Place a certain amount of ammonium chloride in an electric blast drying oven at 100-105°C and dry for 2 hours. Then weigh 0.3146g of dry ammonium chloride and dissolve it in the electrolyte. Transfer the solution to a 100mL volumetric flask and adjust to volume with the appropriate culture medium. The ammonia nitrogen standard stock solution can be stored at 2-5°C for 1 month. To prepare the ammonia nitrogen standard working solution: Transfer 1.00mL of the ammonia nitrogen standard stock solution to a 100mL volumetric flask and adjust to volume with the appropriate culture medium. To prepare the color developer A: Weigh 5.0g of sodium salicylate and 5.0g of potassium sodium tartrate, respectively, and dissolve them in 100mL of 1.0 mol L -1 In KOH solution; Preparation of oxidant B: Prepare 0.05 mol L -1 NaClO solution; Preparation of Catalyst C: Weigh 0.18 g of sodium nitroferricyanide and dissolve it in 18 mL of ultrapure water.

[0082] b. Draw an ammonia nitrogen standard curve: Take six 10 mL colorimetric tubes and add 0.00, 0.04, 0.08, 0.12, 0.16, and 0.20 mL of the ammonia nitrogen standard working solution, respectively. Fill the tube with electrolyte to the 4 mL mark. Then, sequentially add 4 mL of color developer A, 2 mL of oxidant B, and 0.4 mL of catalyst C. Shake well and let stand in the dark for 1 hour. Using a 10 mm cuvette as the sample cell and the electrolyte as the reference solution, measure the absorbance at 655 nm using a UV-visible spectrophotometer. Draw an ammonia nitrogen standard curve with concentration as the horizontal axis and absorbance as the vertical axis.

[0083] c. Sample Testing: Take 4 mL of the sample to be tested and add 4 mL of Color Developer A, 2 mL of Oxidant B, and 0.4 mL of Catalyst C, sequentially. Shake well and let stand in the dark for 1 hour. Using a 10 mm cuvette as the sample cell and the electrolyte as the reference solution, measure the absorbance at 655 nm using a UV-Vis spectrophotometer. Calculate the corresponding ammonia nitrogen concentration using the ammonia nitrogen standard curve.

[0084] (5) 15 N2 verification of electric-driven biological nitrogen fixation: The headspace of the electric-driven biological nitrogen fixation system was repeatedly vacuumed (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 device and analyzed using an isotope mass spectrometer. 15 N abundance.

[0085] (6) Result determination:

[0086] Electricity-driven nitrogen-fixing bacteria Azotobacter vinelandii GDMCC No.1.1487 Ammonia secretion: Determination of NH4 secreted outside the cell 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.

[0087] Under -0.8 V voltage condition, the nitrogen-fixing bacteria GDMCC No.1.1487 in Burk's medium was driven to secrete NH4 outside the cell. + Concentration Figure 2 As shown, after culturing 48h with -0.8 V voltage, NH4 was secreted into the extracellular space. +The concentration was 14.00 μg / mL (0.78 mM), and the NH4 secreted into the extracellular space by the vine nitrogen-fixing bacteria without voltage applied was 0. + The concentration is 1.83µg / mL (0.102 mM). When voltage is 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.

[0088] Furthermore, the elemental analyzer-isotope mass spectrometry was used to detect the activity of brown nitrogen-fixing bacteria when cultured for 60 h under conditions of -0.8 V voltage and no 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). It was calculated that the brown nitrogen-fixing bacteria were cultured under the condition of -0.8V voltage for 60 h. 15 The N abundance is 1.14±0.08% under no voltage applied conditions. 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; Under the condition of adding sucrose, applying -0.8 V voltage and no voltage, the strain was cultured in the electrocatalytic system for 60h and NH4 + Output Figure 4 shown.

[0089] In the electrocatalytic system, the growth of brown nitrogen-fixing bacteria GDMCC No.1.1487 reached the plateau phase at 36 h. At 60 h, the cell density of brown nitrogen-fixing bacteria (OD 600 ) is 2.26 at most, and the cell dry weight is 88.10 mg. Under no voltage condition, the cell density of brown nitrogen-fixing bacteria is 1.71, and the cell dry weight is 56.50 mg (e.g. Figure 5-6After applying voltage, the cell density increased by 1.32 times, and the cell dry weight increased by 1.56 times. This shows that applying voltage in this electrocatalytic system can promote the bacterial growth of vinelandia GDMCC No.1.1487. The bacterial protein content of vinelandia GDMCC No.1.1487 is as high as 71.18%. Due to their nitrogen-fixing properties, diazotrophs express a large amount of nitrogenase (including molybdenum-iron nitrogenase, vanadium-iron nitrogenase, and iron-iron nitrogenase), making them ideal single-cell proteins rich in iron. Single-cell proteins refer to the bacterial proteins of microorganisms such as bacteria and yeast. Microbial cells are rich in protein, and single-cell proteins contain eight amino acids that cannot be synthesized by animal organisms. In particular, they contain high levels of lysine, methionine, and tryptophan, which are lacking in plant feed. Their biological value is far superior to plant protein, with a digestibility of up to 85-90%. Soybeans have the highest protein content among crops, but their protein content is only 35-40%. Therefore, single-cell proteins of vinelandia are an ideal protein feed. Using this system and method, the bacterial protein of vinelandia can be significantly increased.

[0090] Comparative Example 1

[0091] 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 purchased from Guangdong Provincial Microbial Culture Collection) 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 other three brown nitrogen-fixing bacteria to secrete NH4 +， like Figure 4 shown.

[0092] The above scheme shows 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 using an air pump, thereby achieving electric-driven nitrogen fixation in an aerobic environment. In addition, the protein content of the brown nitrogen-fixing bacteria in the electrocatalytic system is significantly increased, which can also provide a good idea for producing feed protein.

[0093] 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 of the above embodiments. Within the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the scope of protection of the present disclosure.

[0094] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0095] In addition, the 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. A biological nitrogen fixation method for simultaneously increasing ammonia production and biomass, characterized in that: The method is carried out by an electrically driven biological nitrogen fixation system, the system comprising an electrochemical workstation and a reactor body; the reactor body comprising 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. The electrodes include a working electrode, a counter electrode, and a reference electrode. The method comprises the following steps: S1. Inoculate brown nitrogen-fixing bacteria into liquid culture medium and culture to obtain bacterial volume 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 to operate 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.1.1487.

2. The method 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; The pH is 6.8-7.0, and steam sterilized at 120-122°C, 101-105kPa for 18-22 minutes; The PBS buffer solution includes 8.5-9.0 g / L Na2HPO4, 4.4-4.6 g / L NaH2PO4, and 0.05-0.15 g / L KCl; has a pH of 6.8-7.0, and is sterilized by steam at 121-125°C and 101-105 kPa for 18-22 minutes.

3. The method according to claim 2, 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 count 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 minutes; 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 under the same conditions for 10-12 minutes to remove residual culture medium and bacterial metabolites to obtain 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.

4. The method according to claim 1, 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.

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

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

Images