Carbon-nitrogen circulating system and application thereof in closed environment

Abstract

The invention relates to the technical field of carbon and nitrogen circulation, and discloses a carbon and nitrogen circulation system and application thereof in a closed environment. A symbiotic system formed by cupriavidus and clostridium pasteurianum has a relatively good carbon and nitrogen co-supply capability, and after the symbiotic system runs in a closed environment for 3 days, the total organic carbon can reach 180 mg / L, and the content of NH4 < + > can reach 22 mg / L. The invention indicates that the symbiotic system formed by the Cupriavidus receptor and the Clostridium pasteurianum has a wide prospect of providing efficient carbon and nitrogen circulation for closed environments such as a human space capsule, a biological closed experiment module, a biosphere and a long-term submarine when being applied to the closed environments such as the human space capsule, the biological closed experiment module, the biosphere and the long-term submarine.

Description

Technical Field

[0001] The present invention relates to the technical field of carbon-nitrogen cycle, and particularly to a carbon-nitrogen cycle system and its application in a closed environment. Background Art

[0002] In natural ecosystems, carbon and nitrogen elements are continuously cycled through various biological and physico-chemical processes to maintain ecological balance. In a closed environment, due to the limited material exchange with the outside world, a system needs to be artificially constructed to simulate the natural carbon-nitrogen cycle. In closed environments such as human spacecraft, biological closed experimental capsules, biospheres, and long-term submarines, constructing an efficient carbon-nitrogen cycle system to achieve the recycling and reuse of essential elements for life such as carbon, oxygen, and nitrogen is of great significance for maintaining life support.

[0003] Current life support systems mostly rely on plants (such as algae and vegetable crops) for photosynthesis to convert CO2 into oxygen and organic matter, and provide nitrogen nutrition through plant absorption or traditional physico-chemical means. However, plant photosynthesis has high requirements for light and space, and its efficiency is limited in a closed capsule; at the same time, under closed environmental conditions, nitrogen mostly exists in the form of inert N2, which cannot be directly utilized by plants, and often relies on carried nitrogen fertilizers or microorganisms in waste recycling to mineralize and nitrify organic nitrogen before absorption, making the system highly dependent on external supplies.

[0004] To improve the autonomy of the life support system in a closed environment, researchers have begun to explore the role of microorganisms in element cycling. For example, there is prior art that proposes using chemolithoautotrophic hydrogen-oxidizing bacteria to convert CO2 into edible single-cell protein for space food production. Hydrogen-oxidizing bacteria (Knallgas bacteria) refer to a class of autotrophic microorganisms that can use hydrogen as an electron donor and oxygen as an electron acceptor to fix CO2 through the Calvin-Benson cycle mediated by the Rubisco enzyme. These hydrogen-oxidizing bacteria can "turn waste into treasure" by synthesizing their own biomass or metabolites, and their protein content can be as high as 70% of the dry weight of the bacterial cells, and the amino acid composition is close to high-quality animal protein. Therefore, in deep space missions with insufficient light, using hydrogen produced by electrolyzing water to drive hydrogen-oxidizing bacteria to produce food and recycle CO2 is an effective solution.

[0005] However, the vast majority of known hydrogen-oxidizing bacteria do not have the ability to fix nitrogen. Under conventional culture conditions, they require an external fixed nitrogen source such as ammonium salts or nitrates to synthesize proteins and nucleic acids. This means that if solely relying on hydrogen-oxidizing bacteria to construct a closed-loop biological regeneration system, additional nitrogen supplementation is still needed. Nitrogen in the closed system mainly exists in the form of N2, accounting for approximately 79% of the air. How to efficiently utilize this nitrogen source is one of the key technical problems. Existing nitrogen-fixing bacteria have the characteristic of strict anaerobiosis, while the gas in the closed environment is a gas that can support the survival of organisms such as humans and plants and has a certain oxygen content. Therefore, the nitrogen-fixing ability of nitrogen-fixing bacteria in the closed environment is limited. Summary of the Invention

[0006] To solve the above technical problems in the construction of the carbon-nitrogen cycle system and further solve the technical problem of the poor carbon-nitrogen cycle ability of the existing carbon-nitrogen cycle system, the present invention provides a carbon-nitrogen cycle system and its application in a closed environment.

[0007] The specific technical solution of the present invention is as follows: In the first aspect, the present invention provides a carbon-nitrogen cycle system, including a symbiotic system formed by hydrogen-oxidizing carbon-fixing bacteria and anaerobic nitrogen-fixing bacteria. The hydrogen-oxidizing carbon-fixing bacteria are Cupriavidus necator, and the anaerobic nitrogen-fixing bacteria are Clostridium pasteurianum.

[0008] Preferably, the symbiotic system is obtained by co-culturing in a sub-region mode. The region is divided into a first region forming a micro-oxic environment and a second region forming an anaerobic environment. The first region and the second region exchange organic carbon and NH4 through a circulating liquid. + .

[0009] Preferably, the first region is used to culture hydrogen-oxidizing carbon-fixing bacteria, and the second region is used to culture anaerobic nitrogen-fixing bacteria.

[0010] Preferably, the culture medium is a mixed medium of LB medium and synthetic medium.

[0011] Preferably, the composition of the LB medium is: 10.0 g / L of tryptone, 5.0 g / L of yeast extract, 10.0 g / L of sodium chloride, and made up to 1000 mL with distilled water.

[0012] Preferably, the composition of the synthetic medium is: 10.0 g / L of glucose, 1.0 g / L of yeast extract powder, 0.1 g / L of peptone, 0.5 - 1.0 g / L of phosphate, 0.2 g / L of magnesium sulfate, and 5.0 g / L of calcium carbonate.

[0013] Preferably, the culture temperature is 35 ± 2 °C.

[0014] In a second aspect, the present invention provides an application of the above carbon-nitrogen cycle system in providing a carbon-nitrogen cycle for a closed environment.

[0015] In a third aspect, the present invention provides an application of the above carbon-nitrogen cycle system in providing a carbon-nitrogen cycle for an artificial simulated closed environment.

[0016] Compared with the prior art, the present invention has the following technical effects: The symbiotic system formed by Cupriavidus necator and Clostridium pasteurianum has a good ability to jointly supply carbon and nitrogen. After operating in a closed environment for 3 days, the total organic carbon can reach 180 mg / L, and the NH4 + content can reach 22 mg / L. This indicates a broad prospect for the symbiotic system formed by Cupriavidus necator and Clostridium pasteurianum to be applied to closed environments such as human spacecraft, biological closed experimental cabins, biospheres, and long-term submarines to provide an efficient carbon-nitrogen cycle for them. Specific Embodiments

[0017] The following further describes the present invention in conjunction with embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are usually only some embodiments of the present invention, rather than all embodiments. Therefore, all other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0018] In the embodiments of the present invention, the symbiotic system formed by carbon-fixing bacteria with hydroxide and nitrogen-fixing anaerobic bacteria is cultured using a medium obtained by mixing LB medium and synthetic medium according to a mass ratio of 1:1. Among them, the composition of the LB medium is: tryptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 10.0 g / L, and distilled water is made up to 1000 mL; the composition of the synthetic medium is: glucose 10.0 g / L, yeast extract powder 1.0 g / L, peptone 0.1 g / L, phosphate 0.5 - 1.0 g / L, magnesium sulfate 0.2 g / L, calcium carbonate 5.0 g / L.

[0019] In the embodiments of the present invention, the single cells of carbon-fixing bacteria with hydroxide are cultured using LB medium, and the gas conditions for culturing are the same as those in the upper culture chamber.

[0020] In the embodiments of the present invention, the single cells of nitrogen-fixing anaerobic bacteria are cultured using synthetic medium, and the gas conditions for culturing are the same as those in the lower culture chamber.

[0021] In the embodiments of the present invention, the analysis method includes: (1) Carbon dioxide (CO2) concentration determination: The gas chromatography (GC) method is used to measure the CO2 concentration in the gas sample. The specific steps are as follows: Sample collection: Take out the gas sample from the reaction system and inject it into a pre-prepared sampling bag or a sealed container. Instrument setting: Use a gas chromatograph equipped with a thermal conductivity detector (TCD), select an appropriate chromatographic column (such as a stainless steel packed column), and helium or nitrogen can be used as the carrier gas. Analysis process: Inject the gas sample into the GC system, record the retention time and peak area of CO2. Calculate the CO2 concentration in the sample by comparing with the standard curve.

[0022] (2) Ammonium ion (NH4 + ) concentration determination: The indophenol blue colorimetric method is used to measure the NH4 + concentration in the solution. The steps are as follows: Reagent preparation: Prepare phenol solution, sodium hypochlorite solution, and sodium nitroprusside solution. Reaction process: Mix the sample with the above reagents. Under alkaline conditions, NH4 + reacts with the reagents to form a blue compound. Determination: Measure the absorbance at a wavelength of about 660 nm on a spectrophotometer. Calculate the NH4 + concentration through the standard curve.

[0023] (3) RuBisCO enzyme activity determination: The colorimetric method is used to measure the RuBisCO enzyme activity. The specific steps include: Enzyme extraction: Extract the crude enzyme solution from plant tissues or microbial cells. Reaction system: Add components such as RuBP, Mg 2+ , NaHCO3, and NADH to the reaction solution. Detection: Monitor the change in absorbance of NADH at 340 nm and calculate the enzyme activity.

[0024] (3) Nitrogenase activity determination (acetylene reduction method): The acetylene reduction method is used to evaluate the nitrogenase activity. The steps are as follows: Sample preparation: Place the nitrogen-fixing microorganism culture in a sealed container and introduce a certain amount of acetylene gas. Reaction process: Incubate under appropriate conditions to allow the nitrogenase to reduce acetylene to ethylene. Detection: Use a gas chromatograph to measure the amount of ethylene generated and calculate the nitrogenase activity. (4) Protein concentration determination (Bradford method): The Bradford method is used to measure the protein concentration. The specific steps are as follows: Reagent preparation: Prepare Bradford reagent using Coomassie Brilliant Blue G-250 dye. Reaction process: Mix the protein sample with the Bradford reagent. After the dye binds to the protein, the color changes from brown to blue. Determination: Measure the absorbance at a wavelength of 595 nm and calculate the protein concentration through the standard curve. (5) Determination of dry cell weight: The microbial biomass was evaluated by measuring the dry cell weight, and the steps were as follows: Sample treatment: Take a certain volume of bacterial liquid and centrifuge to collect the bacteria. Washing: Wash the bacteria with distilled water to remove the residual medium. Drying: Place the bacteria in an oven and dry at 105 °C until constant weight. Weighing: Weigh the dried bacteria using an analytical balance. (6) Determination of poly-β-hydroxybutyrate (PHB) content: The PHB content was determined by high performance liquid chromatography (HPLC), and the specific steps were as follows: Sample treatment: Methanolysis was performed on the bacterial sample to release the monomer of PHB, methyl hydroxybutyrate. HPLC analysis: A reverse-phase chromatographic column was used, the detection wavelength was 210 nm, and the mobile phase was a mixture of methanol and water. Quantification: The PHB content in the sample was calculated by comparing with the standard product.

[0025] Example 1 Table 1 Construct symbiotic systems 1, 2, 3, and 4 formed by carbon-fixing bacteria and anaerobic nitrogen-fixing bacteria as shown in Table 1. The steps include: The above symbiotic systems were cultured using a sub-region co-culture mode. The regions were divided into a first region forming a micro-aerobic environment and a second region forming an anaerobic environment. The first region and the second region exchanged organic carbon and NH4 through a circulating liquid. + Specifically: An integrated double-chamber composite bioreactor that is closed to the outside was constructed. Among them, the double chambers were in an upper and lower structure. The upper part was a carbon-fixing bacteria culture chamber, and the lower part was a nitrogen-fixing bacteria culture chamber. They were connected by a porous membrane or a valve in the middle to make the liquid phase communicate while the gas phase was basically isolated; the material of the upper culture chamber was a pressure-resistant material and was equipped with a gas distributor and a stirring device for efficiently dissolving the H2 / O2 / CO2 mixture in the culture medium; the lower culture chamber was sealed to prevent oxygen from seeping in, and N2 or an inert gas was introduced to maintain a positive pressure anaerobic environment; the diaphragm between the two chambers was an ultrafiltration membrane with an appropriate molecular weight cut-off to allow the free diffusion of small molecule nutrients and metabolites, but restricted the passage of bacteria, physically isolating the two strains to some extent; the culture medium was filled to 75% of the total working volume of the two chambers. The reactor was placed in an incubator, and the temperature was controlled at 35 ± 0.5 °C.

[0026] The carbon-fixing bacteria liquid and the nitrogen-fixing bacteria liquid were mixed at a certain volume ratio of 1:1 and inoculated into the two chambers.

[0027] After inoculation, gas was introduced into the upper carbon-fixing chamber. The initial gas composition volume ratio was H2: air: CO2 = 3:1:1.

[0028] During the cultivation process, the culture solution in the upper chamber is slowly pumped into the lower chamber through a circulation pump, and the same volume of liquid in the lower chamber overflows back into the upper chamber to form a circulation loop. The liquid in the upper and lower chambers circulates at a flow rate of 0.5 L / h. Through the liquid phase circulation, soluble products generated in the upper chamber, such as organic acids and free cell contents (from partial bacterial autolysis or leakage), will flow into the lower chamber and be taken up and utilized by the nitrogen-fixing bacteria; the NH4 + enters the upper chamber through mixing, and a part of it is used by the hydrogen-oxidizing bacteria to synthesize cells, and the rest accumulates in the liquid phase.

[0029] During the cultivation process, after 48 hours of cultivation, CO2 gas is supplemented into the upper chamber to a volume fraction of 65%, that is, the ventilation composition volume ratio is H2: air: CO2 = 48.75: 16.25: 35, to simulate the CO2 generated by human respiration and observe the system processing capacity. At the same time, the circulation liquid flow rate is increased to 1.0 L / h to accelerate nutrient exchange.

[0030] During the cultivation process, after 72 hours of cultivation, the cumulative NH4 + production of the two groups of systems was measured.

[0031] Example 2 The carbon-nitrogen cycle performance of the symbiotic systems 1, 2, 3, and 4 constructed in Example 1 was compared in a closed environment. The test process was carried out according to the steps of Example 1. When the symbiotic system was cultured for 72 hours, the OD 600 values of the upper and lower chambers, as well as the total organic carbon (TOC) in the upper chamber and the NH4 + content in the lower chamber were detected. The results are shown in Table 2.

[0032] As can be seen from Table 2, the symbiotic system 1 constructed in the present invention, that is, the symbiotic system formed by Cupriavidus necator and Clostridium pasteurianum, has good carbon-nitrogen co-supply ability. After operating for 3 days in a closed environment, the total organic carbon can reach 180 mg / L, and the NH4 + content can reach 22 mg / L. This indicates a broad prospect for the symbiotic system formed by Cupriavidus necator and Clostridium pasteurianum to be applied to closed environments such as human space capsules, biological closed experimental cabins, biospheres, and long-term submarines to provide efficient carbon-nitrogen cycles.

[0033] In the present invention, the raw materials and equipment used, unless otherwise specified, are all common raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0034] As described above, it is only a preferred embodiment of the present invention and does not impose any limitation on the present invention. Any simple modification, change, and equivalent transformation made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A carbon-nitrogen cycle system, characterized in that: It includes a symbiotic system formed by carbon dioxide-fixing hydrogen-oxidizing bacteria and anaerobic nitrogen-fixing bacteria, wherein the carbon dioxide-fixing hydrogen-oxidizing bacteria are copper-loving bacteria, and the anaerobic nitrogen-fixing bacteria are Clostridium pasteurianum.

2. The carbon-nitrogen cycle system according to claim 1, wherein: The symbiotic system is obtained by culturing in a sub-region co-culture mode. The region is divided into a first region that forms a micro-oxic environment and a second region that forms an anoxic environment. The first region and the second region exchange organic carbon and NH4 through a circulating liquid + .

3. The carbon-nitrogen circulation system according to claim 2, wherein: The first region is used for culturing carbon dioxide-fixing hydrogen-oxidizing bacteria, and the second region is used for culturing anaerobic nitrogen-fixing bacteria.

4. The carbon-nitrogen circulation system according to claim 2, characterized in that: The culture medium used for the culture is a mixed medium of LB medium and synthetic medium.

5. The carbon-nitrogen circulation system according to claim 4, wherein: The composition of the LB medium is: 10.0 g / L of tryptone, 5.0 g / L of yeast extract, 10.0 g / L of sodium chloride, and the volume is made up to 1000 mL with distilled water.

6. The carbon-nitrogen circulation system according to claim 4, characterized in that: The composition of the synthetic medium is: 10.0 g / L of glucose, 1.0 g / L of yeast extract powder, 0.1 g / L of peptone, 0.5 - 1.0 g / L of phosphate, 0.2 g / L of magnesium sulfate, and 5.0 g / L of calcium carbonate.

7. The carbon-nitrogen cycle system according to claim 2, characterized in that: The temperature for the culture is 35 ± 2 °C.

8. Application of the carbon-nitrogen cycle system according to any one of claims 1 to 7 in providing a carbon-nitrogen cycle for a closed environment.

9. Application of the carbon-nitrogen cycle system according to any one of claims 1 to 7 in providing a carbon-nitrogen cycle for an artificial simulated closed environment.

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