Water body self-bio-nitrogen-fixing bacteria G12, bacterial agent, bacterial bag and application thereof

Abstract

The application belongs to the technical field of water pollution treatment and ecological environment restoration, and relates to development and application of microbial functional resources, and particularly relates to a water self-grown nitrogen-fixing bacteria G12, a bacterial agent, a bacterial bag and application thereof. Ralstonia pickettii The water self-grown nitrogen-fixing bacteria G12 is classified and named as The bacteria is preserved in China Center for Type Culture Collection, located at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, and has a preservation number of CCTCC NO: M 20251602 and a preservation date of July 14, 2025. The strain has wide environmental adaptability, can maintain nitrogenase activity under low-oxygen, weak-light and trace nutrient element fluctuation conditions, and has a self-limiting regulation mechanism of nitrogen-fixing activity in the application process. After the inorganic nitrogen content rises, the nitrogenase activity decreases, which helps to maintain the ecological balance of nitrogen in the system and reduce the risk of secondary pollution.

Description

Technical Field

[0001] This invention belongs to the field of water pollution control and ecological environment restoration technology, and relates to the development and application of microbial functional resources, specifically to a water-borne nitrogen-fixing bacterium G12, a bacterial agent, a bacterial bag, and their applications. Background Technology

[0002] In recent years, green governance technologies based on simulated natural wetland ecosystems have gained widespread attention in the field of water pollution control. Constructed wetlands, low-carbon-nitrogen ratio ecological agricultural wastewater treatment systems, and other plant-microbe coupled environmental remediation systems have demonstrated significant environmental benefits and potential for widespread application in urban and rural wastewater treatment and agricultural non-point source pollution control. These systems effectively reduce major pollutants such as nitrogen, phosphorus, and organic matter through synergistic mechanisms including plant absorption, microbial metabolism, and substrate adsorption, offering advantages such as low energy consumption, high ecological integration, and ease of management.

[0003] In the aforementioned systems, nitrogen removal primarily relies on microbially driven nitrification and denitrification processes. However, this mechanism is highly sensitive to environmental conditions such as dissolved oxygen, organic carbon sources, temperature, and carbon-nitrogen ratios. Especially in the initial stages of system startup, when the nitrogen-phosphorus ratio is imbalanced, or when there are fluctuations in exogenous nitrogen input, a "nitrogen shortage" phenomenon often occurs in the root zone, making it difficult to support plant community building and the stable expression of micro-ecosystem functions, thereby limiting purification efficiency and the speed of ecosystem construction.

[0004] Furthermore, the rhizosphere environment of these plant-microbe synergistic systems is often characterized by low oxygen, weak light, and large fluctuations in micronutrient elements, such as iron. 2+ This poses challenges to the colonization, physiological activity, and nitrogen metabolism of microorganisms. Currently, there is a lack of self-sustaining nitrogen-fixing microbial resources that can function stably under these typical environmental stresses and possess the ability to replenish nitrogen sources in situ. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a water-borne nitrogen-fixing bacterium G12, a bacterial agent, a bacterial bag, and their applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an aquatic autotrophic nitrogen-fixing bacterium G12, which is classified and named as follows: Ralstonia pickettii It is deposited at the China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, with accession number CCTCC NO: M 20251602 and deposit date of July 14, 2025.

[0008] This invention is the first to discover a free-living nitrogen-fixing strain with high environmental adaptability and potential for nitrogen fixation expression, named... Ralstonia pickettii G12, this strain exhibits good adaptability to low oxygen levels and low light tolerance, and can thrive in trace amounts of Fe. 2+ This strain maintains nitrogenase activity under certain conditions and adapts to the rhizosphere environments of various resource-constrained or environmentally fluctuating aquatic ecosystems. Based on these characteristics, this strain can be applied to green environmental remediation projects such as constructed wetlands and agricultural wastewater purification systems. Potential applications include loading the strain onto inert carriers such as highly porous activated carbon and deploying it in the rhizosphere region of plants to form a "microbial-root symbiotic" microenvironment, assisting in the initial establishment of plant communities and rhizosphere nitrogen supply, thereby improving the system's start-up efficiency and operational stability. Furthermore, the nitrogen fixation process of this strain is negatively regulated by environmental nitrogen concentration, reducing its nitrogen load risk in complex environments and demonstrating good ecological safety.

[0009] Secondly, the present invention provides a microbial agent for nitrogen fixation, wherein the microbial agent is formed by adsorbing and attaching the water-borne nitrogen-fixing bacteria G12 onto an adsorption carrier.

[0010] In some specific embodiments, the adsorption carrier is selected from at least one of activated carbon and biochar.

[0011] Thirdly, the present invention provides a microbial bag for nitrogen fixation, which is made of a breathable material encapsulating the microbial agent.

[0012] In some specific embodiments, the breathable material is selected from at least one of nonwoven fabric and nylon fabric.

[0013] Fourthly, the present invention provides the application of the aforementioned microbial bags in constructing a plant-microorganism symbiotic system in water.

[0014] Furthermore, the water body is an artificial wetland or a water body from an ecological agricultural wastewater treatment system.

[0015] Furthermore, the bacterial bags are placed in the rhizosphere core area of ​​plants in the water to achieve local nitrogen source replenishment and synergistic construction of the micro-ecology.

[0016] Furthermore, 4 to 6 of the aforementioned mushroom bags are placed per 1 m² of the rhizosphere core area of ​​the plant.

[0017] The present invention has the following beneficial effects:

[0018] This invention has screened and obtained a free-living nitrogen-fixing strain with broad environmental adaptability that can maintain nitrogenase activity under conditions of low oxygen, low light, and fluctuations in micronutrients. This strain is expected to provide a continuous nitrogen source for plant rhizospheres, enhance plant-microbe synergistic functional expression, improve early colony establishment efficiency and long-term operational stability, and therefore has good engineering application potential in green infrastructure, the initial stage of constructed wetland establishment, and ecological agricultural wastewater treatment. Attached Figure Description

[0019] Figure 1 for Ralstonia pickettii G12 phylogenetic tree.

[0020] Figure 2 This is a colony diagram of the bacteria purified by the streak plate method in Example 1.

[0021] Figure 3 for Ralstonia pickettii G12 cell proliferation under different temperature conditions.

[0022] Figure 4 for Ralstonia pickettii G12 Ammonia Nitrogen Cumulative Changes under Different Temperature Conditions.

[0023] Figure 5 for Ralstonia pickettii Graph showing changes in nitrogenase activity of G12 under different temperature conditions.

[0024] Figure 6 for Ralstonia pickettii G12 cell proliferation under different light conditions.

[0025] Figure 7 for Ralstonia pickettii G12 Ammonia Nitrogen Accumulation Changes under Different Light Conditions.

[0026] Figure 8 for Ralstonia pickettii Graph showing changes in nitrogenase activity of G12 under different light conditions.

[0027] Figure 9 for Ralstonia pickettii Cell proliferation of G12 cells under different concentrations of ferrous ions catalyzed by different concentrations.

[0028] Figure 10 for Ralstonia pickettii Figure 1. Cumulative changes in ammonia nitrogen under different concentrations of ferrous ions catalyzed by G12.

[0029] Figure 11 for Ralstonia pickettii Figure showing the change in nitrogenase activity of G12 under different concentrations of ferrous ions catalyzed by different concentrations. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0031] In the following examples, the nitrogen-fixing bacteria liquid culture medium and nitrogen-fixing bacteria solid culture medium used were the known Ashube nitrogen-free liquid culture medium and Ashube nitrogen-free solid culture medium in the prior art. The formulation of each 1L was: 5.0 g / L mannitol, 0.1 g / L KH2PO4, 0.1 g / L MgSO4·7H2O, 0.1 g / L NaCl, 0.05 g / L CaSO4·2H2O, 2.5 g / L CaCO3, 9.0 g / L agar, and the balance was water; the initial pH was adjusted to 7.0, and the medium was sterilized at 121°C for 30 minutes before use.

[0032] Example 1: Screening and identification of free-living nitrogen-fixing bacteria.

[0033] This embodiment describes a self-growth nitrogen-fixing bacterium, named... Ralstonia pickettii G12 is deposited at the China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, with accession number CCTCCNO: M 20251602 and deposit date of July 14, 2025.

[0034] In this embodiment, the screening and identification method for the free-living nitrogen-fixing bacteria specifically includes the following steps:

[0035] Step 1, enrichment of free-living nitrogen-fixing bacteria:

[0036] 100 mL of sediment from the shallow vegetation zone of Xi'an Lake was filtered through a 0.22 μm polycarbonate membrane. The polycarbonate membrane was then immediately placed on Assumption nitrogen-fixing medium and cultured for 5 days at 30±2℃ using the pure culture method. All samples were cultured in triplicate.

[0037] Step 2, initial screening of free-living nitrogen-fixing bacteria:

[0038] Take single colonies from the nitrogen-fixing bacteria plates obtained from the enrichment culture in step one. Pick single colonies of nitrogen-fixing bacteria that are consistent in color, shape, and size from the culture medium and purify them using the streak plate method on nitrogen-fixing bacteria solid culture medium, such as... Figure 2 As shown in the figure. The inoculated plates were incubated in a 30℃ incubator for 10 days, with three replicates for each colony. The purification was repeated 5 times until the colony morphology was consistent and free of contaminants, thus obtaining several pure strains of nitrogen-fixing bacteria from the initial screening.

[0039] Step 3, secondary screening of free-living nitrogen-fixing bacteria:

[0040] The multiple pure bacterial colonies obtained in step two were inoculated into Ashube nitrogen-fixing liquid medium and cultured at 30℃ for 5 days to obtain seed culture. 2.5 mL of each seed culture was used to detect nitrogenase activity using the acetylene reduction method. The strain with the highest nitrogenase activity was selected as the test strain.

[0041] Step 4, strain identification:

[0042] The DNA of the test strain obtained in step 3 was extracted and subjected to PCR amplification and sequencing. Genomic DNA of the test strain was extracted using an Omega DNA kit. The upstream primer used for PCR identification was 27F, with the sequence 5′-AGTTTGATCMTGGCTCAG-3′, as shown in SEQ ID NO.2; the downstream primer used for PCR identification was 1492R, with the sequence 5′-CGGCCGCGGCTGCTGGCACGT-3′, as shown in SEQ ID NO.3. The 25 μL PCR amplification system consisted of: 20-50 ng / μL genomic DNA template, 12.5 μL PCR Premix, 1 μL of 10 μM primer 27F, 1 μL of 10 μM primer 1492R, and 9.5 μL ddH2O. The PCR program was as follows: 95℃, 5 min; 94℃, 30 s; 57℃, 30 s; 72℃, 90 s; 30 cycles of amplification, followed by extension at 72℃ for 10 min. After PCR, a 16S rDNA fragment was obtained, and its 16S rDNA nucleotide sequence was obtained after sequencing, as shown in SEQ ID NO.1.

[0043]

[0044] The alignment results of the above 16S rDNA nucleotide sequences are as follows: Figure 1 As shown. By Figure 1 It can be seen that the 16S rDNA nucleotide sequence is similar to... Ralstonia pickettii ATCC 27511 Ralstonia pickettii NBRC 102503 has a similarity of over 99% and belongs to the genus Rolstonia. Ralstonia Therefore, the tested strain was identified as *Rolstonia piriformis*. Ralstonia pickettii ( Ralstonia pickettii ), and named it Ralstonia pickettii G12, or simply G12.

[0045] Example 2: Ralstonia pickettii The nitrogenase activity of G12 under different environmental conditions was tested.

[0046] I. Experimental Methods.

[0047] Approximately 1 mL of a suspension of free-living nitrogen-fixing bacteria was inoculated into nitrogen-free liquid culture medium. 140 mL serum bottles were used as reaction vessels, with 70 mL of liquid culture medium added to each bottle. Headspace gas was displaced to achieve an N2:O2 ratio of 95:5. The following variables were set for the experiment: temperature (5℃, 15℃, 25℃, 30℃); light intensity (0 lx, 1000 lx, 2500 lx); Fe... 2+ The concentrations were 0 μM, 0.1 μM, 1 μM, and 10 μM. The cells were cultured for 120 h under each single-factor condition, and the cell count and ammonia nitrogen (NH4) were measured every 12 h during this period. + -N concentration was measured, and nitrogenase activity was determined by sampling every 24 hours. The relevant results are shown below. Figures 3 to 11 As shown.

[0048] II. Experimental Results.

[0049] Depend on Figure 3 It can be seen that the bacteria grow rapidly under suitable temperature conditions, with the 30℃ treatment group showing the fastest growth rate, reaching its maximum cell count at 60 hours, followed by a slight decrease; the peak value in the 25℃ group was slightly lower, approximately 12 × 10⁻⁶. 6 The cells / mL also showed stable proliferation capacity; the cell proliferation rate in the 15℃ group was slower, with a peak value of 6.0 × 10⁻⁶. 6 The cells / mL indicates that within the temperature range of 25~30℃, the bacterial growth activity shows an increasing trend as the temperature rises.

[0050] Depend on Figure 4 , Figure 5The experimental results indicate that different temperature conditions significantly affect the nitrogenase activity and ammonia nitrogen accumulation of autotrophic nitrogen-fixing bacteria. Except for low temperature, both 25℃ and 30℃ promoted the increase of nitrogenase activity and ammonia nitrogen production to varying degrees, with the best performance observed at 30℃. Under 30℃ culture conditions, nitrogenase activity reached its peak value of approximately 75 nmol·mL⁻¹ within 24–48 hours of culture. -1 ·h -1 The ammonia nitrogen concentration also increased significantly, with the highest value approaching 2.3 mg / L.

[0051] Depend on Figure 6 It can be seen that light intensity has an inhibitory effect on the growth of this free-living nitrogen-fixing bacterium. For non-photosynthetic bacteria, prolonged exposure to strong light will inhibit their growth. Compared with the 0 lx dark treatment, the growth rate of the bacteria decreased under 1000 lx and 2500 lx light conditions, but the cells still had the ability to proliferate.

[0052] Depend on Figure 7 , Figure 8 It was found that different light intensities of 0 lx, 1000 lx, and 2500 lx had a significant impact on ammonia nitrogen accumulation and nitrogenase activity in autotrophic nitrogen-fixing bacteria. The experimental results showed that although nitrogenase activity decreased with increasing light intensity, nitrogenase was not completely inactivated. Compared with 0 lx in darkness, nitrogenase activity was significantly inhibited under light intensities of 1000 lx and 2500 lx.

[0053] Depend on Figure 9 It can be seen that adding Fe at concentrations of 0.1 μM, 1 μM, and 10 μM... 2+ It can effectively promote bacterial proliferation, with the bacterial count reaching its peak during the 48-72 hour period. The growth-promoting effect of 10.0 μM is the best, followed by 1.0 μM, while 0.1 μM is close to the control group.

[0054] Depend on Figure 10 , Figure 11 It can be seen that an appropriate amount of Fe 2+ The addition of iron significantly enhanced the generation and release of nitrogen fixation products. 1.0 μM and 10 μM iron effectively enhanced nitrogenase activity, indicating that appropriate amounts of iron ions provide essential cofactors for nitrogen-fixing bacteria, promoting normal nitrogenase function and enhancing bacterial growth and nitrogen product accumulation. At both concentrations, nitrogenase activity showed a clear upward trend, and the generation and release efficiency of ammonia nitrogen also improved, demonstrating the positive effect of iron ions on nitrogenase activation.

[0055] Depend on Figure 4 , Figure 5 , Figure 7 , Figure 8 , Figure 10 , Figure 11The peak nitrogenase activity occurred earlier than the peak ammonia nitrogen accumulation, suggesting a possible feedback regulation mechanism of ammonia nitrogen concentration during nitrogen fixation in this strain. The fact that nitrogenase activity peaked before ammonia nitrogen accumulation indicates that nitrogenase actively reduces nitrogen in the early stages of nitrogen fixation, while ammonia nitrogen accumulation exhibits a certain lag effect; nitrogenase activity begins to decline once ammonia nitrogen reaches its peak. This negative feedback mechanism constitutes a natural ecological self-limiting mechanism for this strain, helping to prevent excessive ammonia nitrogen accumulation in the environment, avoiding potential nitrogen excess problems during nitrogen fixation, thereby reducing its environmental risks in the plant-microbe symbiotic system and enhancing ecological safety.

[0056] Example 3: Ralstonia pickettii G12 root zone mount deployment method and its potential application demonstration.

[0057] Ralstonia pickettii G12 exhibited good performance under low oxygen, different light conditions, and trace amounts of Fe. 2+ The strain exhibits excellent nitrogen-fixing capacity, enzyme activity stability, and environmental adaptability under typical ecological conditions, demonstrating its potential for constructing "microbial-root symbiotic zones" in ecological engineering systems such as constructed wetlands and agricultural wastewater treatment zones. It should be noted that although the strain described in this invention possesses nitrogen-fixing capabilities, its application involves localized attachment to the rhizosphere of constructed wetlands. The released ammonia nitrogen can be directly absorbed by plants or transformed by the surrounding microbial community, without significantly increasing the total nitrogen content in the water. Furthermore, this nitrogen-fixing process is negatively regulated by environmental nitrogen levels; nitrogenase expression is actively inhibited in nitrogen-rich environments, resulting in good ecological safety.

[0058] In this embodiment, a high-porosity inert carrier is used to adsorb and mount the bacterial cells. The high-porosity inert carrier is columnar activated carbon particles, which are deployed in a directional manner in the rhizosphere region. The specific method is as follows:

[0059] 1. Preparation of bacterial culture: ... Ralstonia pickettii G12 was expanded to the logarithmic growth phase, i.e., OD, in Assumption nitrogen-free liquid medium. 600 ≈0.6, centrifuge at 8000 rpm for 10 min to collect bacterial cells, resuspend in sterile PBS buffer, and adjust the bacterial concentration to 10. 8 cells / mL.

[0060] 2. Adsorption and Loading: The bacteria are adsorbed onto the carrier surface and into the pores through a shaking incubation method. The loading time and parameters can be optimized according to actual needs to promote bacterial adsorption into the carrier pores. After loading, gently rinse with PBS to remove unbound bacteria.

[0061] 3. Loading and Fixing: Place the activated carbon loaded with bacteria into a breathable material, such as a non-woven mesh bag, and place it within 10-15cm of the plant root zone to ensure full contact with the rhizosphere microenvironment. This ensures sufficient contact with the root zone's microecological environment, forming a "microbial-root symbiotic zone."

[0062] 4. Deployment density: In the core vegetation area, consider setting 4 to 6 hanging mushroom bags per 1m². After deployment, no external energy or aeration is required, and the bacteria will be fixed with nitrogen under natural conditions.

[0063] Based on the results of Examples 1 and 2, it can be seen that Ralstonia pickettii G12, this strain is resistant to temperature, light, and trace amounts of Fe. 2+ Nitrogenase activity was stably expressed under all single-factor conditions. This was especially true under conditions of 25–30°C, 0–2500 lx of weak light, and 0.1–10 μM low-concentration Fe. 2+ It exhibits strong metabolic activity under typical environmental parameters, demonstrating its good adaptability to complex ecological environments. Furthermore, it offers a potential application scenario through the construction of a symbiotic deployment model of "activated carbon mounting + rhizosphere localization." Ralstonia pickettii G12 can naturally carry out nitrogen fixation in the rhizosphere of aquatic plants under complex environments. The released ammonia nitrogen can be directly absorbed by plants or further transformed by rhizosphere microorganisms, and is expected to provide auxiliary nitrogen source replenishment in the early stages of ecosystem establishment, supporting plant community building and rhizosphere microecological stability. More importantly, the nitrogen fixation process of this strain is regulated by negative feedback of environmental nitrogen concentration. Nitrogenase expression is inhibited under nitrogen-rich conditions, possessing natural "self-limiting" properties and ecological safety guarantees, and will not cause secondary pollution or excessive accumulation of nitrogen.

[0064] In summary, Ralstonia pickettii G12 has advantages such as strong environmental adaptability, stable nitrogen fixation metabolism, flexible deployment methods and controllable ecological risks. It has the potential to be applied in a variety of plant-microbe synergistic systems and is suitable for typical green governance scenarios such as constructed wetlands, agricultural wastewater treatment zones, and ecological floating islands. It has good prospects for engineering promotion and practical value.

[0065] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0066] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A type of aquatic autotrophic nitrogen-fixing bacterium G12, characterized in that, Its classification is named Ralstonia pickettii It is deposited at the China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, with accession number CCTCC NO: M 20251602 and deposit date of July 14, 2025.

2. A microbial agent for nitrogen fixation, characterized in that, The bacterial agent is formed by adsorbing and attaching the water-based nitrogen-fixing bacteria G12 described in claim 1 onto an adsorption carrier.

3. The microbial agent for nitrogen fixation according to claim 2, characterized in that, The adsorption carrier is selected from at least one of activated carbon and biochar.

4. A microbial bag for nitrogen fixation, characterized in that, It is made by encapsulating the microbial agent according to any one of claims 2 to 3 in a breathable material.

5. The microbial bag for nitrogen fixation according to claim 4, characterized in that, The breathable material is selected from at least one of non-woven fabric and nylon fabric.

6. The application of the microbial bags according to any one of claims 4 to 5 in constructing a plant-microorganism symbiotic system in aquatic bodies, characterized in that, The application involves placing the bacterial bags in the rhizosphere core area of ​​plants in water to achieve local nitrogen source replenishment and synergistic construction of the micro-ecology.

7. The application according to claim 6, characterized in that, The water body is either an artificial wetland or the water body of an ecological agricultural wastewater treatment system.

8. The application according to claim 7, characterized in that, Four to six of the aforementioned mushroom bags are placed per 1 m² of the rhizosphere core area of ​​the plant.

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