Nitrifying bacteria and their use in sewage treatment

Abstract

This invention belongs to the field of microbial technology and discloses a nitrifying bacterium and its application in wastewater treatment. The nitrifying bacterium is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO.68388 and deposit date of June 1, 2026. After domestication, its tolerance to uranium is significantly enhanced, and key process parameters are optimized. Under optimal conditions, the removal rates of nitrogen and uranium reach 91.54% and 86.06%, respectively, with rapid kinetic reactions. XHK-1 achieves denitrification through nitrification and assimilation, while simultaneously utilizing the ammonium and phosphate ions generated during denitrification metabolism to fix uranium into a thermodynamically stable ammonium-uranium mica mineral phase through a synergistic mechanism. This invention provides a new microbial resource and technical approach for the efficient and low-cost bioremediation of nitrogen- and uranium-contaminated wastewater.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a nitrifying bacterium and its application in wastewater treatment. Background Technology

[0002] The large amounts of uranium-containing wastewater generated during uranium mining and nuclear industry development are a significant source of radioactive environmental pollution. This type of wastewater typically contains high concentrations of nitrogenous pollutants such as ammonia nitrogen and nitrates, forming a complex nitrogen-uranium composite pollution system. If nitrogen-containing uranium wastewater is discharged directly without effective treatment, it will not only lead to eutrophication of water bodies and damage aquatic ecosystems, but also allow the radioactive nuclide uranium to accumulate through the food chain, ultimately threatening human health.

[0003] Currently, the main methods for treating uranium-containing wastewater include adsorption, ion exchange, membrane separation, and chemical precipitation. Although these methods are fast, they suffer from high costs, complex operations, and a tendency to generate secondary pollution. Furthermore, they are not very effective for treating low-concentration uranium. In contrast, biological treatment methods have become a research hotspot in the field of radioactive wastewater treatment in recent years due to their advantages such as low cost, environmental friendliness, and no secondary pollution.

[0004] However, most existing technologies target single pollutants and struggle to effectively remove both ammonia nitrogen and uranium from nitrogen-containing uranium wastewater simultaneously. Traditional biological denitrification processes require synergistic action of nitrifying and denitrifying bacteria under different oxygen conditions, resulting in complex processes and high operating costs. Furthermore, uranium possesses strong biotoxicity, inhibiting microbial growth and metabolic activity, making it difficult for ordinary microorganisms to survive and function in uranium-containing environments. Therefore, screening for functional bacterial strains with both high uranium tolerance and simultaneous nitrification and denitrification capabilities is crucial for the simultaneous treatment of nitrogen-containing uranium wastewater. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a nitrifying bacterium and its application in wastewater treatment. A nitrifying bacterium with simultaneous nitrification and denitrification capabilities was screened and obtained. After uranium tolerance acclimatization, this strain can grow efficiently in uranium-containing environments and simultaneously remove ammonia nitrogen and uranium from wastewater, exhibiting a synergistic "denitrification-uranium fixation" mechanism. This provides a new microbial resource and treatment method for the bioremediation of complex polluted water bodies.

[0006] To achieve the above objectives, the present invention provides a nitrifying bacterium, wherein the nitrifying bacterium is a nitrifying bacterium ( Bacillus sp. XHK-1, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC NO.68388, deposited on June 1, 2026.

[0007] The application of the nitrifying bacteria in wastewater treatment is also provided.

[0008] Preferably, the wastewater is nitrogen- and uranium-containing composite pollutant wastewater.

[0009] A method for treating nitrogen-containing uranium wastewater using the nitrifying bacteria is also provided, comprising the following steps: S1. Strain activation: Inoculate nitrifying bacteria into the culture medium and culture with shaking; S2. Uranium-tolerant acclimatization: The activated strain is cultured in a uranium-containing medium, then streak purified. The culture and streak purification process is repeated to obtain a stable uranium-tolerant strain. S3. Cell preparation: The domesticated strain is inoculated into the culture medium for expansion culture, the cells are collected by centrifugation, and the cells are resuspended in sterile water to prepare a resuspension solution. S4. Wastewater treatment: The resuspended bacterial solution is added to the nitrogen-containing uranium wastewater, and then the pH and carbon-nitrogen ratio are adjusted for treatment.

[0010] Preferably, in step S1, the culture medium is beef extract peptone medium, specifically: 5 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, pH=7.3; the shaking culture conditions are 30℃, 150 r / min, and culture until OD. 600 The value is 1.

[0011] Preferably, in step S2, the uranium concentration in the uranium-containing culture medium is 2 mg / L, and the culture conditions are 30℃, 150 r / min, and 24 h; the streak purification is carried out by streaking growth on a culture medium containing 2 mg / L uranium using the plate streak method, and after culturing at 30℃ for 72 h, a single colony is picked; the repeated culture and streak purification process specifically involves repeating the culture and streak purification process 6 to 7 times.

[0012] Preferably, in step S3, the culture medium is: glucose 10g / L, beef extract 5g / L, peptone 10g / L, NaCl 5g / L, pH=7.3; centrifugation is performed at 4000r / min for 10min.

[0013] Preferably, in step S4, the amount of resuspended bacterial solution added to the nitrogen-containing uranium wastewater is 1%~5% (V / V); the pH is adjusted to 6.5~7.5; the carbon-nitrogen ratio is 2~7:1; and the temperature during wastewater treatment is 25~35℃.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects: (1) The nitrifying bacteria obtained by screening in this invention have both simultaneous nitrification and denitrification capabilities and high uranium tolerance. They can achieve simultaneous and efficient removal of ammonia nitrogen and uranium in a single oxygen environment, solving the technical problem that traditional bioremediation requires multiple bacterial species and a separate oxygen environment.

[0015] (2) The present invention uses a continuous transfer liquid-solid culture method to acclimate strain XHK-1 to uranium tolerance. After acclimatization, the strain can remove 96% of uranium within 48 hours. Under optimal conditions, the removal rates of ammonia nitrogen and uranium reach 91.54% and 86.06% respectively, with significant remediation effect.

[0016] (3) This invention reveals the unique “denitrification-uranium fixation” synergistic mechanism of nitrifying bacteria XHK-1: through heterotrophic nitrification-aerobic denitrification and assimilation, ammonia nitrogen is converted into nitrogen gas and bacterial biomass. At the same time, the ammonium and phosphate generated by denitrification metabolism are used to fix uranium into a thermodynamically stable ammonium uranium mica mineral phase, thereby achieving the harmlessness and stabilization of pollutants.

[0017] (4) The treatment method of the present invention is simple, low-cost and environmentally friendly. It is applicable to the treatment of nitrogen-containing uranium composite pollutant wastewater generated in uranium mining, nuclear industry and other fields, and has broad application prospects.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of strain XHK-1 on beef extract peptone solid medium. Figure 2 The growth curve of strain XHK-1 and the curves showing the changes in pH and dissolved oxygen (DO) during the culture process are shown. Figure 3 The OD of strain XHK-1 during the 7 rounds of continuous solid-liquid transfer culture for uranium tolerance acclimatization in Example 2. 600 A graph showing the changes in uranium content and uranium removal rate; Figure 4 The graph shows the changes in dissolved oxygen (DO) and uranium concentration over time during the cultivation of strain XHK-1 after uranium tolerance acclimatization. The normal group in the graph represents those without uranium tolerance acclimatization treatment, while the acclimatized group represents those treated with uranium tolerance acclimatization. Figure 5 OD of XHK-1 strain before and after uranium tolerance acclimatization under uranium-containing culture conditions 600 A comparison chart of the changes in pH value and uranium removal rate over time. In the chart, the ordinary group represents those without uranium tolerance acclimatization treatment, and the acclimatized group represents those with uranium tolerance acclimatization treatment. Figure 6 The graph shows the effect of strain XHK-1 on the removal efficiency of ammonia nitrogen and uranium in the system under different carbon-to-nitrogen ratios (C / N). Figure 7 The first-order kinetic exponential decay fitting curve for ammonia nitrogen removal by nitrifying bacteria XHK-1; Figure 8Pseudo-first-order and pseudo-second-order kinetic fitting curves for uranium removal by nitrifying bacteria XHK-1; Figure 9 The image shows the surface morphology of nitrifying bacteria XHK-1 in the control group under normal culture (without uranium tolerance acclimatization). Scale bar: 200 nm. Figure 10 Scale bar: 200 nm; Surface morphology of nitrifying bacteria XHK-1 after uranium tolerance acclimatization. Figure 11 XRD patterns of bacterial cells under different conditions; Figure 12 This is a transmission electron microscope (TEM) image of logarithmic-phase nitrifying bacteria XHK-1 grown under normal culture conditions (without uranium tolerance acclimatization). Scale bar: 500 nm. Figure 13 Transmission electron microscopy (TEM) image of nitrifying bacteria XHK-1 after uranium tolerance acclimation and treatment in a complex contamination system. Scale bar: 500 nm.

[0020] strain preservation Nitrifying bacteria ( Bacillus sp. The sample is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNO.68388, deposit date June 1, 2026, address 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, and named nitrifying bacteria XHK-1. Detailed Implementation

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0022] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0023] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0024] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0025] In the examples, the uranium standard solution of the culture medium was sterilized by filtration through a 0.22 μm filter membrane and then diluted to the required working concentration. All other solutions were effectively sterilized by high-temperature steam sterilization at 121°C for 20 minutes.

[0026] Example 1 I. Screening and identification of nitrifying bacteria XHK-1.

[0027] (1) Sample collection: In the early stage of the experiment, samples were collected from eight sampling points, including the bottom mud of ponds and water accumulation pools around a uranium tailings reservoir in Hunan Province, and uranium tailings 0.5m deep from the surface of the uranium tailings beach. After sampling, the samples were quickly stored in a refrigerator at 4℃.

[0028] (2) Enrichment culture: Take a small amount of water sample, mud sample and uranium tailings sample respectively and add them to beef extract peptone medium (beef extract 5g / L, peptone 10g / L, NaCl 5g / L, pH=7.3), and enrich them at a constant temperature of 30℃ and 150r / min for 24h.

[0029] (3) Separation and screening (dilution plating method): The enriched bacterial culture was subjected to 10... -1 ~10 -6 Dilute the solution, then take 100 μL of the diluted solution and spread it evenly on a nitrification screening solid medium containing 2 mg / L uranium and a high ammonia nitrogen content (approximately 425 mg / L) ((NH4)SO4 2.0 g / L, C4H4Na2O4 8.0 g / L, K2HPO4 0.25 g / L, MgSO4·7H2O 0.125 g / L, NaCl 0.125 g / L, FeSO4·7H2O 0.0025 g / L, MnSO4·4H2O 0.0025 g / L, agar 15 g / L, pH=7.3). Incubate at 30°C for 72 h in a biochemical incubator.

[0030] (4) Purification culture: The dominant colonies in the screening solid medium were picked and cultured in beef extract peptone medium, and then repeatedly inoculated into the screening solid medium for purification. After multiple rounds of enrichment culture, isolation and screening, high-purity and stable strains were obtained. The OD of each strain was measured. 600 and NH4 + -N removal rate was used to select the nitrifying strain with the highest activity, which was named XHK-1.

[0031] II. Morphological observation.

[0032] The purified XHK-1 strain was streaked onto beef extract peptone solid medium and incubated at 30°C for 24 hours before observing the colony morphology.

[0033] The results are as follows Figure 1As shown, the XHK-1 bacterial colony growth bands and single colonies appear pale yellow with smooth surfaces. Single colonies are distributed in a circular or near-circular pattern with relatively neat edges. After dispersing stable colonies, the morphology of the strains was observed under a microscope. The bacteria are distributed in rod-shaped forms, with plump morphologies. The cells are relatively independent, and the cell morphology is diverse, with multiple distributions of independent cells, cells in the dividing phase, and cell aggregates coexisting in the culture environment.

[0034] III. Physiological and biochemical characteristics.

[0035] The purified XHK-1 strain was inoculated onto beef extract peptone medium containing an additional carbon source (10 g / L glucose was added as an additional carbon source) for expansion, and the OD was collected. 600 The culture medium containing 1% of the expanded bacterial culture was inoculated into beef extract peptone medium at a 1% inoculation rate and cultured at 30℃ with constant temperature shaking at 150 rpm. The OD value of the bacterial culture was measured every 1 hour. 600 pH and dissolved oxygen content.

[0036] After 60 hours of continuous culture, the bacterial growth process generally includes four phases: lag phase, logarithmic phase, stationary phase, and death phase. After inoculation, the bacteria proliferate rapidly and quickly enter the logarithmic phase. The growth curve of XHK-1 bacteria is shown below. Figure 2 As shown, bacterial growth was relatively slow after 9 hours of incubation, gradually entering a stationary phase. Over time, the bacterial OD... 600 The concentration reached a maximum of 2.07 at 45 hours, after which the bacterial concentration gradually decreased, and the number of dead cells increased. Regarding pH and DO (dissolved oxygen), both pH and DO decreased rapidly in the early stages of cultivation, compared to OD... 600 The opposite trend indicates that the strain has vigorous growth and metabolism and high oxygen consumption.

[0037] IV. Molecular biological identification.

[0038] Freshly cultured bacteria were selected, and their 16S rDNA genes were extracted and sequenced. The genus of nitrifying bacteria was identified by sequence alignment. DNA was extracted from nitrifying bacteria in the logarithmic growth phase using a bacterial DNA extraction kit. DNA amplification was performed using universal PCR primers, and the purified target band was sequenced after fragment analysis by gel electrophoresis. The obtained 16S rDNA sequence was compared with the NCBI database, and the gene sequence of strain XHK-1 was found to be similar to... Sporosarcina newyorkensis (JX971511.1) is the most similar. A phylogenetic tree was constructed using Mega software to compare the bacterial sequence with that of similar genera, and the results also showed that the most similar strain to XHK-1 was (JX971511.1). Sporosarcina newyorkensisTherefore, strain XHK-1 was identified as Bacillus and its sequence was submitted to GenBank with accession number PV344458.

[0039] Example 2 Uranium tolerance domestication of nitrifying bacteria XHK-1.

[0040] The dominant nitrifying strains tolerant to low-concentration uranium environments were acclimatized using a continuous transfer liquid-solid culture acclimatization method. The specific steps are as follows: (1) Prepare beef extract peptone liquid culture medium with pH 7.3. After sterilizing at 121°C for 20 min, add 1 g / L uranium standard solution that has been sterilized by filtration through a 0.22 μm filter membrane in a clean bench to make the uranium concentration 2 mg / L.

[0041] (2) The activated and expanded XHK-1 bacterial culture (OD) 600 =1) Add 1% (V / V) of the inoculum to the above uranium-containing liquid culture medium and incubate at 30℃ and 150r / min for 24h with shaking.

[0042] (3) After three subcultures, the bacteria were streaked on a screening solid medium containing 2 mg / L uranium using the streak plate method. After culturing at 30°C for 72 h, vigorous single colonies were picked.

[0043] (4) Repeat the above liquid subculturing and solid streak purification process multiple times (7 times in this example) to obtain a stable uranium-resistant strain with high nitrogen-reducing activity.

[0044] (5) Then, stable colonies from the plate were picked and placed in liquid culture medium, and incubated at 30°C and 150 r / min. Their OD values ​​were then measured. 600 Biochemical indicators such as pH, dissolved oxygen, and uranium removal rate.

[0045] The results are as follows Figure 3As shown, low-concentration radioactivity has varying degrees of impact on bacterial growth. Upon initial exposure to a uranium-containing environment, the XHK-1 strain exhibited slow growth and proliferation, as well as low uranium removal efficiency. This is primarily due to the biotoxicity of uranium, which inhibits bacterial growth and significantly threatens its nutrient acquisition and metabolic exchange patterns. However, with increasing acclimatization cycles, the bacterial concentration gradually stabilized after the sixth cycle. This is because the dominant strain gradually adapted to the uranium-containing environment, and its growth and metabolic mechanisms under uranium stress returned to stable operation. The bacterial cells were able to exchange nutrients normally with the culture medium, thus promoting proliferation. After the strain's tolerance improved, bacterial growth and division in the culture medium became more vigorous. Compared to strains after previous acclimatization cycles, later strains possessed more efficient biological activity and uranium removal advantages. A large number of active bacteria removed uranium from the environment through effective biosorption and metabolic accumulation mechanisms, resulting in a continuous decrease in uranium concentration.

[0046] After the first round of acclimatization, the OD of the bacterial culture... 600 The residual uranium concentrations were 0.954 and 1.48 mg / L, respectively, with a uranium removal rate of only 25.8%. Compared with the dominant strains that can stably metabolize and remove uranium, the OD... 600 The improvement reached 49.31%, and the increase in the concentration of XHK-1 strain also led to an increase in uranium removal rate of nearly 60%. During the pH test of each round of acclimatization bacterial solution, it was found that the pH of the bacterial solution increased relatively slowly in the later stage of acclimatization. This was because the metabolism of XHK-1 strain in exchanging nutrients was slowed down under the condition of inhibition, which showed that the proliferation process of XHK-1 strain was inhibited. In the later stage, after the tolerance of XHK-1 strain improved, its ability to obtain carbon and nitrogen sources in the culture environment gradually recovered, and its metabolic growth led to an increase in the pH growth rate.

[0047] The dissolved oxygen content of the strains before and after domestication was measured and found (e.g. Figure 4 As shown in the figure, although the overall trend of dissolved oxygen (DO) in the acclimatized XHK-1 strain was consistent with that of the normal group, the rate of decrease in the initial stage was slightly lower than that of the normal group (XHK-1 strain without acclimatization). This reflects the process that the initial proliferation of XHK-1 strain in the uranium-containing environment was inhibited and it preferentially resisted biotoxicity, resulting in relatively less consumption of dissolved oxygen in the culture medium. However, as time continued, the large-scale reproduction of bacteria increased the concentration of bacterial solution and increased oxygen consumption. From the change of residual uranium concentration in the culture medium, it can be seen that the proliferation of XHK-1 strain was limited in the early stage and it could not effectively remove uranium. With the enhancement of metabolism, the bacteria gradually reduced the concentration of uranium through effective complexation and other effects. This process also increased the consumption of dissolved oxygen. However, in the later stage of culture, XHK-1 strain gradually died, and the rupture and dissolution of bacterial cells caused some uranium to be released again. Therefore, the efficient synchronous uranium removal node within 48 hours is very important, and the residual uranium concentration was reduced to a minimum of 0.07 mg / L.

[0048] Subsequently, the growth characteristics of XHK-1 before and after domestication were measured, and the results are as follows: Figure 5 As shown, the growth trend of bacteria in uranium-containing culture after acclimatization is quite similar to that in ordinary culture (without uranium tolerance acclimatization), and the timeliness is better. In the early stage, the biotoxicity of uranium reduced the bacterial proliferation rate, resulting in a slightly lower bacterial concentration in the first 40 hours compared to the ordinary group. In the later stage, due to the accumulation of metabolic waste in the culture medium of the ordinary group and the accompanying cell death, the OD... 600 The growth rate of the acclimatized group gradually decreased, but it also showed a reduction in the early stages. Later, some usable nutrients remained, and uranium stress may have increased biofilm permeability, accelerating nutrient exchange and transport to some extent. Due to the accelerated energy exchange, metabolic products increased in the culture medium of the acclimatized group bacteria, gradually exceeding those of the control group in terms of pH in the later stages. Within 10 hours, organic acids and other substances produced by bacterial glucose metabolism lowered the pH of the culture medium, reflecting that the bacteria were in a rapid proliferation phase. During this process, the uranium removal rate of the strains in the acclimatized group also increased rapidly, compared to OD... 600 The changes in bacterial concentration and uranium removal rate showed a positive correlation, followed by a gradual decrease in the growth rate. At 48 hours, the uranium removal rate reached 96%, at which point the bacterial solution pH was around 8, indicating alkalinity. Later, bacterial cell death and rupture led to the re-release of uranium, reducing the overall removal efficiency. Simultaneously, the accumulation of metabolic waste further increased the pH of the culture medium; at 60 hours, the pH of the acclimation group was nearly 2% higher than that of the control group. The evolutionary trend of bacterial concentration and uranium removal rate over time revealed that the key lies in the need for sufficient active XHK-1 strains to provide binding sites for effective removal of uranium nuclides. Improving the tolerance of the bacterial solution to uranium-containing environments and controlling the metabolic activity of the target XHK-1 strain are beneficial to the applicability of nitrifying bacteria XHK-1 in remediating nitrogen-containing uranium-contaminated environments.

[0049] Example 3 Optimization of the simultaneous nitrogen reduction and uranium removal performance of nitrifying bacteria XHK-1.

[0050] The concentration of ammonia nitrogen in the system was determined by Nessler's reagent spectrophotometry. The system solution after centrifugation and filtration through a 0.22 μm filter membrane was taken, and potassium sodium tartrate solution was added to the volumetric flask after making up to volume. Nessler's reagent was used as the colorimetric reagent, and the absorbance was measured at a wavelength of 420 nm.

[0051] The uranium concentration of XHK-1 bacterial culture after removal of the system was determined by ultraviolet spectrophotometry. After adding the appropriate reagents and reacting, the absorbance was measured at a wavelength of 578 nm.

[0052] Cell preparation: The XHK-1 bacterial culture after domestication was centrifuged at 4000 r / min for 10 min, the supernatant was discarded, and the culture was resuspended in an equal volume of sterile water to prepare a resuspended bacterial culture.

[0053] I. Optimization of carbon-nitrogen ratio.

[0054] A 1% (v / v) inoculum of resuspended bacterial culture was added to a nitrogen-reducing and uranium-removing culture system containing 100 mg / L ammonia nitrogen (K₂HPO₄ 0.25 g / L, MgSO₄·7H₂O 0.125 g / L, NaCl 0.125 g / L, FeSO₄·7H₂O 0.0025 g / L, MnSO₄·4H₂O 0.0025 g / L, 2 mg / L, pH=7.0, where (NH₄)₂SO₄ and C₄H₄Na₂O₄ were added according to different carbon-nitrogen ratios, C / N ratios of 2:1, 3:1, 4:1, 5:1, 6:1, and 7:1). The system was then subjected to a removal experiment at 30℃ and 150 r / min. The system solution at the corresponding removal time point was centrifuged and filtered to determine its ammonia nitrogen and uranium concentrations, and the removal efficiency was calculated.

[0055] The results are as follows Figure 6 As shown, at the 24-hour stage, the residual ammonia nitrogen content in the system generally decreased with the increase of C / N (carbon-nitrogen ratio). This is because, with a fixed initial inoculum, a higher C / N ratio provides more carbon sources and other energy substances, accelerating the growth and reproduction rate of microorganisms in the removal system. However, when the C / N ratio increased to 7:1, the residual ammonia nitrogen concentration increased to some extent. At this point, the excessive addition of carbon sources led to eutrophication in the system, accelerating the uptake of nutrients by the XHK-1 strain. Competition among the strains reduced the efficiency of ammonia nitrogen removal. Similarly, the trend of ammonia nitrogen content changes in the system at 48 and 72 hours was similar to that at 24 hours, with the highest ammonia nitrogen removal rate at 48 hours under the influence of a higher C / N ratio. When the C / N ratio was 6:1, the residual ammonia nitrogen content in the system at 72 hours was 8.46 mg / L. The overall trend of ammonia nitrogen removal rate was a steady increase followed by a decrease, with the highest removal rate of 91.54% at a C / N ratio of 6:1.

[0056] The uranium removal rate curve in the removal system showed a similar trend to that of ammonia nitrogen, exhibiting a continuous upward trend. The removal rate increased with increasing C / N ratio, with a relatively balanced growth rate. After 72 hours, the uranium removal rate reached its highest at 91.67% in the 7:1 experimental group, representing increases of 33.73% and 5.61% compared to the 2:1 and 6:1 conditions, respectively. Unlike the ammonia nitrogen removal effect, the uranium removal rate continued to increase at a C / N ratio of 7:1, which may be related to microbial activity. With increasing C / N ratio, the metabolic activity of the microorganisms in the system significantly improved, leading to the production of more extracellular polymers on the cell surface. These extracellular polymers contain a large number of negatively charged groups, providing more active sites for XHK-1 to complex uranyl ions. Based on the analysis of the removal efficiency of the dominant XHK-1 strains on ammonia nitrogen and uranium concentrations in the removal system under different C / N ratios, the optimal overall performance was observed at a C / N ratio of 6:1, exhibiting the highest ammonia nitrogen removal capacity and good uranium removal efficiency.

[0057] II. Optimization of action time.

[0058] Under the optimal carbon-to-nitrogen ratio (C / N = 6:1), 1% (V / V) of active XHK-1 strain was introduced into the removal system. The changes in ammonia nitrogen and uranium concentrations in the removal system were measured at different treatment times (0, 6, 12, 24, 48, 72 h). The nitrogen reduction and uranium removal kinetics of the dominant strain were studied on the time scale.

[0059] The fitting results are as follows Figure 7-8 As shown, the kinetics of ammonia nitrogen removal follows a first-order kinetic exponential decay equation fitting process, R0 2 The value was 0.986, much greater than 0.95. From the curve of ammonia nitrogen concentration change over time in the system, it can be seen that in the first 24 hours, the ammonia nitrogen content decreased almost linearly and at a relatively fast rate, with a decrease of 64.84 mg / L. Subsequently, as the removal time continued, the consumption of nutrients in the system and the accumulation of metabolic waste of the XHK-1 strain inhibited its activity to a certain extent, resulting in the removal rate of ammonia nitrogen concentration at 72 hours increasing by only 26.7% compared to 24 hours. The growth rate was relatively slow, but it still continued to rise.

[0060] Regarding the removal efficiency of uranium within the system, the overall trend was one of rapid initial increase followed by a gradual decrease after reaching an extreme value. This is strongly correlated with the physiological activity of the XHK-1 strain. In the early stages, under sufficient carbon source conditions, the XHK-1 strain fixed uranium through metabolic exchange, secretion of large amounts of extracellular proteins and other active substances, and intracellular accumulation, leading to a rapid decrease in uranium concentration. The optimal removal efficiency of 86.06% was achieved within 1440 min. Subsequently, the decay and lysis of the XHK-1 strain released some uranium, resulting in a decrease in the removal rate. Finally, the removal rate decreased to 61.41% at 4320 min, a reduction of 24%. In conclusion, the optimal treatment time was determined to be 48 h.

[0061] By fitting the removal performance with pseudo-first-order and pseudo-second-order dynamics over this period, the correlation coefficient R was found to be... 2 All are above 0.95, with the removal rate k2 in the pseudo-first-order kinetic fitting parameters being 0.086, and the correlation coefficient R... 2 The correlation coefficient R in the pseudo-second-order dynamic fitting parameters is 0.978, while the correlation coefficient R in the pseudo-second-order dynamic fitting parameters is 0.978. 2 The value was 0.981, much larger than the pseudo-first-order model, and the removal rate k3 was 0.227. The experiment showed that the uranium removal kinetics of the XHK-1 strain in the system was more in line with the pseudo-second-order kinetic model, indicating that its uranium removal process was more caused by chemical effects (such as complexation, exchange accumulation, etc.).

[0062] Example 4 Study on the mechanism of simultaneous nitrogen reduction and uranium removal by nitrifying bacteria XHK-1.

[0063] To determine the simultaneous nitrogen and uranium removal mechanism of strain XHK-1, bacterial cells were collected, centrifuged, washed, freeze-dried, ground, and sieved through a 200-mesh sieve for analysis. SEM, TEM, and AFM were used to observe the morphology of the organisms. XRD was used to analyze the mineral phases generated by the bacteria in the removal system, and FTIR was used to analyze the changes in major functional groups in the bacteria, in order to explore the mechanism of simultaneous nitrogen and uranium removal by native nitrifying bacteria under radioactive stress and high-nitrogen environments.

[0064] I. SEM Analysis.

[0065] The morphological characteristics of the strain under different culture conditions were observed and analyzed using biological SEM. For example... Figure 9 As shown, in the control group (without uranium tolerance acclimation), nitrifying bacteria XHK-1 exhibited typical normal cell morphology, with regular rod-shaped cells, smooth surfaces, relatively tight cell arrangement, no obvious adhesion, and no obvious attachments or deposits on the cell membrane. Extracellular polymer production was low, and no visible organic matrix was formed. This indicates that the strain can maintain its normal physiological functions under stress-free conditions.

[0066] In contrast, the nitrifying bacteria XHK-1 in the experimental group (uranium tolerance acclimation) exhibited significant stress response characteristics. For example... Figure 10 As shown, the bacterial cell morphology largely maintains a rod shape, but the surface of the strain becomes rough, and observation reveals a large amount of granular or flocculent material covering the cell surface. Furthermore, significant adhesion and aggregation are observed between cells, and a certain network or membrane-like EPS structure exists around the cell. This reflects the bacteria's self-protective measures against uranium biotoxicity; nitrifying bacteria XHK-1 prevents toxic attack by accelerating the secretion and production of extracellular polymeric substances (EPS). EPS, as a natural biological barrier, can form a protective film on the cell surface, reducing the direct damage of heavy metal ions to the internal structure of the bacteria.

[0067] Nitrifying bacteria XHK-1 rapidly develops tolerance and resistance to radionuclides through the excessive secretion of extracellular polymeric substances (EPS). EPS, composed of polysaccharides, proteins, nucleic acids, and lipids, efficiently chelates heavy metal ions. In uranium-containing environments, EPS not only acts as a physical barrier to isolate UO2... 2+ Direct contact with the cell membrane can also facilitate the transfer of UO2 through coordination. 2+ EPS adsorbs onto the surface, forming stable complexes. Furthermore, EPS enrichment leads to enhanced intercellular adhesion, forming micro-aggregates and further improving the bacterial community's resilience. Changes in the microstructure of nitrifying bacteria XHK-1 reflect its multiple adaptation mechanisms in uranium-contaminated environments, including changes in cell surface structure, dynamic regulation of EPS, and adjustments in physiological metabolism, through excessive EPS secretion and UO2... 2+ The adsorption and complexation of uranium by the XHK-1 strain creates a highly efficient biological barrier, enabling the effective fixation and removal of uranium.

[0068] II. XRD Analysis.

[0069] The structural changes of the cell phase composition of XHK-1 strain under conventional culture and uranium-containing culture (uranium tolerance acclimatization) in a nitrogen-reducing and uranium-removing system for a medium to long duration under different experimental environmental conditions were analyzed. The results are as follows: Figure 11 As shown, the XRD pattern of strain XHK-1 under conventional culture conditions was relatively smooth, without characteristic sharp diffraction peaks. The broad peaks at 2θ ranges of 7–12° and 17.5–22.5° were caused by the amorphous biomass of the bacteria. The XRD patterns of the cells in the other experimental groups were quite similar, indicating that strain XHK-1 maintained good cell integrity under the corresponding environments, without obvious damage or distortion.

[0070] Under uranium tolerance acclimation, the sample test results showed a weak diffraction peak related to uranyl phosphate (limited by uranium concentration), which is due to the interaction between the bacterial cells and UO2. 2+ An interaction occurred, with the extracellular polymers on the bacterial cell surface rich in functional groups, and UO2 adsorbed on the bacterial surface. 2+ Compounds were formed by combining with phosphate and carboxyl groups from teichoic acid and proteins. Comparison with standard PDF cards indicated that the resulting compound phase was likely uranyl phosphate crystals (UO₂HPO₄·H₂O). In the nitrogen and uranium removal system, the acclimated strains and ordinary strains showed slight differences in XRD patterns. Because the acclimated strains had a certain amount of uranyl ions loaded on their cell surface before resuspension, the relative intensity of their diffraction peaks increased after being added to the contamination system. This is partly due to the enhanced environmental adaptability of the acclimated strains, enabling them to rapidly perform related metabolic processes within a certain time, thus increasing the amount of compounds produced through complexation and transformation. Furthermore, to resist the biotoxicity of pollutants, the acclimated strains exhibited a relatively higher EPS content on their surface. Since bacterial EPS carries a strong negative charge, it can chelate and electrostatically adsorb with metal cations in the system, and the large number of active functional groups also facilitates the immobilization of uranyl ions. Overall, the diffraction patterns of the strains in the removal system showed more pronounced broad peaks in the amorphous characteristic region compared to the control group, with improved diffraction intensity. Due to the abundant sodium succinate provided as a carbon source within the contaminated system, the bacteria's metabolic rate and nutrient utilization efficiency accelerated, leading to the secretion of more EPS (extracellular polymeric substances), thus enhancing the biomass content in the amorphous characteristic region. Analysis revealed that the phase of the solidified uranium deposits generated within the system was ammonium uranium mica ((NH4)(UO2)(PO4)·3H2O). In conclusion, adsorption and complexation by the XHK-1 strain are important uranium removal mechanisms within the contaminated system, particularly evident in the nitrogen reduction and uranium removal process of domesticated strains. Amino, carboxyl, hydroxyl, and phosphate groups play crucial roles in pollutant removal, and specific functional enzymes such as phosphatases from the XHK-1 strain actively participate in the removal process during the formation of the post-uranium removal deposits.

[0071] III. TEM analysis.

[0072] The microstructure of the logarithmic-phase strain grown under normal culture conditions (without uranium tolerance acclimatization) is as follows: Figure 12As shown, under macroscopic observation, the bacterial cells exhibit the typical ultrastructure of Gram-positive bacteria. The strains are elliptical and short rod-shaped, with an overall length ranging from 1 to 3 μm and a width from 0.5 to 0.8 μm. There is no obvious aggregation, damage, or shrinkage among the bacterial cells, and the bacteria can divide and proliferate normally. Among the selected target bacterial cells, the cell size is 1.95 μm × 0.75 μm, and a dense cell wall and cell membrane can be clearly observed. The measured cell wall thickness is approximately 23.8 μm, and the biomass within the cells is uniformly distributed, exhibiting an ideal growth state.

[0073] After uranium tolerance acclimation culture, the morphological characteristics of strain XHK-1 underwent certain changes. For example... Figure 13 As shown, the morphology of bacteria in the field of view has changed from a predominantly ellipsoidal shape to a stable distribution of both rod-shaped and ellipsoidal shapes, with obvious cell division, indicating that the strain can still carry out normal metabolism under pollution stress and has good stability. Structural damage such as invagination and breakage within the bacterial cells has decreased, but plasmolysis still exists. The presence of particulate matter within the cells indicates that the XHK-1 strain has a significantly improved intracellular fixation capacity for uranium compared to the unacclimated group, and the structural stability of the bacteria also contributes to the long-term effectiveness of uranium removal. The size of the target strain has changed, decreasing to 1.56 μm × 0.53 μm. The biomass distribution within the bacteria is relatively uniform, but dense structures still exist near the cell, indicating that the bacteria have developed a certain degree of tolerance after acclimation. Under uranium stress, the strain can still carry out corresponding metabolism, synthesizing a large amount of soluble proteins and other organic matter. This is used for the synthesis and secretion of extracellular EPS on the one hand, and for the encapsulation and stabilization of the uranium nuclide transported into the cell on the other, ultimately forming a stable uranyl phosphate mineral phase.

[0074] IV. Mechanism of synergistic nitrogen and uranium reduction by microorganisms.

[0075] Based on the microstructure and phase analysis of nitrifying bacteria XHK-1 in the nitrogen reduction and uranium removal system, it was found that its nitrogen reduction and uranium removal mechanism mainly includes multiple synergistic mechanisms, including the conversion and utilization of ammonia nitrogen and the simultaneous adsorption and fixation of uranium.

[0076] There are two metabolic pathways in biological nitrogen reduction mechanisms: ①NH4 + In terms of dissimilar processes, bacteria diffuse NH4 into the cell from within the system through heterotrophic nitrification and aerobic denitrification pathways. + Transformation and removal are carried out. Previous studies have shown that multiple specific functional enzymes participate in and are specifically expressed during heterotrophic nitrification-aerobic denitrification. Under sufficient electron donor conditions, NH4+... + After crossing the cell membrane and entering the cytoplasm, NH4+ is processed by various functional enzymes within the system.+ The removal of.

[0077] ② Compared with the dissimilar pathway, NH4 + It also plays an important role in assimilation; bacteria need sufficient carbon and nitrogen sources to maintain growth and metabolism, and NH4+... + As the core inorganic nitrogen source, it enters the cell and acts as an electron donor, which can be assimilated by bacteria. Under metabolic action, it synthesizes nitrogen-containing life substances such as proteins, nucleic acids (DNA / RNA), and peptidoglycans, which serve as the material basis for the proliferation and biomass increase of nitrifying bacteria XHK-1.

[0078] Based on microscopic and phase analysis results, nitrifying bacteria XHK-1 inhibits UO2. 2+ The removal of nitrifying bacteria is not a single mechanism; it involves a synergistic mechanism of biosorption and bio-induced mineralization deposition. Regarding bio-surface adsorption, the extracellular polymeric substances (EPS) secreted by nitrifying bacteria XHK-1 onto the cell wall are rich in various active functional groups, such as carboxyl (-COOH), hydroxyl (-OH), amino (-NH), and phosphate groups (-PO4). 3- These active functional groups, such as UO2, are related to... 2+ It has a strong binding affinity and can effectively adsorb and fix UO2 through electrostatic attraction, coordination complexation, and hydrogen bonding. 2+ In terms of bio-induced deposition, due to the large number of negative charges on the surface of bacterial cells, UO2 is adsorbed... 2+ During the process, nitrifying bacteria XHK-1 mainly utilizes its cell membrane and other surface structures such as glycocalyx to form an osmotic barrier to repel radioactive heavy metals and reduce the susceptibility of related groups to UO2. 2+ Nitrifying bacteria XHK-1 resist the biotoxicity of radioactive heavy metals through methods such as sensitivity testing. However, this resistance mechanism also burdens the metabolic rate and proliferation of the nitrifying bacteria XHK-1 itself, resulting in some UO2... 2+ Due to structural damage to the cell membrane or through metabolic channels, the substance diffuses into the bacterial cell and is bound and encapsulated by endobiotics, thus generating some deposits.

[0079] XRD analysis revealed that the uranium deposits were primarily composed of uranyl phosphate and uranium ammonium phosphate rock mineralization, demonstrating the crucial role of phosphate groups in uranium fixation. The inorganic phosphorus source provided within the culture system was converted to PO4 through membrane transport proteins and intracellular phosphorus metabolism. 3- On the one hand, it serves as a cell membrane element in the phospholipid molecular layer, contributing to UO2. 2+ On the one hand, it is immobilized by surface adsorption, and on the other hand, it interacts with the diffused UO2 inside the cell. 2+ Complex deposition occurs as a uranyl phosphate mineral phase. This is due to the presence of NH4 in the nitrogen reduction and uranium removal system. + High content; nitrifying bacteria XHK-1 partially releases NH4 during nitrification to reduce nitrogen.+ It exists inside the cell, along with intracellular PO4. 3- UO2 2+ The combination resulted in thermodynamically more stable uranium ammonium phosphate mineralized deposits, thus achieving intracellular mineralization and fixation of uranium. TEM results from nitrifying bacteria XHK-1 showed that the biomass within the bacteria exhibited certain aggregation and particulate matter, further indicating the formation of solidified uranium mineral phases.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A nitrifying bacterium, characterized in that, Nitrifying bacteria are nitrifying bacteria ( Bacillus sp. XHK-1, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC NO.68388, deposited on June 1, 2026.

2. The application of nitrifying bacteria in wastewater treatment according to claim 1.

3. The application according to claim 2, characterized in that, The wastewater is a complex pollutant containing nitrogen and uranium.

4. A method for treating nitrogen-containing uranium wastewater using the nitrifying bacteria described in claim 1, characterized in that, Includes the following steps: S1. Strain activation: Inoculate nitrifying bacteria into the culture medium and culture with shaking; S2. Uranium-tolerant acclimatization: The activated strain is cultured in a uranium-containing medium, then streak purified. The culture and streak purification process is repeated to obtain a stable uranium-tolerant strain. S3. Cell preparation: The domesticated strain is inoculated into the culture medium for expansion culture, the cells are collected by centrifugation, and the cells are resuspended in sterile water to prepare a resuspension solution. S4. Wastewater treatment: The resuspended bacterial solution is added to the nitrogen-containing uranium wastewater, and then the pH and carbon-nitrogen ratio are adjusted for treatment.

5. The method according to claim 4, characterized in that, In step S1, the culture medium is beef extract peptone medium, specifically: 5 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, pH=7.3; the shaking culture conditions are 30℃ and 150 r / min.

6. The method according to claim 4, characterized in that, In step S2, the uranium concentration in the uranium-containing culture medium is 2 mg / L, and the culture conditions are 30℃, 150 r / min, and 24 h. The streak purification is carried out by streaking the culture medium containing 2 mg / L uranium using the plate streak method, and after culturing at 30℃ for 72 h, a single colony is picked. The repeated culture and streak purification process is specifically repeated 6 to 7 times.

7. The method according to claim 4, characterized in that, In step S3, the culture medium is specifically: glucose 10g / L, beef extract 5g / L, peptone 10g / L, NaCl 5g / L, pH=7.3; centrifugation is performed at 4000r / min for 10min.

8. The method according to claim 4, characterized in that, In step S4, the amount of resuspended bacterial solution added to the nitrogen-containing uranium wastewater is 1%~5% (V / V); the pH is adjusted to 6.5~7.5; the carbon-nitrogen ratio is 2~7:1; and the temperature during wastewater treatment is 25~35℃.

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