Citrobacter freundii and its use

Abstract

The present application relates to the field of microbial technology, in particular to Citrobacter werkmanii and its application. The present application provides Citrobacter werkmanii SDT3 with dual functions of denitrification and cobalt (III) reduction under aerobic conditions, and the preservation number is CCTCC NO: M 2025956. The strain can efficiently realize aerobic denitrification reduction of nitrate and nitrite under aerobic conditions without additional ammonia nitrogen, and simultaneously complete the synchronous reduction of cobalt (III) -histidine complex (Co(III)His). The strain is suitable for treating flue gas denitration failure absorption liquid containing Co(III)His, nitrate and nitrite, has the advantages of high absorption liquid regeneration efficiency, mild process conditions, wide application range of organic carbon source, no secondary pollution, easy engineering application, etc.; can provide an efficient and stable microbial technology solution for efficient regeneration of flue gas denitration absorption liquid, long-term continuous and stable operation of the denitration system.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to Citrobacter velutipes and its applications. Background Technology

[0002] Nitrogen oxides (NOx) are significant air pollutants that contribute to acid rain and photochemical smog, with coal-fired flue gas being a major source of NOx emissions. Chemical denitrification technology is currently the mainstream industrial process, but it generally suffers from high investment costs and demanding operating conditions. Biological flue gas denitrification, on the other hand, has significant application prospects due to its advantages such as operating at ambient temperature and pressure, simple process, low energy consumption, and minimal secondary pollution.

[0003] Biological NOx purification relies on microbial metabolic processes to convert NOx into harmless substances, typically involving mass transfer from the gas phase to the liquid phase and bioreduction in the liquid phase. Since NOx in flue gas is primarily poorly water-soluble NO, the effectiveness of biological methods alone is limited. Therefore, researchers have combined bioreduction with chemical NO absorption, developing the Chemical Absorption-Bioreduction Flue Gas Denitrification (CABR) technology.

[0004] CABR technology typically uses Fe(II)EDTA complexes as NO absorbents, followed by the complete elimination of captured NO by denitrifying bacteria. However, industrial flue gas usually contains 3%–8% O2, which easily causes Fe(II)EDTA to oxidize and deactivate. Although the generated Fe(III)EDTA can be reduced simultaneously with the captured NO during the bioreduction stage, this simultaneous reduction process is severely inhibited by O2. To address the inhibition of CABR technology by O2, researchers have focused on Co(II)His complexes, which have better O2 tolerance than Fe(II)EDTA, and aerobic denitrifying bacteria, which have better O2 tolerance than traditional denitrifying bacteria. Co(II)His can simultaneously complex and absorb NO and O2 under neutral conditions, significantly reducing the rate of oxidative deactivation, while also promoting the conversion of NO to nitrates and nitrites. During the bioreduction stage, aerobic denitrifying bacteria are used to simultaneously reduce Co(III)His, nitrates, and nitrites in the absorbent, thus regenerating the absorbent.

[0005] Previous studies have confirmed that the aerobic denitrifying strain *Paracoccus versutus* LYM can simultaneously reduce Co(III)His, nitrate, and nitrite under aerobic conditions, providing a research basis for the bioregeneration of Co(II)His absorbents and the construction of hyperaerobic-tolerant CABR systems. However, the engineering application of strain LYM has significant limitations: firstly, the absorbent regeneration process must rely on external ammonia nitrogen supply, increasing reagent costs and process complexity; secondly, this strain has a rapid growth rate and enters the stationary phase prematurely, with metabolic flux biased towards cell proliferation, leading to a premature decline in reductive metabolic activity and a short duration of high Co(II) effective concentration, making it difficult to ensure continuous and stable absorbent regeneration and limiting its engineering application. Therefore, there is an urgent need to provide a new strain that can simultaneously reduce nitrate, nitrite, and Co(III)His under aerobic conditions. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide *Citrobacter wormii* and its applications. The present invention provides a strain of *Citrobacter wormii* capable of simultaneously reducing nitrate, nitrite, and cobalt(III) under aerobic conditions, which can be applied in the regeneration of flue gas denitrification failure absorbent. Addressing the problems of existing chemical absorption-bioreduction flue gas denitrification technologies, such as the dependence on ammonia nitrogen, insufficient growth and metabolic stability, and poor economic and convenient engineering applications of aerobic denitrification strains, the present invention aims to provide a strain of *Citrobacter wormii* SDT3 capable of simultaneously reducing nitrate, nitrite, and Co(III)His under aerobic conditions. This strain can grow and function normally without ammonia nitrogen, has a broad carbon source utilization spectrum, stable growth and metabolism, and can efficiently achieve the biological regeneration of flue gas denitrification failure absorbent, thus providing excellent strain resources and technical support for constructing a stable, simple, and low-cost chemical absorption-bioreduction flue gas denitrification system.

[0007] This invention provides Citrobacter werkmanii with accession number CCTCC NO: M 2025956.

[0008] The screening and identification method for the strain described in this invention includes: The *Citrobacter werkmanii* SDT3 strain provided by this invention was screened from aerobic pool water samples from a wastewater treatment plant. Precise species identification was completed through joint sequence analysis of 31 housekeeping genes, confirming its taxonomic name as *Citrobacter werkmanii*, and its strain number as SDT3. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on May 6, 2025, with accession number CCTCC NO: M 2025956.

[0009] The strain has the following characteristics:

[0010] (1) The colonies are round, milky white, with neat edges and a smooth, moist surface;

[0011] (2) Heterotrophic bacteria can simultaneously reduce Co(III)His, nitrate and nitrite under aerobic conditions;

[0012] (3) It can grow normally and maintain high efficiency in reduction and denitrification without the need for exogenous ammonia nitrogen;

[0013] (4) Sodium lactate, sodium succinate, sodium acetate, and sodium citrate can be used as organic carbon sources, and the carbon source adaptability is wide;

[0014] (5) It can grow normally in the 20 mM Co(III)His system and is tolerant of high concentration of cobalt complex environment;

[0015] (6) After about 36 hours, the bacteria enter the stable growth period, with full proliferation, stable metabolism, and strong continuous function.

[0016] The method for culturing the strain described in this invention includes: inoculating *Citrobacter wormi* SDT3 at an inoculum of 0.1-0.2 g DCW / L into a liquid culture medium, and incubating under aerobic conditions for 30 days. o C. Incubate at 150 rpm under constant temperature and vibration.

[0017] The culture medium consisted of: Co(III)His 2-20 mM, MgSO4·7H2O 0.1 g / L, Na2HPO4·12H2O 5.73 g / L, KH2PO4 0.54 g / L, NaNO3 or NaNO2 5-20 mM, and trace element solution 1 mL / L. One or more of sodium lactate, sodium succinate, sodium acetate, and sodium citrate were used as organic carbon sources. The C / N ratio (the ratio of the mass of carbon in the organic carbon source to the mass of nitrogen in the substrate nitrate and / or nitrite) was controlled at 20:1-40:1, and the initial pH was 6.5-7.5.

[0018] The composition and concentration of the trace element solution are as follows: EDTA 50 g / L, ZnSO4 22 g / L, CaCl2 5.54 g / L, MnCl2·4H2O 5.06 g / L, FeSO4·7H2O 50.00 g / L, (NH4)6Mo7O 24 ·4H2O 1.10 g / L, CuSO4·5H2O 1.57 g / L, CoCl2·6H2O 1.61 g / L.

[0019] The *Citrobacter worm* SDT3 and strain LYM screened and identified in this invention can efficiently utilize various organic carbon sources such as sodium lactate, sodium citrate, sodium succinate, and sodium acetate, and have a wide range of substrate applicability. Based on these commonalities, strain SDT3 of this invention possesses outstanding technical advantages: it can complete simultaneous denitrification and Co(III)His reduction under aerobic conditions without added ammonia nitrogen, resulting in a simple and economical process; the strain exhibits a slow growth rhythm, a delayed arrival at the stationary phase, and a metabolic flux more biased towards the reduction and regeneration pathway, maintaining high Co(II) effective concentrations for a longer period and demonstrating excellent regeneration stability; simultaneously, it shows good tolerance to high concentrations of Co(III)His, nitrates, and nitrites, with no nitrite residue during denitrification, meeting the requirements for long-term continuous and stable operation of CABR systems and effectively compensating for the shortcomings of existing strains and regeneration technologies.

[0020] The strain of this invention can simultaneously reduce Co(III)His, nitrates and nitrites in the failed absorbent of flue gas denitrification under aerobic conditions using organic carbon sources as electron donors, thereby regenerating the Co(II)His absorbent. The regenerated absorbent can be recycled for flue gas NO absorption, ensuring the continuous and stable operation of the denitrification system and significantly improving the regeneration efficiency of the denitrification absorbent and the reliability of the entire process.

[0021] This invention provides a microbial inoculant, comprising at least one of the following (1) to (3) and an acceptable adjuvant:

[0022] (1) The Citrobacter werkmanii as described in claim 1.

[0023] (2) The inactivated Citrobacter werkmanii as described in claim 1;

[0024] (3) Metabolites, derivatives, fermentation broths, cultures, exosomes, lysates or extracts of Citrobacter werkmanii as described in claim 1.

[0025] This invention provides the application of the aforementioned Citrobacter werkmanii and / or the aforementioned microbial agents in the treatment of pollutants containing nitrogen and / or cobalt complexes.

[0026] In some embodiments, the nitrogen-containing pollutants include wastewater containing nitrates and / or nitrites and / or coal-fired flue gas containing nitrogen oxides;

[0027] The cobalt complex includes Co(III)His.

[0028] In some embodiments, the treatment uses an organic carbon source as an electron donor to reduce nitrates and / or nitrites to nitrogen and / or Co(III)His to Co(II)His under aerobic conditions.

[0029] In some embodiments, the organic carbon source includes at least one of sodium lactate, sodium succinate, sodium citrate, and sodium acetate.

[0030] In some specific embodiments, the organic carbon source is sodium lactate.

[0031] The present invention provides a method for treating pollutants containing nitrogen and / or cobalt complexes, comprising inoculating the pollutants containing nitrogen and / or cobalt complexes with the aforementioned Citrobacter werkmanii and / or the aforementioned microbial agent, and then culturing them.

[0032] In some embodiments, the concentration of the inoculation is 0.1~0.2 g DCW / L.

[0033] In some embodiments, the carbon source for cultivation includes at least one of sodium lactate, sodium succinate, sodium citrate, and sodium acetate;

[0034] The culture temperature is 20-40°C. o C, the rotation speed of the culture is 120~200 rpm.

[0035] In some specific embodiments, the culture temperature is 30°C. o C, the rotation speed of the culture is 150 rpm.

[0036] In some embodiments, the carbon source for cultivation is sodium lactate, and the C / N ratio of the contaminant is greater than or equal to 30.

[0037] In some embodiments, the C / N ratio of the pollutant is 30 or 40.

[0038] Those skilled in the art can control the C / N ratios to be 0, 10, 20, 30, and 40, and further experimental results show that a C / N ratio of 30 or 40 can achieve a better degradation effect.

[0039] In some embodiments, the C / N ratio of the pollutant is 30.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. No ammonia nitrogen dependence, adapted to low ammonia nitrogen conditions: The strain SDT3 of this invention does not require exogenous inorganic ammonia nitrogen and can grow normally and efficiently complete aerobic denitrification under ammonia nitrogen-free conditions; while the control strain LYM basically loses its growth and denitrification metabolic activity under ammonia nitrogen-free conditions. This strain completely eliminates ammonia nitrogen constraints, significantly broadening the practical engineering application scenarios of flue gas denitrification and other low-ammonia-nitrogen and ammonia-free processes.

[0042] 2. It does not rely on histidine ligands for nitrogen supply, maintaining the stability of the denitrification system: Histidine serves only as a key ligand for denitrification absorption and should not be consumed by microorganisms as a nutrient nitrogen source. The control strain LYM requires histidine for nitrogen supply when ammonia nitrogen is absent, which easily leads to unnecessary ligand loss and increased reagent replenishment costs. SDT3 metabolism does not rely on histidine as a nitrogen source, maintaining the stability of the absorbent composition for a long time and ensuring the continuous operation of the denitrification process.

[0043] 3. Wide carbon source adaptability and flexible engineering selection: SDT3 can efficiently utilize a variety of commonly used organic carbon sources such as sodium lactate, sodium succinate, sodium citrate, and sodium acetate, without the need for special customized carbon sources. The carbon source selection is wide-ranging, which can be flexibly selected according to the site conditions and operating costs, significantly improving the practicality and economy of the process.

[0044] 4. Stable growth and metabolism, and strong tolerance to cobalt stress: SDT3 has sufficient proliferation and high biomass accumulation. Under high concentration Co(III)His stress, its metabolism is stable and its function is not easily inactivated. The cell growth cycle is reasonable and the steady-state metabolism is good. It can be directly adapted to the treatment needs of high concentration flue gas denitrification failure absorbent liquid and is suitable for long-term continuous operation.

[0045] 5. Complete denitrification pathway with no risk of secondary pollution: The nitrite intermediates generated during the SDT3 denitrification process can be completely converted and degraded under suitable and sufficient carbon source conditions, resulting in no nitrite residue in the final system. This effectively avoids the risk of secondary pollution caused by the accumulation of intermediates, and the treatment process is environmentally friendly.

[0046] 6. Simple and green process with low operating costs: The entire process can operate stably under aerobic conditions without the need for anaerobic / anoxic units. The process is simple and requires less equipment investment. At the same time, no additional ammonia nitrogen is needed, which significantly reduces energy consumption and chemical maintenance costs and makes the project highly feasible.

[0047] Biological Preservation Instructions

[0048] Citrobacter werkmanii SDT3 was deposited on May 6, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025956. Attached Figure Description

[0049] Figure 1The colony morphology of the strains after five generations of purification is shown, from left to right: SDT3, SDT4 and SDT5;

[0050] Figure 2 This is a phylogenetic tree showing the housekeeping gene sequence of the strain SDT3 of the present invention;

[0051] Figure 3 The effects of different carbon sources on SDT3 growth (a), nitrate denitrification (b, c) and Co(III) His reduction (d) under aerobic and ammonia-free conditions were shown.

[0052] Figure 4 The effects of different C / N ratios on SDT3 growth (a), nitrate denitrification (b, c) and Co(III) His reduction (d) under aerobic conditions with no ammonia nitrogen and sodium lactate carbon source were shown.

[0053] Figure 5 The effects of different concentrations of Co(III)His on SDT3 growth (a), nitrate denitrification (b, c) and Co(III)His reduction (d) under aerobic ammonia-free nitrogen-sodium lactate carbon source were shown.

[0054] Figure 6 The effects of different carbon sources on the growth (a), nitrate denitrification (b, c), and Co(III)His reduction (d) of control strain LYM under aerobic and ammonia-free conditions were shown. Detailed Implementation

[0055] This invention provides *Citrobacter wiltii* and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0056] The test materials used in this invention are all commercially available products. The invention will be further illustrated below with reference to specific embodiments.

[0057] Example 1: Strain Screening and Identification

[0058] 1.1 Culture medium preparation

[0059] NO3 -Basic culture medium: MgSO4·7H2O·0.1 g / L, Na2HPO4·12H2O·5.73 g / L, KH2PO4·0.54 g / L, NaNO3 10 mM, sodium lactate (70%) 18.70 g / L, trace element solution 1 mL / L, adjust pH to around 7.2, 121 o Sterilize at 20 min.

[0060] NO3 - Mixed medium with Co(III)His: to NO3 - To prepare NO3, add 10 mM CoCl2·6H2O and 40 mM histidine (His) to the basal culture medium. - Mix with Co(II)His culture medium; place the prepared culture medium in a shaker at 30°C. o C. After shaking at 150 rpm for more than 12 hours, the dissolved oxygen in the system oxidizes Co(II)His to Co(III)His, thus producing NO3. - Mixed with Co(III)His medium.

[0061] Solid culture medium: Add 1.5%-2.0% agar to the corresponding liquid culture medium and boil at 121°C. o Sterilize at C for 20 min, then pour into plates after sterilization.

[0062] 1.2 Strains Isolation and Screening

[0063] The mud-water mixture taken from the aerobic tank was inoculated at a rate of 5% until NO3 was reached. - In the basal medium, the medium was changed every 1-2 days, and Co(III)His was gradually added, with the concentration increasing from 2 mM to 10 mM; subsequently, NO3 was used. - Enrichment and acclimatization culture was continued using a mixed medium with Co(III)His. The medium was changed every 1-2 days, for a total of 4-5 changes, to obtain an enriched and acclimatized bacterial culture. Samples were taken periodically during the acclimatization process to determine the concentrations of nitrate nitrogen, nitrite nitrogen, and Co(II). The results showed that this mixed bacterial community could completely degrade 10 mM NO3 within 12 h. - NO2, an intermediate product of metabolic accumulation - It can also be completely degraded within 24 hours, and the Co(II) concentration increases significantly at 24 hours, confirming that the bacterial community with highly efficient Co(II)His absorbent regeneration ability has been successfully domesticated and enriched.

[0064] Take the enriched and domesticated bacterial solution, dilute it, and spread it on NO3. - Basic solid culture medium plates, 30 oIncubate at an inverted temperature for 2-3 days. After colonies have grown, wash the plates with sterile PBS buffer. Inoculate the eluent onto NO3. - Mixed with Co(III)His medium, at 30 o C. The system was incubated aerobically with constant temperature shaking at 150 rpm, and the concentrations of nitrate nitrogen, nitrite nitrogen, and Co(II) were measured periodically. Results showed that 10 mM NO3 in the system... - It can be completely degraded within 12 hours, and the accumulated NO2 - It can be completely removed within 24 hours, and at the same time, a significant increase in Co(II) concentration can be detected at 24 hours, indicating that the target functional strain can grow and reproduce normally on this solid culture medium.

[0065] The colony eluent was further diluted and coated onto NO3. - After culturing on basic solid culture plates for 2-3 days, based on apparent characteristics such as colony size, color, morphology, and transparency, 12 single colonies with significant morphological differences were selected and inoculated using a sterile inoculation loop on NO3. - Purification was performed by streak plating on basal solid culture medium plates; the plates were then incubated at 30°C. o After incubation at an inverted temperature for 2-3 days, 12 purified strains were obtained, which were numbered SDT1 to SDT12.

[0066] SDT1~SDT12 were inoculated into NO3, respectively. - In a mixed medium with Co(III)His, 30 o C. The culture was carried out under constant temperature and aerobic shaking at 150 rpm, and the concentrations of nitrate nitrogen, nitrite nitrogen, and Co(II) were measured periodically. The experimental results showed that only strains SDT3, SDT4, and SDT5 could simultaneously meet the requirement of 10 mM NO3. - Complete degradation within 12 hours, metabolizing intermediate NO2 - Complete reduction was achieved within 24 hours, and the Co(II) concentration in the system significantly increased at 24 hours. Based on this, SDT3, SDT4, and SDT5 were identified as the superior strains with highly efficient Co(II) His absorbent regeneration function.

[0067] SDT3, SDT4, and SDT5 were diluted and coated onto NO3. - Single colonies grew on basal solid culture plates after 2-3 days of incubation. After five consecutive streak purifications, pure strains with uniform colony morphology and stable genetic traits were finally obtained. The colony morphologies of SDT3, SDT4, and SDT5 are shown in the figure. Figure 1 .

[0068] 1.3 Strain Identification

[0069] The purified strains SDT3, SDT4, and SDT5 were sent to Majorbio (Shanghai) Co., Ltd. for housekeeping gene sequencing. A phylogenetic tree was constructed using the neighbor-joining method with MEGA software to analyze the housekeeping gene sequences. The results for strain SDT3 are as follows: Figure 2 As shown.

[0070] Sequencing and evolutionary analysis results showed that the amino acid and nucleotide sequences of the 31 housekeeping genes of strains SDT3, SDT4, and SDT5 were completely identical, proving that the three isolated strains were indeed the same strain. Sequence alignment showed that the nucleotide sequence of the housekeeping genes of this strain had a homology similarity of 99.5% with that of Citrobacter werkmanii (GCA 000759755.1).

[0071] Based on this, the strain was identified as Citrobacter werkmanii and named SDT3; the strain was deposited at the China Center for Type Culture Collection on May 6, 2025, with accession number CCTCC NO: M2025956.

[0072] Example 2: Carbon source adaptability experiment of the present invention strain SDT3

[0073] To investigate the broad-spectrum utilization of different organic carbon sources by strain SDT3, sodium lactate, sodium succinate, sodium acetate, and sodium citrate were used as the sole carbon sources. Experiments were conducted under conditions of no exogenous ammonia nitrogen, simultaneously setting up a Co(III)His-free aerobic denitrification system and a simultaneous reduction system containing 10 mM Co(III)His. Experimental procedure: NO3 was prepared according to the formula in Example 1, using sodium lactate, sodium succinate, sodium acetate, and sodium citrate as the sole carbon sources. - basal culture medium and NO3 - In the mixed medium with Co(III)His, the carbon source addition was controlled at C / N=30, with specific concentrations of sodium lactate (70%) 18.7 g / L, sodium succinate (C4H4Na2O4·6H2O) 33.6 g / L, sodium acetate (CH3COONa·3H2O) 33.9 g / L, and sodium citrate (C6H5O7Na3·2H2O) 24.4 g / L. The bacterial suspension of strain SDT3 was inoculated into each of the above media at an inoculum of 0.1–0.2 g DCW / L and cultured aerobically at 30 ℃ and 150 rpm with constant temperature shaking. The concentrations of nitrate nitrogen, nitrite nitrogen, and Co(II) were measured periodically. The experimental results are shown below. Figure 3 .

[0074] Results from a Co(III)His aerobic denitrification system showed that strain SDT3 exhibited good adaptability to all four tested carbon sources. Specifically, with sodium lactate and sodium succinate as carbon sources, the strain grew rapidly, quickly entering the stationary phase, exhibiting high nitrate degradation rates, and only transient accumulation of nitrite which was quickly and completely eliminated. With sodium acetate and sodium citrate as carbon sources, the initial growth and substrate degradation rates were relatively slow, but the final biomass reached a high level, nitrate was completely degraded, and nitrite accumulation was low and gradually and completely eliminated.

[0075] Results of the simultaneous reduction system containing Co(III)His showed that strain SDT3 could achieve stable growth, complete nitrate degradation, and effective Co(III)His reduction under four carbon source conditions. Sodium lactate showed the best overall performance, with the fastest growth, denitrification, and Co(III)His reduction rates, and rapid and complete removal of accumulated nitrite. Sodium citrate was the second best, with excellent overall performance and a complete denitrification pathway. Sodium succinate performed well with complete metabolic function, but some nitrite residue remained at the endpoint. Sodium acetate had the slowest overall rate, higher nitrite residue at the endpoint, and the lowest Co(III)His reduction efficiency, but still met the basic process requirements for simultaneous denitrification and Co(III)His reduction. The overall performance of strain SDT3 in simultaneous aerobic denitrification and Co(III)His reduction under different carbon source conditions is shown in Table 1.

[0076] Table 1. Comprehensive performance of strain SDT3 in simultaneous aerobic denitrification and Co(III)His reduction under different carbon source conditions.

[0077]

[0078] In summary, strain SDT3 can effectively utilize four carbon sources—sodium lactate, sodium succinate, sodium acetate, and sodium citrate—under conditions without exogenous ammonia nitrogen, and can stably complete aerobic denitrification and simultaneous reduction of Co(III) and His. The differences between different carbon sources are only in growth rate, denitrification process, and nitrite accumulation. The overall metabolic function is complete, and the range of carbon source selection is wide.

[0079] Example 3: Experiment on the effect of C / N ratio on the strain SDT3 of the present invention

[0080] To investigate the effect of C / N ratio on the simultaneous denitrification and Co(III)His reduction performance of strain SDT3, experiments were conducted using sodium lactate as the sole carbon source under aerobic conditions without exogenous ammonia nitrogen, with different C / N gradients. Experimental procedure: NO3 was prepared using different amounts of sodium lactate as the carbon source, following the formulation in Example 1. -The culture medium was mixed with Co(III)His, and the C / N ratio was adjusted to 0, 10, 20, 30, and 40 respectively. The bacterial suspension of strain SDT3 was inoculated into each of the above culture media at an inoculum size of 0.1–0.2 g DCW / L, and cultured aerobically with constant temperature shaking at 30 ℃ and 150 rpm. The concentrations of nitrate nitrogen, nitrite nitrogen, and Co(II) were measured periodically. The results are as follows: Figure 4 As shown.

[0081] The results showed that the C / N ratio significantly regulated the growth of strain SDT3 in the synchronization system, and the strain's metabolic proliferation was highly dependent on the organic carbon source. Under a C / N=0 condition, the strain showed almost no proliferation, and OD... 660 The C / N ratio was consistently no higher than 0.3; bacterial growth was significantly limited when C / N=10; the optimal growth state of the strain was achieved when C / N≥30, and the carbon source was no longer a limiting factor for proliferation. Regarding denitrification performance: nitrate degradation was weak when C / N=0, with a large accumulation of nitrite; when C / N≥10, nitrate could be completely degraded within 7.5 h, with a peak nitrite accumulation of 125.5~130.8 mg / L; nitrite could be completely reduced under C / N≥20 conditions, while a significant nitrite endpoint remained at C / N=10, and the nitrite reduction rate decreased with increasing C / N ratio. The Co(III)His reduction pattern showed that reduction was extremely weak at C / N=0 and C / N=10; reduction performance was significantly improved at C / N≥20, with the best results at C / N=30 and C / N=40, where the Co(II) peak reached 5.0~5.1 mM, and the apparent formation rate was 0.22 mM / h.

[0082] In summary, considering both the regeneration effect of the absorbent and the operating cost, the optimal C / N ratio for strain SDT3 with sodium lactate as the carbon source was determined to be 30.

[0083] Example 4 Performance study of strain SDT3 in simulated flue gas denitrification failure absorbent system

[0084] To investigate the regeneration performance of strain SDT3 on simulated flue gas denitrification failure absorbent, experiments were conducted using sodium lactate as the sole carbon source under aerobic conditions without added ammonia nitrogen, with different concentrations of Co(III)His. Experimental procedure: Referring to the formulation in Example 1, the initial concentrations of CoCl2·6H2O were adjusted to 0 mM, 5 mM, 10 mM, 15 mM, and 20 mM, respectively, with corresponding initial His concentrations of 0 mM, 20 mM, 40 mM, 60 mM, and 80 mM. NO3 was prepared... -Mixed with Co(III)His medium; inoculate the bacterial suspension of strain SDT3 into each of the above media at an inoculum of 0.1–0.2 g DCW / L, and culture aerobically with constant temperature shaking at 30 ℃ and 150 rpm; measure the concentrations of nitrate nitrogen, nitrite nitrogen, and Co(II) at regular intervals. Results are shown in […]. Figure 5 .

[0085] As shown in the figure, under aerobic conditions without ammonia nitrogen, strain SDT3 entered the stationary phase after approximately 36 hours, with OD... 660 The concentrations reached 2.4, 4.3, 5.1, 5.3, and 5.0 respectively; strain SDT3 could reduce 10 mM NO3 within 10 h. - The degradation was complete. Nitrite reached its peak at 8-10 h and then gradually degraded, leaving no residue within 24 h. At different Co(III)His concentrations of 5-20 mM, the Co(II) concentration first increased and then decreased, reaching the maximum values ​​of 2.4 mM, 4.4 mM, 5.2 mM and 6.1 mM at 32 h, respectively.

[0086] The results show that under aerobic conditions without ammonia nitrogen, strain SDT3 can grow and metabolize normally, simultaneously achieving aerobic denitrification of nitrate and Co(III)His reduction. The strain tolerates Co(III)His concentrations up to 20 mM, and no nitrite intermediates remain during the denitrification process. The simulated failed absorbent can be effectively regenerated after being processed by the strain and can be recycled for NO absorption in flue gas, ensuring the continuous and stable operation of the denitrification process.

[0087] Example 5: Performance verification of control strain LYM and comparative analysis with SDT3

[0088] To highlight the technical advantages of the SDT3 strain of this invention, a functionally similar LYM strain was selected, and a parallel control experiment was conducted under the same experimental conditions and procedures as in Example 2. The experiment was conducted under the baseline condition of no added ammonia nitrogen, with a control group containing 10 mM ammonia nitrogen in a sodium lactate system. The experimental results are shown below. Figure 6 .

[0089] The results of the simple aerobic denitrification system showed that under the conditions of no ammonia nitrogen and no Co(III)His, LYM grew very weakly in the four carbon source environments of sodium lactate, sodium succinate, sodium acetate and sodium citrate. Nitrate nitrogen was basically not degraded and the denitrification function was basically lost. Only after the addition of ammonia nitrogen could LYM grow normally and complete the aerobic denitrification process.

[0090] Results of the simultaneous Co(III)His reduction system showed that, without added ammonia nitrogen, LYM could utilize histidine in the system to maintain its growth and metabolism, achieving cell proliferation, nitrate degradation, and Co(III)His reduction under all four carbon source conditions. Overall, sodium lactate showed the best performance, sodium citrate had a good effect, sodium succinate had moderate performance, and sodium acetate showed weaker performance, with higher nitrite residue and relatively insufficient Co(III)His reduction capacity. Table 2 shows the comprehensive performance of simultaneous aerobic denitrification and Co(III)His reduction of strain LYM under different carbon source conditions.

[0091] Table 2. Comprehensive performance of strain LYM in simultaneous aerobic denitrification and Co(III)His reduction under different carbon source conditions.

[0092]

[0093] Comparative analysis of Tables 1 and 2 Figure 3 , Figure 6 It can be seen that in the Co(III)His synchronous system, both SDT3 and LYM can utilize various organic carbon sources such as sodium lactate, sodium citrate, sodium succinate, and sodium acetate to simultaneously achieve the reduction and conversion of Co(III)His and nitrate under aerobic conditions. The core differences between the two strains are mainly reflected in their nitrogen source and ligand dependence, growth and metabolic rhythm, and synchronous reduction adaptability.

[0094] LYM is highly dependent on external ammonia nitrogen or histidine ligands for regeneration metabolism. Without external nitrogen source, the bacterial growth is weak and the denitrification function is basically lost. At the same time, the growth rate of this strain is relatively fast, and it is easy to enter the stationary phase too early. The metabolic flux is preferentially biased towards the bacterial cell proliferation, the reductive metabolic activity decays prematurely, and the effective concentration of high Co(II) in the system is maintained for a limited time.

[0095] The strain SDT3 described in this invention requires no external ammonia nitrogen and does not depend on histidine ligands. It can grow stably and independently complete aerobic denitrification in an environment without an external nitrogen source, making it applicable to a wider range of operating conditions. Its growth rhythm is slow, and it enters the stationary phase later. Its metabolic allocation is more inclined towards Co(III)-His reduction and regeneration, maintaining a high effective Co(II) concentration for a longer period, demonstrating excellent reductive metabolism sustainability. Simultaneously, it exhibits good tolerance to high substrate stress, resulting in better adaptability and long-term operational stability, demonstrating significant overall technical advantages. To provide a clear and quantitative comparison of the functional characteristics of the two strains, key indicators are summarized in Table 3.

[0096] Table 3 Comparison of key characteristics between strain SDT3 and LYM

[0097]

[0098] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Citrobacter werkmanii with accession number CCTCC NO: M 2025956.

2. A microbial inoculant, characterized in that, Includes at least one of the following (1) to (3) and an acceptable adjuvant: (1) The Citrobacter werkmanii as described in claim 1. (2) The inactivated Citrobacter werkmanii as described in claim 1; (3) Metabolites, derivatives, fermentation broths, cultures, exosomes, lysates or extracts of Citrobacter werkmanii as described in claim 1.

3. The use of Citrobacter werkmanii as described in claim 1 and / or the microbial agent as described in claim 2 in the treatment of pollutants containing nitrogen and / or cobalt complexes.

4. The application according to claim 3, characterized in that, The nitrogen-containing pollutants include wastewater containing nitrates and / or nitrites and / or coal-fired flue gas containing nitrogen oxides; The cobalt complex includes Co(III)His.

5. The application according to claim 4, characterized in that, The treatment uses an organic carbon source as an electron donor to reduce nitrates and / or nitrites to nitrogen and / or Co(III)His to Co(II)His under aerobic conditions.

6. The application according to claim 5, characterized in that, The organic carbon source includes at least one of sodium lactate, sodium succinate, sodium citrate, and sodium acetate.

7. A method for treating pollutants containing nitrogen and / or cobalt complexes, characterized in that, This includes inoculating a contaminant containing nitrogen and / or cobalt complexes with the Citrobacter werkmanii of claim 1 and / or the microbial agent of claim 2, and then culturing it.

8. The processing method according to claim 7, characterized in that, The concentration of the inoculation is 0.1~0.2g DCW / L.

9. The processing method according to claim 7 or 8, characterized in that, The carbon source for cultivation includes at least one of sodium lactate, sodium succinate, sodium citrate, and sodium acetate. The culture temperature is 20-40°C. o C, the rotation speed of the culture is 120~200 rpm.

10. The processing method according to any one of claims 7 to 9, characterized in that, The C / N ratio of the pollutant is greater than or equal to 30.

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