Denitrification bacterial agent under high-salt and low carbon-nitrogen ratio conditions and application thereof

Abstract

A denitrification bacterial agent under high salt and low carbon-nitrogen ratio conditions and application thereof relate to the field of microbial water treatment technology. The bacterial agent is composed of Marinobacterium sp. Marinobacter shengliensis subsp. alexandrii YZC-3 and Stutzeri sp. Stutzerimonas degradans YZC-8, and the preservation numbers are GDMCC NO:68289 and 68290 respectively. The bacterial agent maintains high and stable denitrification activity under the conditions of salinity of 2% to 4% and C / N of 4 to 8, and has strong salt stress resistance. When treating simulated wastewater under the conditions of salinity of 4% and C / N of 4, the total nitrogen removal rate is more than 90%, and when treating actual mariculture tail water, the total nitrogen removal rate is stably maintained at 85% to 93% in long-term operation. The bacterial agent is salt-tolerant, stable in operation, low in cost and environmentally friendly.

Description

Technical Field

[0001] This invention relates to the field of microbial water treatment technology, and in particular to a denitrifying agent and its application under high salt and low carbon-nitrogen ratio conditions. Background Technology

[0002] Nitrogen pollution, as one of the major environmental challenges facing the world today, severely restricts the sustainable development of mariculture. Nitrogen pollution mainly includes organic nitrogen and inorganic nitrogen, the latter being primarily ammonia nitrogen (NH4+). + -N), nitrite (NO2) - -N) and nitrate (NO3) - -N) is the main component, of which NO3 is the main component. - The environmental pollution problems caused by excessive nitrogen (N) are quite widespread. It not only causes certain toxicity to farmed organisms, but also leads to the continuous eutrophication and red tides in marine ecosystems.

[0003] Currently, nitrogen removal technologies for marine aquaculture wastewater can be mainly divided into physical, chemical, and biological methods. Physical methods are effective in removing large suspended solids, while chemical methods excel in rapid oxidation and decomposition. However, considering long-term ecological benefits and operating costs, biological treatment technology is increasingly demonstrating stronger comprehensive application value. Compared to traditional physicochemical methods, biological treatment technology offers advantages such as lower cost, no secondary pollution, and no disruption to the ecological balance, thus finding widespread application in aquaculture wastewater nitrogen removal. Biological denitrification, as the core process for removing total nitrogen, is easily constrained by environmental factors. Marine aquaculture wastewater is characterized by high salinity and a low carbon-to-nitrogen ratio, and its water quality fluctuates with the aquaculture cycle and seasonal changes. These characteristics create a dual inhibition on the biological denitrification process: the high salinity environment damages microbial cell structure through osmotic pressure stress, inhibiting the activity of key denitrification enzymes, leading to a decrease in the metabolic activity of denitrifying bacteria and even their death; the low carbon-to-nitrogen ratio results in a severe shortage of electron donors, hindering the denitrification process due to insufficient energy supply, leading to increased NO3- content. - -N is not completely restored, NO2 - The accumulation of nitrogen (N-O) leads to a sharp decline in total nitrogen removal efficiency. Under the combined pressure of high salinity and limited carbon sources, existing denitrifying agents struggle to maintain efficient and stable nitrogen removal performance, resulting in poor system stability and failing to meet the engineering requirements for nitrogen removal in marine aquaculture wastewater. Therefore, developing specialized microbial agents capable of efficient nitrogen removal under high salinity and low C / N ratio conditions has become a pressing technical bottleneck in this field. Summary of the Invention

[0004] The purpose of this invention is to address the problems of poor salt tolerance and low nitrogen removal efficiency of traditional bacterial agents under low carbon-nitrogen ratio conditions, and to provide a denitrifying bacterial agent that can operate under high salt and low carbon-nitrogen ratio conditions.

[0005] A second objective of this invention is to provide the application of the bacterial agent in biological denitrification of nitrogen-containing wastewater and in biological denitrification of mariculture.

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

[0007] A denitrifying agent that operates under high salt and low carbon-to-nitrogen ratio conditions, composed of *Gastrobacterium* (…). Marinobacter shengliensis subsp. alexandrii YZC-3 and Sturgeonia ( Stutzerimonas degradans It is a compound of YZC-8; the seabacterium ( Marinobacter shengliensis subsp. alexandrii The accession number for YZC-3 is GDMCC NO: 68289, and the accession number for YZC-8 is GDMCC NO: 68290; *Hymenobacterium* ( Marinobacter shengliensis subsp. alexandrii YZC-3 and YZC-8 are both deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China; the deposit date is May 15, 2026.

[0008] Furthermore, the denitrifying agent contains *Hymenobacterium* (…). Marinobacter shengliensis subsp. alexandrii YZC-3 and Sturgeonia ( Stutzerimonas degradans The volume ratio of YZC-8 is 1:2 to 2:1; the ratio within this range can achieve complete reduction and denitrification of nitrate nitrogen, with 1:1 being the preferred ratio. This ratio has the best synergistic effect, the highest denitrification efficiency, and the lowest accumulation of nitrite nitrogen.

[0009] Furthermore, the suitable operating conditions for the denitrifying bacteria agent are: salinity of 2%~4% (corresponding to 20~40 g / L based on NaCl), and carbon-nitrogen ratio (C / N) of 4~8 for the nitrogen-containing simulated wastewater; the optimal operating conditions are salinity of 3% and C / N of 4; the preferred salinity of the nitrogen-containing simulated wastewater is 30 g / L.

[0010] The morphological and biological characteristics of the strain of this invention are as follows:

[0011] The seabacteria ( Marinobacter shengliensis subsp. alexandrii YZC-3 (hereinafter referred to as strain YZC-3): After being cultured on isolation medium at 28℃ for 48 h, the colonies were grayish-white, smooth, slightly raised, with regular edges, and opaque, with a colony diameter of approximately 1 mm. The 16S rRNA gene sequence of strain YZC-3 is shown in SEQ ID NO.1 of the sequence listing. Based on 16S rRNA sequencing comparison, the strain was identified as *Hymenobacterium* (…). Marinobacter shengliensis subsp. alexandrii It showed 99.93% similarity to the known type strain LZ-6.

[0012] The Stuzelella ( Stutzerimonas degradansYZC-8 (hereinafter referred to as strain YZC-8): After being cultured on isolation medium at 28 ℃ for 48 h, the colony surface was relatively smooth, grayish-white or dark milky white, and the colony diameter was approximately 2 mm; the 16S rRNA gene sequence of strain YZC-8 is shown in SEQ ID NO.2 of the sequence listing. Based on 16S rRNA sequencing comparison, the strain was identified as *Stuzeria stuzonii*. Stutzerimonas degradans It showed 99.22% similarity to the known type strain DSM 50238.

[0013] This invention also provides the application of the microbial agents described in the above technical solution in the actual denitrification treatment of marine aquaculture tailwater; it can achieve efficient removal of total nitrogen without the need for a large amount of external carbon source, and complete the conversion of nitrate nitrogen to gaseous nitrogen.

[0014] The application involves using seabacteria ( Marinobacter shengliensis subsp. alexandrii YZC-3 and Sturgeonia ( Stutzerimonas degradans YZC-8 was activated separately and then mixed and added to saline wastewater for biological denitrification under a micro-aerobic environment with dissolved oxygen (DO) of 0.2–1.0 mg / L. The specific steps are as follows:

[0015] (1) Bacillus subtilis ( Marinobacter shengliensis subsp. alexandrii YZC-3 and Sturgeonia ( Stutzerimonas degradans YZC-8 was activated and expanded separately. The activation medium was an enriched medium, cultured at 28 ℃ and 150 r / min, and then autoclaved at 121 ℃ for 30 min before use. The culture was continued until the bacterial OD reached the target value. 600 It reached around 1.0;

[0016] (2) Inoculation and treatment: The activated seabacteria ( Marinobacter shengliensis subsp. alexandrii YZC-3 and Sturgeonia ( Stutzerimonas degradans YZC-8 was mixed at a volume ratio of 1:2 to 2:1 and inoculated into the reactor. The total inoculation amount was 30% (v / v). Biological denitrification was carried out using the bacterial agent under microaerobic conditions.

[0017] Compared with the prior art, the present invention has the following outstanding technical effects:

[0018] 1. High nitrogen removal efficiency and strong salt tolerance. The bacterial agent is formulated by combining strains YZC-3 and YZC-8. This bacterial agent can effectively remove nitrogen in a salinity range of 2% to 4%. After an adaptation period at a salinity of 4%, the TN removal rate can reach more than 90%.

[0019] 2. High carbon source utilization efficiency. Under the condition of a low carbon-nitrogen ratio of C / N of 4, no additional carbon source is required, and the TN removal rate still reaches 91.38% within 12 hours, reducing the cost of external carbon sources for aquaculture wastewater treatment.

[0020] 3. Less accumulation of intermediate products. NO2 is produced during denitrification. - -N has a low peak value and degrades quickly, dropping to a low level within 12 hours, posing little environmental risk and effectively avoiding secondary pollution caused by toxic intermediate product residues.

[0021] 4. Good stability in practical applications. After 60 days of continuous treatment of actual aquaculture wastewater, the TN removal rate remained at 85%~93% during the stable period. The process is highly stable and can adapt to fluctuations in aquaculture wastewater quality, making it suitable for large-scale engineering applications. Attached Figure Description

[0022] Figure 1 The images show the morphological characteristics of strains YZC-3 and YZC-8. (a) represents YZC-3, and (b) represents YZC-8.

[0023] Figure 2 Phylogenetic trees for strains YZC-3 and YZC-8. (a) represents YZC-3, and (b) represents YZC-8.

[0024] Figure 3 This is a graph showing the carbon and nitrogen metabolism correlation between strains YZC-3 and YZC-8.

[0025] Figure 4 The effects of different compound ratios of microbial agents on nitrogen removal from simulated wastewater are shown. Among them, (a) the total nitrogen removal rate versus reaction time under different compound ratio systems, (b) the residual nitrate nitrogen concentration versus reaction time under different compound ratio systems, (c) the accumulation concentration of nitrite nitrogen versus reaction time under different compound ratio systems, and (d) the concentration of ammonium nitrogen versus reaction time under different compound ratio systems.

[0026] Figure 5 The effect of different carbon-nitrogen ratios on the nitrogen removal efficiency of the microbial agent is shown. Among them, (a) the change of nitrate nitrogen concentration with reaction time under different carbon-nitrogen ratios, (b) the change of nitrite nitrogen accumulation concentration with reaction time under different carbon-nitrogen ratios, (c) the change of ammonium nitrogen concentration with reaction time under different carbon-nitrogen ratios, and (d) the change of total nitrogen removal rate with reaction time under different carbon-nitrogen ratios.

[0027] Figure 6 The effect of different salinities on the removal of nitrates by bacterial agents.

[0028] Figure 7 The long-term operational effect of microbial agents on actual aquaculture wastewater treatment. Detailed Implementation

[0029] The following embodiments, in conjunction with the accompanying drawings, will further illustrate the present invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, all experimental methods of the present invention can employ conventional microbial culture and water quality testing methods; the reagents and culture media used can all be commercially available raw materials.

[0030] Example 1: Isolation, purification and identification of strains

[0031] (1) Sample source:

[0032] The isolated samples were taken from natural ocean sediments in the Indian Ocean, and native denitrification strains adapted to high-salt environments were screened.

[0033] (2) Culture medium composition

[0034] a. Enrichment medium: C4H4Na2O4·6H2O 5.4333 g / L, (NH4)2SO4 0.25 g / L, K2HPO4·3H2O 1.5 g / L, KH2PO4 0.45 g / L, MgSO4·7H2O 0.05 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·4H2O 0.01 g / L, NaCl 30 g / L, trace elements 1 mL, vitamins 1 mL, pH 7.5.

[0035] b. Isolation medium: C4H4Na2O4·6H2O 5.4333 g / L, (NH4)2SO4 0.25 g / L, K2HPO4·3H2O 1.5 g / L, KH2PO4 0.45 g / L, MgSO4·7H2O 0.05 g / L, FeSO4·7H2O 0.01 g / L, MnSO4·4H2O 0.01 g / L, NaCl 30 g / L, trace elements 1 mL, vitamins 1 mL, agar 15 g / L, pH adjusted to 7.5.

[0036] c. Compound vitamin solution: Biotin (Vh) 0.05 g, folic acid (Vb9) 0.50 g, pyridoxine (Vb6) 0.50 g, thiamine (Vb1) 1.00 g, niacin (Vb3) 1.00 g, calcium pantothenate (Vb5) 0.50 g, cobalamin (Vb12) 0.01 g, riboflavin (Vb2) 0.10 g, lipoic acid 0.10 g, EDTA-Na 0.40 g, dissolved in 1000 mL of water, pH 7.5, filtered through a 0.22 μm membrane for sterilization.

[0037] d. Trace element mixture: ZnSO4·7H2O 0.10 g, MnCl2·4H2O 0.40 g, H3BO3 1.24 g, CoCl2·2H2O 0.50 g, CuCl2·2H2O 0.50 g, NiSO4·6H2O 0.02 g, NaMoO4·2H2O 0.40 g, KI 0.20 g, CaCl2 5.00 g, FeSO4·7H2O 1.10 g, EDTA-Na 10.00 g, dissolved in 1000 mL of water, pH 7.4, and sterilized by filtration through a 0.22 μm filter membrane.

[0038] (3) Isolation and purification steps: Weigh about 3 g of ocean sediment sample and inoculate it into 100 ml of enrichment medium. Incubate at 28℃ and 150 r / min for 6 days, and replace the medium with fresh medium every 48 h. After 3 rounds of enrichment, dilute and spread it on isolation medium. Pick single colonies of different morphologies and streak them on the isolation medium again for purification to obtain two pure strains, labeled as YZC-3 and YZC-8, respectively.

[0039] (4) 16S rRNA sequencing and morphological characteristics: Genomic DNA of the two strains was extracted using a bacterial genomic DNA extraction kit (Cyber-Tech, Shanghai, China). After PCR amplification with universal primers 27F and 1492R, the DNA was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing sequence of strain YZC-3 is shown in SEQ ID NO.1 of the sequence listing, and it has 99.93% similarity to the model strain LZ-6. The sequencing sequence of strain YZC-8 is shown in SEQ ID NO.2 of the sequence listing, and it has 99.22% similarity to the model strain DSM 50238. Both strains meet the criteria for species identification and have the same morphology.

[0040] The sequencing results of strain YZC-3 are as follows:

[0041]

[0042] Incubate at 28°C for 48 h on the isolation medium, such as... Figure 1 As shown in (a), the colony is grayish-white, smooth, slightly raised, with regular edges, and opaque, with a diameter of about 1 mm.

[0043] The sequencing results of strain YZC-8 are as follows:

[0044]

[0045] Incubate at 28°C for 48 h on the isolation medium, such as Figure 1 As shown in (b), the colony has a relatively smooth surface, is grayish-white or dark milky white, and has a diameter of about 2 mm.

[0046] (5) Construction of the phylogenetic tree of strains

[0047] After obtaining the 16S rRNA gene sequence of the strain, it was compared with the BLAST database on the website of the National Center for Biotechnology Information (NCBI) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Based on the comparison results, the 16S rRNA gene sequence of the strain with high homology was obtained. Sequence alignment was performed using the MUSCLE program in MEGA 11.0 software. Based on the alignment results, a phylogenetic tree of the strains was constructed using the neighbor-joining method, with the bootstrap parameter set to 1000, to explore the species relationships among the strains. Figure 2 As shown in (a), *Gymnobacterium* was identified by phylogenetic tree as... Marinobacter shengliensis subsp. alexandrii LZ-6 has a similarity of 99.93% to existing type strains; such as Figure 2 As shown in (b), *Stuzeria* was identified by phylogenetic tree as Stutzerimonas degradans DSM 50238 has a similarity of 99.22% to the existing type strain.

[0048] The accession number of strain YZC-3 is GDMCC NO: 68289, and the accession number of YZC-8 is GDMCC NO: 68290. Both YZC-3 and YZC-8 are deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on May 15, 2026. The two strains were tested by the collection center and found to be viable and physiologically stable.

[0049] Example 2: Strain genome draft sequencing and carbon and nitrogen metabolism association analysis

[0050] Genomic DNA was extracted from strains YZC-3 and YZC-8 using standard methods and sent to Shanghai Meiji Genomics Co., Ltd. Draft genome sequencing was performed using the HiSeq platform, followed by quality control, assembly, and annotation of the obtained sequences. Annotation revealed that the genomes of both strains YZC-3 and YZC-8 carry complete denitrification metabolic pathways coupled with carbon metabolism pathways, such as... Figure 3 As shown, all of these can achieve the effect of NO3 - The complete process of conversion to N2. Nitrate uptake and reduction: The periplasmic nitrate transporter Nrt mediates extracellular NO3. -The nitrate is taken up across the membrane, and then reduced to NO2 by the nitrate reductase Nap / Nxr. - Nitrite metabolism: Cytochrome cd1 nitrite reductase NirS (dominant NO2) - The conversion to NO; complete removal of nitrogen oxides: Nitric oxide reductase Nor catalyzes NO to N2O, which is then reduced to N2 by nitrile oxide reductase Nos, completing the final denitrification process. At the carbon metabolism level, the strain uses acetate as its core carbon source, and its metabolic network is coupled with the denitrification process: acetate is activated by acetyl-CoA synthase (ACS) to generate acetyl-CoA (acetyl-CoA synthase). Figure 3 In the process of Acetate → ACS → Acetyl-CoA, the latter enters the tricarboxylic acid cycle (TCA cycle) to complete oxidative decomposition, while the reducing power generated (such as NADH) provides energy support for electron transfer in denitrification.

[0051] Example 3: Optimization of the compound ratio of microbial agents

[0052] (1) Prepare simulated wastewater 1: KNO3 0.36 g / L ， KH₂PO₄ 22 mg / L, MgSO₄·7H₂O 75 mg / L, FeSO₄·7H₂O 5 mg / L, CaCl₂ 5 mg / L, NaCl 30 g / L, CH₃COONa 640 mg / L, autoclaved at 121 ℃ for 30 min.

[0053] (2) Strains YZC-3 and YZC-8 were inoculated into enrichment medium and cultured with shaking at 28 ℃ and 150 rpm until the logarithmic growth phase (OD). 600 The seed culture was prepared by adding 70 mL of simulated tailwater (approximately 1.0 g / mL). A 50 mL sealed cell culture flask was used as a small-scale reactor. The seed culture was inoculated into the reaction system at a total inoculum of 30% (v / v). Five experimental groups were set up according to the strain volume ratios: a single strain YZC-3 group, a single strain YZC-8 group, and mixed groups with YZC-3 and YZC-8 in volume ratios of 1:1, 1:2, and 2:1, respectively. All reactors were placed in a shaker, with the rotation speed controlled at 90 rpm to maintain the anoxic / micro-oxygen environment caused by the sealing and to ensure mass transfer. During the experimental period, 5 mL samples were taken every 4 h to determine inorganic nitrogen (NH4+). + -N, NO2 - -N, NO3 - The concentrations of -N) and TN were sampled, and an equal volume (5 mL) of fresh simulated wastewater 1 was immediately added to the system after sampling to maintain a constant reaction volume.

[0054] Among them, NH4 +-N was determined using Nessler's reagent spectrophotometry (HJ 535-2009); NO2 - -N and NO3 - -N was determined by ion chromatography.

[0055] Figure 4 This study compares the nitrogen removal efficiency of bacterial agents under different compound ratios. Analysis of the TN removal rate results shows that ( Figure 4 In (a) of the study, the compound groups exhibited a synergistic biological advantage in treating high-salinity wastewater. Among them, the compound system constructed at a 1:1 ratio showed the best denitrification efficiency. This group demonstrated denitrification advantage after 4 hours of reaction, and its TN removal rate reached 75.39% by 12 hours, gradually stabilizing at around 79.03% after 16 hours. In contrast, the denitrification function of the single-strain YZC-3 group was almost stagnant throughout the entire reaction cycle, with a TN removal rate of only 13.99% at 16 hours. Although the single-strain YZC-8 group initiated denitrification after 8 hours, its 16-hour removal rate (52.70%) was still lower than all the compound groups, indicating that the denitrification effect of the single-strain system under high-salinity conditions has certain limitations. Figure 4 As shown in (b), all three compound groups (1:1, 1:2, 2:1) exhibited strong nitrate reducing ability, with NO3 at 12 h. - -N concentration has been reduced below the detection limit (<0.5 mg / L), achieving rapid substrate transformation. Meanwhile, NO3 in strain YZC-3 single-cell group... - -N degradation was relatively slow, with a residual concentration of 37.63 mg / L after 16 hours, indicating a conversion rate of less than 25%. In strain YZC-8, the residual concentration was 2.92 mg / L after 12 hours, and complete conversion was only achieved after 16 hours, suggesting that while its nitrate reductase activity could gradually adapt to salt stress, the overall reaction rate was relatively limited. Figure 4 As shown in (c) in the figure, NO2 was observed during the experiment. - NO2 accumulation was observed in three treatment groups, with significant accumulation peaks at 8 h. The 2:1 mixture group showed the highest peak (19.5 mg / L), followed by strain YZC-8 (15.23 mg / L) and the 1:1 mixture group (13.67 mg / L). However, the mixture groups exhibited relatively rapid NO2 accumulation. - The reduction rate of -N decreased at 12 h. Small amounts of NH4 were detected in all groups during the reaction. + -N (<1 mg / L) Figure 4 (d) in the figure, but all were eventually reduced to below the detection limit. In summary, under this experimental system, the 1:1 compound group showed relatively superior denitrification performance. In this invention, the 1:1 compound group not only shortened the reaction cycle and improved the TN removal efficiency, but also may have reduced the intermediate product NO2.- The potential environmental risks posed by the long-term accumulation of -N show certain application potential under these high-salt denitrification conditions.

[0056] Experiments show that within a volume ratio range of 1:2 to 2:1, both strains can form a metabolically complementary system and complete denitrification from nitrate nitrogen to nitrogen gas. Among them, the synergistic complementary effect between strains reaches its peak at a 1:1 ratio, with the fastest denitrification rate, the highest total nitrogen removal rate, and the lowest accumulation of nitrite intermediate products, which is the optimal ratio for implementation in this invention. Complete denitrification can also be achieved at ratios of 1:2 and 2:1, but the accumulation of nitrite nitrogen is higher and the denitrification rate is slightly lower than that at a 1:1 ratio, but it can still meet the denitrification requirements of conventional saline wastewater.

[0057] Example 4: Effect of different C / N ratios on the denitrification performance of microbial agents

[0058] (1) Prepare simulated wastewater 2: KNO3 0.36 g / L, KH2PO4 22 mg / L, MgSO4·7H2O 75 mg / L, FeSO4·7H2O 5 mg / L, CaCl2 5 mg / L. Adjust the salinity and C / N ratio by adjusting the amount of NaCl and CH3COONa respectively. NaCl 20 g / L during the start-up stage.

[0059] (2) Strains YZC-3 and YZC-8 were inoculated into enrichment medium and cultured with shaking at 28 ℃ and 150 rpm until the logarithmic growth phase. The OD of the strains was monitored. 600 The bacterial concentration was approximately 1.0. 450 ml of bacterial culture was placed in centrifuge tubes and centrifuged at 4800 rpm for 5 min to collect the bacterial cells. After discarding the supernatant, the bacterial precipitate was washed with sterile physiological saline (to remove residual organic carbon sources). Finally, the bacterial cells were resuspended in an appropriate amount of simulated wastewater 2 to prepare an inoculated bacterial suspension.

[0060] (3) Add 10% polyurethane biological packing to the sequencing batch reactor (3L), introduce the prepared bacterial suspension into the reactor, and fill it with simulated wastewater 2. Then seal the reactor (cover it with a lid) to create a micro-aerobic denitrification environment. Turn on the peristaltic pump to build a closed-loop hydraulic circulation system and adjust the flow rate to drive the liquid flow in the reactor.

[0061] (4) Using simulated wastewater 2 as the substrate, simulated wastewater with C / N ratios of 8, 6, 4, 3, and 2 were prepared by adjusting the concentration of added CH3COONa. Batch experiments were conducted, with each batch lasting for 7 days. After the effluent quality stabilized, the effects of different electron donor levels on the denitrification rate and intermediate products (NH4+) were comprehensively evaluated by monitoring the tri-nitrogen conversion pattern, DO and TN removal during the reaction cycle. + -N, NO2- The accumulated effects of -N) are used to determine the optimal operating parameters.

[0062] like Figure 5 As shown in (a), as time progresses, the NO3 in each group... - -N concentrations all showed a decreasing trend, with the high C / N groups (8 and 6) showing the lowest NO3 concentrations. - The reduction rate of -N was relatively fast, decreasing to 0 mg / L by the 6th hour of the reaction; the groups with C / N ratios of 4 and 3 achieved NO3 reduction by the 8th hour. - The removal of -N was largely achieved, while the low-carbon group with a C / N ratio of 2 still had 19.01 mg / L of NO3 at 8 h. - -N residue indicates that insufficient carbon source addition affected NO3 in the early stages of the reaction. - -N reduction processes are inhibited. In NO2 - -N transformation dynamics ( Figure 5 In (b) of the study, the systems with C / N ratios of 8 and 6 exhibited a characteristic of initial accumulation followed by degradation, with their concentrations peaking at 4 h (17.75 mg / L) and 5 h (18.08 mg / L), respectively. They were then gradually reduced, reaching undetectable levels at 9 h and 11 h, respectively. The NO2 concentration in the group with a C / N ratio of 4... - The -N peak appeared later, around hour 6, and the degradation rate was relatively slow in the later stages; in contrast, NO2 in the groups with C / N ratios of 3 and 2... - -N exhibited a continuous accumulation state, with concentrations rising to 22.65 mg / L and 17.89 mg / L respectively by the end of the 12-hour reaction, indicating the presence of significant NO2. - -N accumulation phenomenon. Figure 5 As shown in (c), trace amounts of NH4 were present in each group during the reaction. + -N was generated, and its concentration did not exceed 2 mg / L, indicating the presence of a small amount of dissimilar nitrate reduction to ammonium (DNRA) process in the system, but its interference with the overall inorganic nitrogen change was small. Under the combined effect of the transformation of various forms of inorganic nitrogen, the TN removal rate decreased with the decrease of C / N ratio. Figure 5 (d) After 12 hours of reaction, the TN concentrations in groups with C / N ratios of 8, 6, and 4 decreased to 2.89 mg / L, 3.50 mg / L, and 4.18 mg / L, respectively, with TN removal rates reaching 94.09%, 92.93%, and 91.38%, respectively, achieving a good overall nitrogen removal level. However, the groups with C / N ratios of 3 and 2 showed reduced NO2 removal. - A large amount of -N is retained, along with some unreduced NO3. -The presence of -N resulted in the final TN concentrations remaining at relatively high levels of 27.37 mg / L and 39.01 mg / L, revealing the crucial regulatory role of carbon source supply in maintaining the integrity of the denitrification reaction and the TN removal efficiency. In summary, controlling the C / N ratio to 4 can serve as a reference condition for maintaining a relatively complete denitrification process in this reaction system while ensuring good economic efficiency.

[0063] Example 5: Effect of different salinities on the denitrification performance of bacterial agents

[0064] (1) Prepare simulated wastewater 3: KNO3 0.36 g / L, KH2PO4 22 mg / L, MgSO4·7H2O 75 mg / L, FeSO4·7H2O 5 mg / L, CaCl2 5 mg / L, CH3COONa 256 mg / L

[0065] (2) The experiment maintained the influent NO3 - With the -N concentration and C / N ratio kept constant, the salinity of the simulated wastewater was adjusted using NaCl, with three gradients of 2%, 3%, and 4% set sequentially, and the reactor was operated in a sequencing batch reactor (SBR) mode. The reactor was maintained for 30 batches under each salinity condition, with real-time monitoring of effluent quality changes. Once the system effluent indicators stabilized, samples were collected at the reaction endpoint (24 h) to measure effluent TN and NO3. - -N, NO2 - -N and DO concentrations.

[0066] This invention examines NO3 - Under the condition that -N is the only nitrogen source and the influent C / N ratio is 4, the dynamic response characteristics of the reactor to the increase in salinity gradient over a continuous 90 days are as follows: Figure 6 As shown. By Figure 6 It can be seen that during the 1-30 day operation period at 2% salinity, the system's TN removal rate fluctuated around 95%, and the effluent NO3... - -N and NO2 - The nitrogen (TN) concentrations were all at low levels (<0.01 mg / L), and the dissolved oxygen in the influent, which was as high as 7 to 8 mg / L, was consumed. The dissolved oxygen in the effluent stabilized at around 1.0 mg / L, indicating that the microbial agents in the reactor could fully carry out denitrification at this salinity level and had not been significantly inhibited by salinity. When the influent salinity was increased to 3% on the 31st day of operation, the change in osmotic pressure had a certain stress effect on the microbial cells, leading to a short-term degradation in the system's nitrogen removal performance. The TN removal rate rapidly dropped to around 85%, and obvious nitrogen accumulation was detected in the effluent, including NO2. - -N concentration surged to 4.25 mg / L, higher than NO3 concentration during the same period. --N residual amount (1.52 mg / L). After a fluctuating adaptation period of about 5 days, the concentrations of various forms of nitrogen in the effluent gradually decreased, with NO3... - -N was 100% removed, and the TN removal rate recovered and stabilized at around 95%. On day 61, with the salinity further increasing to 4%, the denitrification system experienced more severe initial inhibition, and the TN removal rate dropped to 77.62%, resulting in effluent NO2... - -N and NO3 - The accumulation peaks of -N further increased to 7.11 mg / L and 3.55 mg / L, but after 9 days of adaptation, the concentrations of both decreased to below the detection limit, and the TN removal rate recovered to about 94%.

[0067] Example 6: Denitrification effect of compound bacterial agent on actual marine aquaculture wastewater

[0068] (1) Approximately 3 L of aquaculture water was collected from aquarium No. 1 at the aquaculture base of the Third Institute of Oceanography, Ministry of Natural Resources, as the system influent. A microbial reactor was used to treat the water for denitrification. Actual wastewater baseline water quality: NO3 - -N 19.52±2.85 mg / L, NO2 - -N 0.0251±0.0014 mg / L, NH4 + -N 2.52±0.85 mg / L, TN 21.32±0.13 mg / L, COD 105±20 mg / L, salinity 2.85%, pH 7.35±0.28.

[0069] (2) The hydraulic retention time (HRT) of the reactor system was set to about 24 h. During the treatment period, influent and effluent samples were collected at 24 h intervals, and TN and NO3 in the water samples were measured systematically. - -N, NO2 - -N, NH4 + The nitrogen content was used to comprehensively evaluate the nitrogen removal performance of the denitrification system.

[0070] Figure 7 The nitrogen removal performance characteristics of the microbial reactor in treating actual wastewater were demonstrated during 60 days of continuous operation. In the initial stage of reactor operation (days 1-7), the system was in an adaptation phase to the wastewater, and NO3... - -N and NO2 - -N concentration fluctuated to some extent. Effluent NO3 - - The highest NO2 concentration in the initial stage reached approximately 3.7 mg / L. -The initial NO3- concentration was approximately 1.2 mg / L. Over time, the concentrations of both nitrogen (TN) and nitrogen (N) showed a significant decreasing trend, while the TN removal rate gradually increased from approximately 73% initially, reaching and stabilizing at a high level of over 90% around day 7. After entering the stable operating period (days 8-60), the reactor exhibited relatively stable nitrogen removal efficiency. Although the influent NO3-... - -N concentration fluctuated between 17 and 21 mg / L, effluent NO3 - -N concentration remained relatively stable at a low level close to 0 mg / L, with NO2 in the effluent... - -N also remained in a low range of around 0.1~0.3 mg / L, indicating that the system achieved relatively sufficient NO3- concentration. - -N reduction, and no obvious NO2 reaction occurred. - -N accumulation phenomenon. In addition, NH4 in the effluent... + The NO-N concentration remained relatively stable within a low range of approximately 0.2–0.5 mg / L throughout the entire operating cycle, indicating that denitrification was the dominant nitrogen metabolism pathway within the reactor. During the 60-day stable period, the system's TN removal rate remained consistently high, between 85% and 93%. Overall, this reactor effectively reduces NO3- in the influent when treating actual aquaculture wastewater. - It controls the accumulation of intermediate products and exhibits good process stability and denitrification potential during long-term continuous operation.

[0071] Experimental data show that the present invention is made from seabacteria ( Marinobacter shengliensis subsp. alexandrii YZC-3 and Sturgeonia ( Stutzerimonas degradans The YZC-8 compound microbial agent system exhibits excellent resistance to salinity shocks and carbon-limited adaptability under high salinity and low C / N ratio conditions, enabling stable and efficient nitrogen removal from nitrogen-containing wastewater. Compared to the conventional limitation of maintaining a C / N ratio greater than 5 in traditional denitrification processes, this microbial agent, even under conditions with a C / N ratio as low as 4 and salinity as high as 4%, allows the system to recover and stabilize the TN removal rate at over 90% after a short period of adaptation. During 60 days of continuous operation treating actual marine aquaculture wastewater, the TN removal rate remained at 85%–93% during the stable period, with effluent NO3... - -N is close to 0 mg / L, NO2 - -N is controlled at 0.1~0.3 mg / L. This characteristic effectively overcomes the limitations of traditional biological denitrification processes for treating high-salinity aquaculture wastewater, which are highly dependent on external carbon sources and have poor salt tolerance. In practical engineering applications, it can reduce the amount of carbon source added and the overall operating cost.

[0072] Experimental data show that strains YZC-3 and YZC-8 of this invention can form a synergistic system when combined: YZC-3 secretes compatible solutes to improve the osmotic pressure environment of the system and protects the stability of the metabolic enzyme activity of YZC-8; YZC-8 can continuously provide a complete denitrification metabolic pathway and efficiently reduce nitrate nitrogen. The carbon metabolic pathways of the two are coupled with each other to achieve a synergistic denitrification effect of 1+1>2. The metabolic complementarity between the strains reaches its peak when the volume ratio is 1:1.

[0073] Both strains have genomes annotated with complete denitrification metabolic pathways and efficient carbon metabolism pathways. Under high-salt stress, they exhibit differential expression characteristics of functional genes: although the YZC-3 genome carries a complete set of denitrification-related genes, the expression level of its denitrification reductase encoding genes may be downregulated under high-salt conditions, making it difficult to achieve efficient nitrate reduction when acting alone. Analysis suggests that the core functional advantage of this strain lies in the large-scale synthesis and secretion of betaine-compatible solutes, reducing osmotic pressure stress and ensuring stable expression of various key denitrification enzymes in YZC-8. YZC-8 can completely and stably express all denitrification functional enzymes within a salinity range of 2%–4%. Carbon metabolism is highly coupled with the denitrification reaction: both strains use acetate as the preferred carbon source, which is activated by acetyl-CoA and undergoes the tricarboxylic acid cycle, continuously generating reducing power to provide electrons and energy for the stepwise denitrification reaction. The system completes NO3 reduction through the stepwise catalytic synergy of nitrate transport proteins, nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase. - →NO2 - The complete denitrification pathway →NO→N2O→N2 eliminates nitrogen residues; the carbon metabolism pathways of the two strains cooperate and couple with each other to jointly construct a composite synergistic denitrification system of 1+1>2.

[0074] The above embodiments are only used to illustrate the technical solutions and beneficial effects of the present invention in detail, and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make conventional modifications, equivalent substitutions, and simple optimizations to the technical solutions according to actual needs within the scope of the technology disclosed in the present invention. Any changes that do not depart from the core technical concept and technical solutions of the present invention should fall within the scope of protection of the present invention.

Claims

1. A denitrifying bacterial agent operating under high salt and low carbon-to-nitrogen ratio conditions, characterized in that, The denitrifying agent is composed of seabacteria ( Marinobacter shengliensis subsp. alexandrii YZC-3 and Sturgeonia ( Stutzerimonas degradans The two strains were combined with YZC-8. Both strains were deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 15, 2026. The accession number of strain YZC-3 was GDMCC NO: 68289, and the accession number of strain YZC-8 was GDMCC NO: 68290.

2. The denitrifying bacteria agent under high salt and low carbon-to-nitrogen ratio conditions according to claim 1, characterized in that, The seabacteria ( Marinobacter shengliensis subsp. alexandrii The 16S rRNA gene sequence of YZC-3 is shown in SEQ ID NO.1 of the sequence listing, and the *Stuzeria* strain ( Stutzerimonas degradans The 16S rRNA gene sequence of YZC-8 is shown in the sequence listing SEQ ID NO.

2.

3. The denitrifying bacteria agent under high salt and low carbon-to-nitrogen ratio conditions according to claim 1, characterized in that, The seabacteria ( Marinobacter shengliensis subsp. alexandrii YZC-3 and Sturgeonia ( Stutzerimonas degradans The volume ratio of YZC-8 is 1:2 to 2:1, with a preferred volume ratio of 1:

1.

4. The denitrifying bacteria agent under high salt and low carbon-to-nitrogen ratio conditions according to claim 1, characterized in that, The denitrifying bacteria agent is suitable for saline wastewater environments with the following conditions: salinity of 2% to 4% based on sodium chloride and carbon-nitrogen ratio (C / N) of 4 to 8; the optimal operating conditions are salinity of 3% based on sodium chloride and C / N of 4.

5. The application of the denitrifying bacteria agent according to any one of claims 1 to 4 in the biological denitrification treatment of saline wastewater with high salt content and low carbon-to-nitrogen ratio.

6. The application according to claim 5, characterized in that, The saline wastewater includes mariculture tailwater, coastal industrial saline wastewater, and tidal flat aquaculture saline wastewater.

7. The application according to claim 5, characterized in that, Denitrification treatment methods include: removing seabacteria ( Marinobacter shengliensis subsp. alexandrii YZC-3, Sturgeonia ( Stutzerimonas degradans YZC-8 was activated and cultured separately, then mixed and added to saline wastewater to carry out biological denitrification in a micro-aerobic environment, without the need for additional large amounts of external carbon sources.

8. The application according to claim 7, characterized in that, The dissolved oxygen concentration in the micro-oxygen environment is controlled at 0.2–1.0 mg / L.

9. The application according to claim 7, characterized in that, The activation culture conditions for the strain were: culture temperature 28℃, shaking speed 150 r / min, and continuous culture until the bacterial culture OD reached the target value. 600 It reached around 1.

0.

10. The application according to claim 7, characterized in that, The total inoculum volume fraction of the two strains after mixing was 30%.

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