Biological hybrid, construction method thereof and application of biological hybrid in denitrification nitrogen removal of high-salt nitrogen-containing wastewater

Abstract

The invention belongs to the technical field of water pollution treatment, and discloses a biological hybrid, a construction method thereof and application of the biological hybrid in denitrification nitrogen removal of high-salt nitrogen-containing wastewater, the biological hybrid is constructed by an H.titanicae BH1 strain and a MoS2 nano material, and the nitrate reduction rate of the constructed H.titanicae BH1 / MoS2 biological hybrid is remarkably improved compared with that of a single H.titanicae BH1 strain; meanwhile, the hybrid can enhance the denitrification process by improving the activity of a denitrification key enzyme, promoting the secretion of extracellular secretions (EPS) and enhancing the electron transfer performance, and shows high salt adaptability superior to that of a single strain within the salinity range of 2%-7%. The invention provides a novel microbial preparation and a technical scheme for efficient treatment of high-salt nitrogen-containing wastewater, has the advantages of high treatment efficiency, strong environmental adaptability, no secondary pollution and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of water pollution control technology, and relates to a biohybrid and its construction method and its application in denitrification of high-salt nitrogen-containing wastewater, especially to a H. titanicae BH1 / MoS2 biohybrid and its construction method and its application in denitrification of high-salt nitrogen-containing wastewater. Background Technology

[0002] With the development of industry and agriculture, the discharge of nitrogen-containing wastewater has been increasing year by year. Among them, high-salinity nitrogen-containing wastewater (such as wastewater from chemical, seafood processing, and pickled food production) has become a challenge in the field of wastewater treatment due to its high salinity and high nitrogen concentration, which strongly inhibits microbial metabolism. Traditional biological denitrification technology suffers from problems such as osmotic pressure imbalance and enzyme activity inhibition in high-salinity environments, resulting in low denitrification efficiency and poor stability. Physicochemical methods (such as ion exchange and reverse osmosis) have drawbacks such as high energy consumption and easy generation of secondary pollution.

[0003] To improve denitrification efficiency, researchers screened and cultivated denitrifying microorganisms with inherent salt tolerance or halophilic abilities, such as certain moderately halophilic or salt-tolerant strains (Halomonas titanicae BH1, H. titanicae BH1). These strains maintain basic physiological functions by synthesizing compatible solutes to balance the osmotic pressure inside and outside the cell. However, relying solely on such strains often results in limited denitrification rates and insufficient adaptability to high salt and high nitrogen shock loads.

[0004] Therefore, how to achieve a synergistic improvement in denitrification efficiency and adaptability under high-salt environments is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing high-salt nitrogen-containing wastewater treatment technologies, and to provide a biohybrid, its construction method, and its application in denitrification of high-salt nitrogen-containing wastewater. By constructing a biohybrid of H. titanicae BH1 strain and MoS2 nanomaterials, the problem of low efficiency and poor adaptability of traditional denitrification technology in high-salt environments is solved.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: The first aspect of this invention provides a method for constructing a biological hybrid, comprising the following steps: S1. Activation culture of H. titanicae BH1 strain: Streak H. titanicae BH1 strain on agar solid medium containing 2-5% salinity and incubate at 30-37℃ for 14-18 h; pick a single colony and inoculate it into liquid medium containing 3% salinity and incubate at 30-37℃ in a shaker for 14-18 h; take 5 mL of the above bacterial solution and inoculate it into sterile denitrification expansion medium and continue to incubate at 30-37℃ for 14-18 h to obtain the logarithmic growth phase bacterial solution; S2. Pretreatment of bacterial culture: The logarithmic phase bacterial culture obtained in step S1 is centrifuged and resuspended with PBS buffer, and the OD value is adjusted to 0.5; S3. Hybrid construction: The logarithmic-phase bacterial culture after pretreatment in step S2 was transferred to a serum bottle containing denitrification expansion medium. MoS2 nanomaterials with a final concentration of 0.1-0.7 mM and cysteine ​​solution with a mass concentration of 0.08-0.12 wt% were added (to protect the activity of the strain). The mixture was then incubated in a constant temperature shaking incubator at 30-37℃ for 40-50 h to obtain a homogeneous H. titanicae BH1 / MoS2 biohybrid.

[0007] Preferably, the denitrification expansion medium comprises: CH3COONa 1-2 g / L, KNO3 0.1-0.5 g / L, MgSO4 0.01-0.1 g / L, NaCl 20-40 g / L, CaCl2 0.1-0.5 g / L, K2HPO4 0.01-0.03 g / L, tryptone 1-2 g / L, yeast extract 0.1-0.8 g / L, trace element solution 0.8-1.2 mL / L, and vitamin solution 0.8-1.2 mL / L; and is autoclaved at 121℃ for 20 min. More preferably, the denitrification expansion medium comprises: CH3COONa 1.46 g / L, KNO3 0.35 g / L, MgSO4 0.05 g / L, NaCl 30.00 g / L, and CaCl2 0.20 g / L. The solution contained 0.022 g / L K2HPO4, 1.50 g / L tryptone, 0.50 g / L yeast extract, 1 mL / L trace element solution, and 1 mL / L vitamin solution; and was autoclaved at 121℃ for 20 min.

[0008] Preferably, the PBS buffer comprises 0.5-0.8 g / L Na3PO4·12H2O, 0.4-0.8 g / L NaH2PO4·2H2O, 0.01-0.08 g / L KCl, and 0.4-0.8 g / L NaCl, and is autoclaved at 121°C for 20 min. More preferably, the PBS buffer comprises 0.76 g / L Na3PO4·12H2O, 0.62 g / L NaH2PO4·2H2O, 0.0745 g / L KCl, and 0.5265 g / L NaCl, and is autoclaved at 121°C for 20 min.

[0009] Preferably, the agar solid culture medium containing 2-5% salinity in step S1 comprises: NaCl 20-50 g / L, tryptone 5-15 g / L, yeast extract 1-10 g / L, and agar 15-20 g / L; more preferably, the agar solid culture medium containing 2-5% salinity comprises: NaCl 30.00 g / L, tryptone 10.00 g / L, yeast extract 5.00 g / L, and agar 15-20 g / L, and is autoclaved at 121°C for 20 min.

[0010] Preferably, the trace element solution in step S3 comprises: FeCl3·6H2O 1-2 g / L, MnCl2·4H2O 0.1-0.5 g / L, CuSO4·5H2O 0.01-0.05 g / L, H3BO3 0.1-0.3 g / L, KI 0.1-0.5 g / L, Na2MoO4 0.02-0.08 g / L, CoCl2·6H2O 0.1-0.3 g / L, ZnSO4·7H2O 0.1-0.4 g / L, and EDTA 5-15 g / L; and is autoclaved at 121℃ for 20 min. The composition of the trace element solution is further optimized to include: FeCl3·6H2O 1.50 g / L, MnCl2·4H2O 0.12 g / L, CuSO4·5H2O 0.03 g / L, H3BO3 0.15 g / L, KI 0.18 g / L, Na2MoO4 0.06 g / L, CoCl2·6H2O 0.15 g / L, ZnSO4·7H2O 0.12 g / L, and EDTA 10.00 g / L; and sterilized by autoclaving at 121℃ for 20 min. The vitamin solution consists of (100× concentrated mother liquor): thiamine 0.1 g / L, riboflavin 0.05 g / L, niacin 0.1 g / L, calcium pantothenate 0.1 g / L, pyridoxine 0.15 g / L, cyanocobalamin 0.01 g / L, biotin 0.05 g / L, folic acid 0.05 g / L, para-aminobenzoic acid 0.05 g / L, and lipoic acid 0.05 g / L.

[0011] Preferably, the final concentration of MoS2 nanomaterials is 0.2 mM, at which point the hybrid has the highest denitrification efficiency and does not inhibit the activity of the strain.

[0012] Preferably, the volume ratio of the logarithmic phase bacterial culture to the cysteine ​​solution is 100:0.5-1.5.

[0013] Preferably, the H. titanicae BH1 strain is a moderately halophilic bacterium, which grows fastest at a salinity of 3% and can maintain osmotic pressure balance in a high-salt environment by secreting tetrahydropyrimidine; the MoS2 nanomaterial has a nanosheet-like structure, low cytotoxicity, low genotoxicity and good biocompatibility, and can be used as an electron transport medium to promote denitrification electron transport.

[0014] Another aspect of the present invention provides the application of the H. titanicae BH1 / MoS2 biohybrid in enhanced denitrification in high-salt nitrogen-containing wastewater.

[0015] Preferably, the salinity of the high-salinity nitrogen-containing wastewater is 2%-7%, and the NO3 content is [missing information]. - -N concentration 50-200 mg / L.

[0016] Preferably, the application method includes the following steps: (1) Hybrid activation: Take the H. titanicae BH1 / MoS2 biohybrid and dilute it with denitrification medium to OD. 600 =0.5, 30-37℃, 150 rpm oscillation recovery for 20-40 min, to obtain the activated biological hybrid; (2) Wastewater treatment: The activated bio-hybrid was inoculated into high-salt nitrogen-containing wastewater at a volume ratio of 1:8-12 and treated at 30-37℃ under static anaerobic conditions for 4-8 h. The denitrification efficiency was then tested.

[0017] Preferably, the denitrification medium comprises: CH3COONa 1-2 g / L, KNO3 0.5-1 g / L, MgSO4 0.01-0.08 g / L, NaCl 20-40 g / L, CaCl2 0.1-0.3 g / L, K2HPO4 0.01-0.03 g / L, trace element solution 0.8-1.2 mL / L, and vitamin solution 0.8-1.2 mL / L; more preferably, the denitrification medium comprises: CH3COONa 1.46 g / L, KNO3 0.72 g / L, MgSO4 0.05 g / L, NaCl 30.00 g / L, CaCl2 0.20 g / L, K2HPO4 0.022 g / L, trace element solution 1 mL / L, and vitamin solution 1 mL / L.

[0018] This invention is the first to construct a biohybrid of H. titaniumicae BH1 strain and MoS2 nanomaterial. H. titaniumicae BH1 (a moderately halophilic bacterium with an optimal salinity range of 2%-7%) can secrete tetrahydropyrimidine to cope with high salt stress and has excellent denitrification capacity. The nanosheet-like MoS2 is low in toxicity and has good electron transport performance, which can promote electron transport during denitrification. The nanomaterial MoS2 enhances the denitrification process of the strain. Its core mechanism lies in the excellent electron transport performance of MoS2, which can accelerate the electron transfer efficiency during denitrification and improve the activity of key enzymes. The combination of the two achieves a synergistic improvement in microbial salt tolerance and the electron transport performance of nanomaterials, resulting in high denitrification efficiency.

[0019] Compared with the prior art, the advantages and beneficial effects of the present invention are: 1. The H. titanicae BH1 / MoS2 biohybrid of this invention enhances the denitrification process by increasing the activity of key denitrification enzymes, promoting the secretion of extracellular secretions (EPS), and enhancing electron transport performance. It also exhibits superior high-salt adaptability compared to single strains within a salinity range of 2%-8%, with a nitrogen removal rate 4.26 times higher than that of single strains. This solves the problem of low nitrogen removal efficiency in high-salt environments, and has a wide salinity adaptability range (optimal 2%-7%), making it suitable for various high-salt nitrogen-containing wastewaters.

[0020] 2. The construction process of the H. titanicae BH1 / MoS2 biohybrid of the present invention is simple, requires a small amount of MoS2 (0.2mM), and has low processing cost.

[0021] In summary, the H. titanicae BH1 / MoS2 biohybrid and its application provided by this invention offer a novel technical solution for the treatment of high-salt nitrogen-containing wastewater, and have significant theoretical and practical application value. Attached Figure Description

[0022] Figure 1 The images show the SEM and EDS spectra of the hybrid in Example 1. Figure 2 Hybrids with different MoS2 concentrations for NO3 - The effect of -N removal rate; where (a) is the denitrification efficiency diagram, and (b) is the biological removal rate of nitrate under different hybrids for 3 h; Figure 3 The graph shows the denitrification efficiency of the hybrid in Example 1 and the single H. titanicae BH1 bacterial culture in Comparative Example 1 under different salinity conditions. Figure 4 NO3- content of the hybrid strain of Example 1 and the single H. titanicae BH1 bacterial culture of Comparative Example 1 under different salinity conditions - -N removal rate graph; Figure 5 To verify the denitrification performance of different biological hybrids. Detailed Implementation

[0023] The present invention will be further described in detail below through specific embodiments. The following embodiments are merely descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0024] The Halomonas titanicae BH1 strain used in this embodiment of the invention is from the American Type Culture Collection (ATCC), accession number ATCC BAA-1257, website: https: / / www.atcc.org.

[0025] MoS2 nanomaterials: MoS2 nanomaterials were prepared by hydrothermal synthesis. First, 1.96 g of thiourea and 1.064 g of ammonium molybdate tetrahydrate were added to 30 mL of ultrapure water, and the mixture was vigorously stirred at 300 r / min for 35 min to obtain a homogeneous solution. Then, the solution was transferred to a 100 mL Teflon-lined stainless steel high-pressure reactor and heated in an oven at 180 °C for 24 h. After cooling, the product was collected and washed several times alternately by centrifugation at 30000 r / min for 5 min, respectively. Finally, it was vacuum dried at 60 °C for 12 h to obtain MoS2 nanomaterials.

[0026] Pd nanoparticles were synthesized by reducing sodium tetrachloropalladium with sodium formate. A 50 mM phosphate buffer solution was dispensed into 100 mL serum bottles, and the system was aerated with nitrogen for 15 min to ensure an anaerobic environment. Then, 1 mM sodium formate and 50 mg / L sodium tetrachloropalladium were added, and the mixture was shaken and incubated for 24 h to obtain Pd nanoparticles. The precursor sodium tetrachloropalladium was purchased from Sigma-Aldrich (product code 205818-5G).

[0027] Cysteine ​​was purchased from Sigma, product code C5360-100G.

[0028] Example 1 A method for constructing a *H. titanicae* BH1 / MoS2 biohybrid, comprising the following steps: Activation of S1. H. titanicae BH1 strain Take the lyophilized powder of *H. titanicae* BH1, dissolve it in sterile physiological saline, and streak it on 3% salinity agar solid medium (NaCl 30.00 g / L, tryptone 10.00 g / L, yeast extract 5.00 g / L, agar 18.00 g / L, autoclaved at 121℃ for 20 min). Incubate in a constant temperature biochemical incubator at 30-37℃ for 16 h. Pick a single colony with uniform morphology from the plate and inoculate it into 250 mL of 3% salinity liquid medium. Incubate in a constant temperature shaker at 30-37℃ for 16 h to obtain the primary seed culture. Take 5 mL of the primary seed culture and transfer it to 500 mL of sterile denitrification expansion medium (CH3COONa 1.46 g / L, KNO3 0.35 g / L, MgSO4 0.05 g / L, NaCl 30.00 g / L, CaCl2 0.20 g / L). In a solution containing (g / L of K2HPO4, 0.022 g / L of K2HPO4, 1.50 g / L of tryptone, 0.50 g / L of yeast extract, 1 mL / L of trace element solution, and 1 mL / L of vitamin solution), cultured at 30-37℃ for 16 h to obtain the logarithmic growth phase bacterial culture (OD200). 600=1.0±0.1). The trace element solution composition includes: FeCl3·6H2O 1.50 g / L, MnCl2·4H2O 0.12 g / L, CuSO4·5H2O 0.03 g / L, H3BO3 0.15 g / L, KI 0.18 g / L, Na2MoO4 0.06 g / L, CoCl2·6H2O 0.15 g / L, ZnSO4·7H2O 0.12 g / L, EDTA 10.00 g / L; sterilized by autoclaving at 121℃ for 20 min. The vitamin solution consists of (100× concentrated mother liquor): thiamine 0.1 g / L, riboflavin 0.05 g / L, niacin 0.1 g / L, calcium pantothenate 0.1 g / L, pyridoxine 0.15 g / L, cyanocobalamin 0.01 g / L, biotin 0.05 g / L, folic acid 0.05 g / L, para-aminobenzoic acid 0.05 g / L, and lipoic acid 0.05 g / L.

[0029] S2. Pretreatment of bacterial culture Transfer the logarithmic-phase bacterial culture to centrifuge tubes, centrifuge at 4000 g for 5 min, and discard the supernatant. Resuspend the bacterial cells in PBS buffer (Na3PO4·12H2O 0.76 g / L, NaH2PO4·2H2O 0.62 g / L, KCl 0.0745 g / L, NaCl 0.5265 g / L, autoclaved at 121℃ for 20 min). Repeat the centrifugation-resuspending process three times. Finally, adjust the OD of the bacterial culture. 600 =0.5, reserved.

[0030] Construction of S3. H. titanicae BH1 / MoS2 hybrid Take 100 mL of pretreated logarithmic-phase bacterial culture and transfer it to a 250 mL serum bottle. Add MoS2 nanomaterials (dispersed by ultrasonication in ultrapure water for 30 min) to make the final MoS2 concentration 0.2 mM. Then add 1 mL of 0.1 wt% cysteine ​​solution (to protect bacterial activity). After sealing the serum bottle, place it in a constant temperature shaking incubator at 30-37℃ for 48 h. During this period, take samples every 12 h to observe the homogeneity of the system. Finally, obtain a homogeneous H. titanicae BH1 / MoS2 biohybrid.

[0031] Characterization of the biohybrid: A small sample of the hybrid was observed using a scanning electron microscope. MoS2 nanosheets were found uniformly attached to the surface of *H. titanicae* BH1 cells. EDS analysis showed the presence of Mo (18.50 wt%) and S (15.07 wt%) on the cell surface, confirming successful hybrid construction. SEM and EDS images are shown below. Figure 1 .

[0032] Viable cell count: The CCK8 method was used to calculate the viable cell count as 1.2 × 10⁻⁶. 8 cfu / mL.

[0033] Example 2 The only difference from Example 1 is that the final concentration of MoS2 is 0.1 mM.

[0034] Example 3 The only difference from Example 1 is that the final concentration of MoS2 is 0.3 mM.

[0035] Example 4 The only difference from Example 1 is that the final concentration of MoS2 is 0.5 mM.

[0036] Example 5 The only difference from Example 1 is that the final concentration of MoS2 is 0.7 mM.

[0037] Comparative Example 1 A single strain, H. titanicae BH1, was used.

[0038] Comparative Example 2 Only 0.2 mM MoS2 was used.

[0039] Comparative Example 3 A method for constructing a BH1&Pd hybrid involves taking 100 mL of pretreated logarithmic-phase bacterial culture, transferring it to a 250 mL serum bottle, adding Pd nanomaterials (dispersed ultrasonically in ultrapure water for 30 min) to achieve a final Pd nanoparticle concentration of 0.2 mM, then adding 1 mL of 0.1 wt% cysteine ​​solution (to protect bacterial activity), sealing the serum bottle, and culturing it in a constant-temperature shaking incubator at 30-37℃ for 48 h. Samples are taken every 12 h to observe the homogeneity of the system, ultimately obtaining a homogeneous H. titanicae BH1&Pd biohybrid.

[0040] Comparative Example 4 A method for constructing a BH1&Pd&MoS2 hybrid involves taking 100 mL of pretreated logarithmic-phase bacterial culture and transferring it to a 250 mL serum bottle. MoS2 nanomaterials (dispersed ultrasonically in ultrapure water for 30 min) are added to achieve a final MoS2 concentration of 0.1 mM. Then, Pd nanomaterials (dispersed ultrasonically in ultrapure water for 30 min) are added to achieve a final Pd nanoparticle concentration of 0.1 mM. Finally, 1 mL of 0.1 wt% cysteine ​​solution is added (to protect bacterial activity). The serum bottle is sealed and incubated in a 30-37 ℃ constant temperature shaking incubator for 48 h. Samples are taken every 12 h to observe the homogeneity of the system, ultimately obtaining a homogeneous BH1&Pd&MoS2 biohybrid.

[0041] Effect verification (1) Verification of the enhanced denitrification performance of H. titanicae BH1 / MoS2 hybrid Simulated high-salinity nitrogen-containing wastewater, formula: NaCl 30.00 g / L (salinity 3%), KNO3 0.72 g / L (NO3 - -N=100mg / L), CH3COONa 1.46 g / L, prepared with ultrapure water, and autoclaved at 121 ℃ for 20 min; The experimental group was divided into 5 groups, corresponding to Examples 1-5 respectively, with 3 replicates per group and a treatment volume of 250 mL per group, as detailed below: Experimental group 1: Simulated high-salt nitrogen-containing wastewater + 0.1 mM MoS2 hybrid (OD) 600 =0.5); Experimental Group 2: Simulated high-salt nitrogen-containing wastewater + 0.2 mM MoS2 hybrid (OD) 600 =0.5); Experimental Group 3: Simulated high-salt nitrogen-containing wastewater + 0.3 mM MoS2 hybrid (OD) 600 =0.5); Experimental Group 4: Simulated high-salt nitrogen-containing wastewater + 0.5 mM MoS2 hybrid (OD) 600 =0.5); Experimental Group 5: Simulated high-salt nitrogen-containing wastewater + 0.7 mM MoS2 hybrid (OD) 600 =0.5); Non-biological blank control group (comparative example 2): simulated high-salt nitrogen-containing wastewater, 0.2 mM MoS2, no bacterial strain; Biological control group (comparative example 1): simulated high-salt nitrogenous wastewater + single H. titanicae BH1 bacterial culture (OD) 600 =0.5); All groups were placed in a 37℃ constant temperature biochemical incubator and anaerobically cultured for 8 h. Samples were taken every 2 h to detect NO3. - -N concentration, NO2 - -N concentration, nitrate reductase (Nar) activity, and nitrite reductase (Nir) activity.

[0042] The denitrification performance of the materials in the examples and comparative examples is as follows: Figure 2 As shown in (a), it can be seen that after 8 h of cultivation, the NO3 levels in each group... --N removal rates were as follows: non-biological blank control group (0%) < biological control group (26.17%) < experimental group 5 (70.84%) < experimental group 4 (76.73%) < experimental group 3 (78.84%) < experimental group 1 (85.84%) < experimental group 2 (89.84%). The nitrate reduction rate of experimental group 2 (Example 1) was 4.26 times that of the control group, and the nitrate reduction rate was 3-5 times higher than that of the single H. titanicae BH1 strain.

[0043] Key enzyme activity The Nar activity in experimental group 2 was 0.14 pg / mL, which was 101.45% higher than that in the control group (0.07 pg / mL); the Nir activity was 0.15 pg / mL, which was 78.87% higher than that in the control group (0.08 pg / mL), proving that the hybrid enhances denitrification by increasing the activity of key enzymes.

[0044] (2) Verification of high-salt adaptability of H. titanicae BH1 / MoS2 hybrid The salinity gradient for simulating high-salinity nitrogen-containing wastewater was set at 2%, 3%, 5%, and 7% (based on NaCl concentration), corresponding to NaCl addition amounts of 20.00 g / L, 30.00 g / L, 50.00 g / L, and 70.00 g / L, respectively. The remaining components were: KNO3 0.72 g / L (NO3... - -N=100 mg / L), CH3COONa 1.46 g / L, prepared with ultrapure water, and autoclaved at 121℃ for 20 min; The experimental group consisted of 8 groups, with 3 replicates per group and a treatment volume of 250 mL per group. Control group: Simulated wastewater with different salinities + single H. titanicae BH1 bacterial culture (OD) 600 =0.5); Experimental group: Simulated wastewater with different salinities + 0.2 mM Example 1: MoS2 hybrid (OD) 600 =0.5); Treatment conditions: Anaerobic incubation at 30-37℃ for 4 hours; NO3 levels were measured after incubation. - -N removal rate and tetrahydropyrimidine content.

[0045] Test results are as follows Figure 3 and Figure 4 As shown, by Figure 2 It can be seen that NO3 in the control group --N removal rate decreased continuously with increasing salinity (48.67% at 2% salinity → 5.42% at 7% salinity); the removal rate in the experimental group showed a "first increase, then decrease" trend, and the denitrification performance of the *H. titanicae* BH1 / MoS2 hybrid was improved at different salinities. Figure 4 It can be seen that NO3 at a salinity of 3% - -N removal rate was the highest (89.34%), reaching 65.21% even at 7% salinity, both higher than the control group at the same time.

[0046] (3) Verification of denitrification performance of different biological hybrids Simulated wastewater: salinity 3% (NaCl 30.00 g / L), NO3 - -N concentration 100 mg / L (KNO3 0.72 g / L), carbon source CH3COONa 1.46 g / L, prepared with ultrapure water, and autoclaved at 121℃ for 20 min; The experiment was divided into 4 groups, with 3 replicates in each group, and a treatment volume of 250 mL. The specific groupings are as follows: Control group: Simulated wastewater + single bacterial strain (OD) 600 =0.5); Experimental Group 1: Simulated wastewater + BH1 & MoS2 hybrid (OD) 600 =0.5); Experimental Group 2: Simulated wastewater + BH1 & Pd hybrid (OD) 600 =0.5); Experimental Group 3: Simulated wastewater + BH1 & Pd & MoS2 hybrid (OD) 600 =0.5); Treatment conditions: Anaerobic culture at 30-37℃ for 10 hours, with NO3 samples taken every hour. - -N concentration, calculate NO3 - -N removal rate and relative removal rate (in C t / C0 means, C t For real-time NO3 - -N concentration, C0 is the initial NO3- concentration. - -N concentration).

[0047] A comparison of the denitrification performance of different biohybrids is shown in the figure below. Figure 5As shown, the denitrification efficiency of a single bacterium is relatively low, but it improves when the bacteria are combined with materials (such as MoS2 and Pd). The H. titanicae BH1 & MoS2 system exhibits the fastest denitrification rate and highest efficiency. MoS2 tends to slightly aggregate in high-salt bacterial solutions due to van der Waals forces, resulting in uneven local attachment, which gradually disperses with subsequent shaking culture. The bacterial survival rate is high, and the system's OD600 is stable without significant fluctuations. The nanomaterial MoS2 enhances the denitrification process of the strain; its core mechanism lies in the excellent electron transfer properties of MoS2, which accelerates electron transfer efficiency during denitrification and enhances the activity of key enzymes. Pd nanoparticles exhibit some toxicity to BH1 strain, leading to a decrease in strain activity. Pd nanoparticles have a spherical structure, high surface energy, and require long ultrasonic dispersion times, making them prone to agglomeration and resulting in low hybridization efficiency. Metallic Pd inhibits the denitrification performance of the BH1 strain, while the addition of MoS2 can restore and improve denitrification efficiency by adsorbing metal ions and mitigating its toxic inhibitory effect on strain metabolism. When Pd and MoS2 are mixed, electrostatic adsorption and aggregation easily occur due to the difference in surface charge between the two, resulting in poor material dispersion; poor synergy among multiple materials, uneven co-attachment, decreased cell survival rate, and poor system stability; the strengthening effect of composite nanomaterial (Pd+MoS2) on BH1 strain is lower than that of single material MoS2.

[0048] The BH1&MoS2 hybrid constructed in this invention exhibits the best denitrification rate and removal efficiency compared to other biohybrids. This is attributed to the excellent salt tolerance and denitrification capacity of the *H. titaniumicae* BH1 strain, combined with the highly efficient synergy of the electron transport properties of MoS2, further validating the advantages of this hybrid in the treatment of high-salt nitrogenous wastewater. MoS2 shows low cytotoxicity to the *H. titaniumicae* BH1 strain, and the hybridization exhibits relatively good uniformity, material dispersibility, and system stability. Furthermore, the construction cycle of the BH1&MoS2 hybrid is relatively short.

[0049] High denitrification efficiency: The nitrate reduction rate of the 0.2 mM MoS2 hybrid strain was 4.26 times higher than that of the single strain, and NO3... - -N removal rate reaches 89.84%; strong adaptability to high salt: stable operation within a salinity range of 2%-7%, tetrahydropyrimidine secretion increases by 20%-40%; clear mechanism: enhanced denitrification is achieved by strengthening electron transfer, improving enzyme activity, and promoting EPS secretion; environmentally friendly: MoS2 is low in toxicity, has no secondary pollution, and the treatment cost is lower than that of physicochemical methods.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several modifications and improvements can be made without departing from the inventive concept, and these all fall within the protection scope of the present invention.

Claims

1. A method for constructing a biological hybrid, characterized in that, Includes the following steps: S1. Activation culture of H. titanicae BH1 strain: Streak H. titanicae BH1 strain on agar solid medium containing 2-5% salinity and incubate at 30-37℃ for 14-18 h; pick a single colony and inoculate it into 250 mL of liquid medium containing 2-5% salinity, and incubate at 30-37℃ in a shaker for 14-18 h; take the above bacterial solution and inoculate it into sterile denitrification expansion medium, and continue to incubate at 30-37℃ for 14-18 h to obtain the logarithmic growth phase bacterial solution; S2. Pretreatment of bacterial culture: The logarithmic phase bacterial culture obtained in step S1 is centrifuged and resuspended with PBS buffer, and the OD value is adjusted to 0.5; S3. Hybrid construction: The logarithmic phase bacterial culture after pretreatment in step S2 was transferred to a serum bottle containing denitrification expansion medium. MoS2 nanomaterials with a final concentration of 0.1-0.7 mM and cysteine ​​solution with a mass concentration of 0.08-0.12 wt% were added. The mixture was then placed in a constant temperature shaking incubator at 30-37℃ and cultured for 40-50 h to obtain a homogeneous H. titanicae BH1 / MoS2 biohybrid.

2. The construction method according to claim 1, characterized in that, The denitrification expansion medium comprises: CH3COONa 1-2 g / L, KNO3 0.1-0.5 g / L, MgSO4 0.01-0.1 g / L, NaCl 20-40 g / L, CaCl2 0.1-0.5 g / L, K2HPO4 0.01-0.03 g / L, tryptone 1-2 g / L, yeast extract 0.1-0.8 g / L, trace element solution 0.8-1.2 mL / L, and vitamin solution 0.8-1.2 mL / L; and is autoclaved at 121℃ for 20 min.

3. The construction method according to claim 1, characterized in that, The PBS buffer solution comprises: Na3PO4·12H2O 0.5-0.8 g / L, NaH2PO4·2H2O 0.4-0.8 g / L, KCl 0.01-0.08 g / L, and NaCl 0.4-0.8 g / L, and is autoclaved at 121℃ for 20 min.

4. The construction method according to claim 1, characterized in that, The agar solid culture medium containing 2-5% salinity mentioned in step S1 comprises: NaCl 20-50 g / L, tryptone 5-15 g / L, yeast extract 1-10 g / L, and agar 15-20 g / L; the trace element solution mentioned in step S3 comprises: FeCl3·6H2O 1-2 g / L, MnCl2·4H2O 0.1-0.5 g / L, CuSO4·5H2O 0.01-0.05 g / L, H3BO3 0.1-0.3 g / L, KI 0.1-0.5 g / L, Na2MoO4 0.02-0.08 g / L, CoCl2·6H2O 0.1-0.3 g / L, ZnSO4·7H2O 0.1-0.4 g / L, and EDTA. 5-15g / L; autoclaved at 121℃ for 20min.

5. The construction method according to claim 1, characterized in that, The final concentration of MoS2 nanomaterials was 0.2 mM.

6. A biohybrid prepared by the construction method according to any one of claims 1-5.

7. The application of a bio-hybrid as described in claim 6 in enhanced denitrification in high-salt nitrogen-containing wastewater.

8. The application according to claim 7, characterized in that, The salinity of the high-salinity nitrogen-containing wastewater is 2%-7%, and the NO3 content is [missing information]. - -N concentration 50-200 mg / L.

9. The application according to claim 7, characterized in that, The application method includes the following steps: (1) Hybrid activation: Take the H. titanicae BH1 / MoS2 biohybrid and dilute it with denitrification medium to OD. 600 =0.5, 30-37℃, 150 rpm oscillation recovery for 20-40 min, to obtain the activated biological hybrid; (2) Wastewater treatment: The activated bio-hybrid was inoculated into high-salt nitrogen-containing wastewater at a volume ratio of 1:8-12 and treated at 30-37℃ under static anaerobic conditions for 4-8 h. The denitrification efficiency was then tested.

10. The application according to claim 9, characterized in that, The denitrification medium comprises: CH3COONa 1-2 g / L, KNO3 0.5-1 g / L, MgSO4 0.01-0.08 g / L, NaCl 20-40 g / L, CaCl2 0.1-0.3 g / L, K2HPO4 0.01-0.03 g / L, trace element solution 0.8-1.2 mL / L, and vitamin solution 0.8-1.2 mL / L.

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