Salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain for producing acyl homoserine lactone and application

Abstract

This invention discloses a heterotrophic nitrifying-aerobic denitrifying bacterial strain producing acyl-homoserine lactones and its applications. The strain belongs to the genus *Vibrio* (…). Vibrio The bacterium LV-Q1, with accession number CGMCC NO.28696, can grow in saline and saline (10–80 g NaCl / L) environments using ammonia and nitrate nitrogen as the sole or mixed nitrogen source. It can also utilize organic carbon sources for simultaneous nitrification and denitrification under a single aerobic condition, indicating that strain LV-Q1 possesses the ability to simultaneously remove nitrogen and carbon. Furthermore, during high-salt denitrification, this strain produces acylhomoserine lactones (AHLs) signaling molecules. Exogenous enhancement or inhibition of the secretion of these signaling molecules affects the growth, biofilm formation, and denitrification performance of strain LV-Q1. This is of great significance for regulating the biofilm formation and denitrification performance of functional bacteria and for the bioaugmented treatment of high-salt nitrogen-containing wastewater.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain that produces acyl homoserine lactone and its application. Background Technology

[0002] The discovery of heterotrophic nitrifying-aerobic denitrifying bacteria provides a biological basis for novel simultaneous nitrification-denitrification nitrogen removal processes. These strains can directly convert ammonia nitrogen into gaseous end products under aerobic conditions alone, overturning the traditional view that nitrification can only be completed by autotrophic bacteria and denitrification can only occur under anaerobic conditions. Therefore, understanding and studying these heterotrophic nitrifying-aerobic denitrifying strains has become an important research hotspot in environmental microbiology. Some researchers have isolated halophilic heterotrophic nitrifying-aerobic denitrifying bacteria from high-salt environments and used them to treat high-salt nitrogen-containing wastewater, which has enhanced the denitrification effect of reactors in high-salt environments (Bioresour. Technol., 2015, 179: 421-428; Bioresour. Technol., 2012, 113: 280-287). Therefore, strengthening the screening and cultivation of halophilic denitrifying bacteria and studying their application and mechanism in degrading pollutants under high-salt environments is particularly necessary for improving the treatment of high-salt wastewater.

[0003] Although current reports confirm the feasibility of inoculating halophilic heterotrophic nitrifying-aerobic denitrifying bacteria for bioaugmentation of high-salinity wastewater denitrification and have yielded a batch of halophilic nitrifying-denitrifying bacteria, many factors still limit the application of these bacteria in bioaugmentation technology. For example, biotic factors such as competition from indigenous microorganisms and predation by protozoa, as well as abiotic factors such as environmental temperature, pH, and pollutant concentration, all affect the activity and survival of the added bacteria, leading to common problems such as bacterial loss or difficulty in long-term colonization. Therefore, achieving colonization of the inoculated bacteria in polluted environments and enhancing their persistent effect in polluted environments is key to constructing bioaugmentation systems for stably improving the treatment effect of high-salinity wastewater.

[0004] Quorum sensing (QS) is a communication mechanism among microorganisms that coordinates population behavior and controls gene expression in response to changes in community density. Bacteria regulate various physiological behaviors through QS, including bioluminescence, antibiotic biosynthesis, bacterial motility, and biofilm development. QS is mediated by the synthesis, release, and sensing of signaling molecules. Different microorganisms possess signaling molecules with different structures and functions. Among them, acyl homoserine lactones (AHLs) are used by most Gram-negative bacteria for communication. Exogenous signaling molecules or inoculation of quorum-sensing bacteria play an important role in promoting colonization of inoculated bacteria and the formation of systemic biofilms (Sci. Total Environ., 2020, 735: 139449; Chem. Eng. J., 2016,302: 172-186; RSC Adv., 2018, 8(54): 30783-30793; J Hazard. Mater., 2022,437: 129277; Sci. Total Environ., 2022, 807: 150589).

[0005] Numerous studies have shown that AHLs and other signaling molecules are widely present in wastewater denitrifying bacteria such as nitrifying and denitrifying bacteria, and regulate biological denitrification genes such as nitrification and denitrification to varying degrees (Chemosphere, 2021, 274:129970). Meanwhile, the stability of these signaling molecules in the environment directly determines their effectiveness (Bioresour.Technol., 2014, 169C: 229-235. Sci. Pollut. R., 2013, 20(9): 6201-6209). Therefore, if we can screen and cultivate microorganisms (or groups) that can produce AHLs and also possess salt tolerance and even denitrification capabilities, and strengthen research on the effects of these signaling molecules on the colonization and biofilm formation of salt-tolerant denitrifying bacteria, this will help solve the problem of loss of highly efficient degrading bacteria in high-salt biologically enhanced denitrification systems and find methods to maintain the activity of inoculum bacteria for a prolonged period. Summary of the Invention

[0006] This invention overcomes the shortcomings of the prior art and provides a salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain that produces acyl homoserine lactone.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain that produces acyl homoserine lactone, classified and named as Vibrio ( ). VibrioThe bacterium LV-Q1 (sp.) was deposited on October 20, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; its accession number is CGMCC NO. 28696.

[0008] The screening method for the salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain LV-Q1, which produces acyl homoserine lactone, is as follows: (1) Sludge water samples were obtained from the aerobic section of a pharmaceutical wastewater, municipal sewage and coking wastewater treatment plant in Taiyuan, Shanxi Province. The samples were first enriched and cultured at 30℃ and 120rpm for 2 days.

[0009] (2) Then, a portion of the enriched culture medium was inoculated into a high-salt nitrification medium with a salinity of 50 g NaCl / L and an ammonia nitrogen concentration of 100 mg / L, and the salt-tolerant denitrifying bacteria were selectively cultured at 30 °C and 120 rpm for 2 days.

[0010] (3) The mixed bacterial solution from step (2) is subjected to 10 −1 -10 −9 Serial dilutions were performed, and the diluted solutions were spread onto solid nitrification medium (salinity 0) with an ammonia nitrogen concentration of 100 mg / L. The medium was then incubated in an incubator at 30°C for 3–5 days. Individual colonies of different morphologies were extracted and cultured, and the process was repeated three times to obtain more than 100 purified strains.

[0011] (4) More than 100 salt-tolerant denitrifying bacteria obtained in step (3) were initially screened using the biosensor method to obtain 9 strains of AHLs signal molecules. Then, these 9 strains of AHLs signal molecules were inoculated into a high-salt (30g NaCl / L) nitrification and denitrification medium. Taking into account the nitrification and denitrification performance and signal molecule production ability of each strain under high-salt conditions, LV-Q1 strain was selected as the target strain.

[0012] This strain exhibits the following phenotypic characteristics: colonies are milky white, round, raised, with regular edges, smooth spots, slightly raised cells, smooth surface, moist texture, and opaque. It is Gram-negative under a microscope.

[0013] The gene sequence of this strain is SEQ ID NO: 1, and the gene sequence characteristics are shown in the sequence listing. The base sequence length is 1469 bp.

[0014] Based on its morphology and analysis of the GenBank database on the NCBI website, BLAST homology analysis showed that strain LV-Q1 is closely related to the genus Vibrio and strains Vibrio fluvialis strain BBS 2087 and Vibrio fluvialis The strain EP27-13-11 showed a sequence homology of up to 99%, therefore strain LV-Q1 was identified as a Vibrio species. Vibrio sp.).

[0015] In addition, the present invention also provides the application of the above-mentioned salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain that produces acyl homoserine lactone in the treatment of saline and nitrogenous wastewater.

[0016] Furthermore, the wastewater salinity is calculated in terms of NaCl, and the salinity range is 10 g / L to 80 g / L. The Vibrio LV-Q1 bacteria described in this invention can perform aerobic heterotrophic nitrification denitrification using ammonia nitrogen as the sole nitrogen source under both salt-free and high-salt conditions (10 g NaCl / L–80 g NaCl / L). Moreover, when the salinity is not higher than 50 g NaCl / L, strain LV-Q1 achieves optimal ammonia nitrogen removal performance within 24 hours. Therefore, a salinity of 50 g NaCl / L is selected as the preferred salinity for strain LV-Q1.

[0017] As mentioned above, the Vibrio LV-Q1 bacteria can grow using ammonia nitrogen as the sole nitrogen source under a salinity of 50 g NaCl / L and can utilize organic carbon sources for aerobic nitrification and denitrification, indicating that strain LV3 has heterotrophic nitrification capabilities for simultaneous denitrification and carbon removal.

[0018] As mentioned above, the Vibrio LV-Q1 bacteria can grow using nitrate nitrogen as the sole nitrogen source under a salinity of 50 g NaCl / L, and can also utilize organic carbon sources for aerobic denitrification, indicating that strain LV3 has the ability to simultaneously remove nitrogen and carbon.

[0019] The above wastewater formulation uses 4.28 g / L sodium pyruvate as the sole carbon source, 0.47 g / L ammonium sulfate or 0.61 g / L sodium nitrate as the sole nitrogen source, NaCl concentration of 50 g / L, and the remaining components are KH2PO4 0.4 g / L, MnSO4•H2O 0.01 g / L, MgSO4•7H2O 0.05 g / L, FeSO4•7H2O 0.01 g / L, pH 7.0.

[0020] As described above, the Vibrio LV-Q1 bacterium can grow using ammonia nitrogen and nitrate nitrogen as a mixed nitrogen source under a salinity of 50 g NaCl / L, and can simultaneously nitrify and denitrify using an organic carbon source under a single aerobic condition, indicating that strain LV3 possesses simultaneous nitrification and denitrification capabilities. The wastewater formulation uses 4.28 g / L sodium pyruvate as the sole carbon source, 0.24 g / L ammonium sulfate or 0.31 g / L sodium nitrate as a mixed nitrogen source, NaCl concentration of 50 g / L, and the remaining components are KH2PO4 0.4 g / L, MnSO4•H2O 0.01 g / L, MgSO4•7H2O 0.05 g / L, FeSO4•7H2O 0.01 g / L, pH 7.0.

[0021] As described above, the Vibrio LV-Q1 bacterium exhibits secretion of AHL-like signaling molecules, particularly during its logarithmic growth phase, during high-salt denitrification at a salinity of 50 g NaCl / L. Within the scope of signaling molecule research, strain LV-Q1 can secrete C8-HSL, C10-HSL, 3OC6-HSL, 3OC8-HSL, 3OC10-HSL, and 3OC12-HSL, with 3OC10-HSL and C10-HSL being the predominant signaling molecules. The wastewater formulation uses 3.67 g / L sodium pyruvate as the sole carbon source and 0.47 g / L ammonium sulfate as the sole nitrogen source, with a NaCl concentration of 50 g / L. The remaining components are KH2PO4 0.4 g / L, MnSO4•H2O 0.01 g / L, MgSO4•7H2O 0.05 g / L, and FeSO4•7H2O 0.01 g / L, with a pH of 7.0.

[0022] The present invention also provides the application of the above-mentioned salt-tolerant heterotrophic nitrifying-aerobic denitrifying strains that produce acyl homoserine lactones in regulating biofilm denitrification.

[0023] As described above, Vibrio LV-Q1 bacteria can be promoted or inhibited in high-salt denitrification processes by exogenously applying certain concentrations of signaling molecules (C10-HSL and 3OC10-HSL) or the inhibitor vanillin. The concentration range of the signaling molecules C10-HSL or 3OC10-HSL is 10–200 nmol / L; or the concentration range of the signaling molecule inhibitor vanillin is 10–500 mg / L.

[0024] Exogenous application of certain concentrations of the signaling molecules C10-HSL or 3OC10-HSL enhances their denitrification and biofilm formation performance under high-salt conditions, with exogenous application of 3OC10-HSL showing a more significant promoting effect. The culture medium formulation is as follows: the wastewater formulation uses 3.67 g / L sodium pyruvate as the sole carbon source and 0.47 g / L as the sole nitrogen source, with a NaCl concentration of 50 g / L, and the concentration range of C10-HSL or 3OC10-HSL is 10–200 nmol / L. The remaining components are KH2PO4 0.4 g / L, MnSO4•H2O 0.01 g / L, MgSO4•7H2O 0.05 g / L, FeSO4•7H2O 0.01 g / L, pH 7.0.

[0025] The signaling molecule inhibitor vanillin was applied externally at a certain concentration to inhibit its denitrification and biofilm formation performance under high-salt conditions. The culture medium formulation was as follows: the wastewater formulation used 3.67 g / L sodium pyruvate as the sole carbon source and 0.47 g / L as the sole nitrogen source, with a NaCl concentration of 50 g / L, a vanillin concentration of 10–500 mg / L, and the remaining components being KH2PO4 0.4 g / L, MnSO4•H2O 0.01 g / L, MgSO4•7H2O 0.05 g / L, FeSO4•7H2O 0.01 g / L, and a pH of 7.0.

[0026] Compared with the prior art, the present invention has the following beneficial effects: The strain of this invention can grow in saline and salt-free environments using ammonia nitrogen and nitrate nitrogen as the sole or mixed nitrogen source, and exhibits good simultaneous nitrification and denitrification denitrification effects. Simultaneously, during high-salt denitrification, this strain produces acylhomoserine lactone (AHL) signaling molecules. Exogenous enhancement or inhibition of the secretion of these signaling molecules affects the strain's growth, biofilm formation, and denitrification performance, providing a valuable bacterial resource for the bioaugmented treatment of high-salt nitrogen-containing wastewater. This is of great significance for regulating the biofilm formation and denitrification performance of functional bacteria and for the bioaugmented treatment of high-salt nitrogen-containing wastewater.

[0027] This invention is suitable for denitrification of biofilms and treatment of saline and nitrogenous wastewater. It has broad application prospects and good practical application value and social benefits. Attached Figure Description

[0028] Figure 1 This indicates the nitrification performance of different strains under high-salt conditions with ammonia nitrogen as the sole nitrogen source.

[0029] Figure 2 This indicates the denitrification performance of different strains under high-salt conditions with nitrate nitrogen as the sole nitrogen source.

[0030] Figure 3This indicates the signal molecule secretion performance of four different salt-tolerant nitrifying and denitrifying strains.

[0031] Figure 4 This indicates the colony morphology of strain LV-Q1.

[0032] Figure 5 This represents the phylogenetic tree of strain LV-Q1.

[0033] Figure 6 This indicates the effect of different salinities on the growth of strain LV-Q1.

[0034] Figure 7 This indicates the effect of different salinities on the nitrification performance of strain LV-Q1.

[0035] Figure 8 This indicates the aerobic heterotrophic nitrification performance of strain LV-Q1 under high-salt conditions with ammonia nitrogen as the sole nitrogen source.

[0036] Figure 9 This indicates the aerobic denitrification performance of strain LV-Q1 under high-salt conditions with nitrate nitrogen as the sole nitrogen source.

[0037] Figure 10 This indicates the simultaneous nitrification and denitrification performance of strain LV-Q1 under high-salt conditions with ammonia nitrogen and nitrate nitrogen as mixed nitrogen sources.

[0038] Figure 11 This indicates the growth and ammonia nitrogen degradation of exogenous strain LV-Q1 during high-salt denitrification.

[0039] Figure 12 This indicates the secretion of AHL-type signaling molecules by exogenous strain LV-Q1 during high-salt denitrification.

[0040] Figure 13 This indicates the effect of the exogenous signaling molecule C10-HSL on the high-salt denitrification performance of strain LV-Q1.

[0041] Figure 14 This indicates the effect of the exogenous signaling molecule 3OC10-HSL on the high-salt denitrification performance of strain LV-Q1.

[0042] Figure 15 This indicates the effect of the exogenous signaling molecule inhibitor vanillin on the high-salt denitrification performance of strain LV-Q1.

[0043] Figure 16 This indicates the effect of exogenous signaling molecules C10-HSL and 3OC10-HSL on biofilm formation performance during high-salt denitrification of strain LV-Q1.

[0044] Figure 17 This indicates the effect of the exogenous signaling molecule inhibitor vanillin on the biofilm formation performance of strain LV-Q1 during high-salt denitrification. Detailed Implementation

[0045] The present invention will be further described below with reference to specific embodiments.

[0046] Unless otherwise specified, all methods described in the following examples are conventional. In these examples, nitrate nitrogen was determined using the salicylic acid colorimetric method, ammonia nitrogen using Nessler's reagent spectrophotometry, nitrite nitrogen using the N-(1-naphthyl)-ethylenediamine spectrophotometric method, total nitrogen using the alkaline potassium persulfate ultraviolet spectrophotometric method, COD using the potassium dichromate method, TOC using the combustion oxidation-nondispersive infrared absorption method, dissolved oxygen using a portable dissolved oxygen meter (HQ30D, HACH), pH using a pH meter (Seven2Go pro, Mettler Toledo, Switzerland), and OD... 600 Measurements were taken at a wavelength of 600 nm using a visible spectrophotometer. pH was adjusted using 2 mol / L hydrochloric acid and 2 mol / L NaOH. The biosensor method used in this example primarily employed… Agrobacterium tumefaciens A136 and Agrobacterium tumefaciens Preliminary detection of signal molecule production types in quorum sensing reporter strains such as KYC55 was conducted. Signal molecules in the supernatant were extracted using organic solvents and a solid-phase extraction column. Combined with standard quorum sensing signal molecules, LC / MS was used to further characterize the signal molecule types in the crude extract of halophilic bacteria. In this example, biofilm formation was primarily measured using crystal violet staining at OD0.05. 570 Measurement. All units used in the examples conform to national standards. Example 1

[0047] The screening of salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain LV-Q1, which produces acyl homoserine lactone, is as follows: (1) Sludge water samples were obtained from the aerobic stage of a pharmaceutical wastewater treatment plant, municipal sewage, and coking wastewater treatment plant in Taiyuan, Shanxi Province. First, 10 mL of the water sample was placed in a 250 mL Erlenmeyer flask containing 90 mL of enrichment medium. The enrichment medium consisted of 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl, with a pH of 7.0. The flask was then sealed with a sterile breathing membrane and placed in a shaker at 30 °C and 120 rpm to enrich the bacterial suspension for 2 days.

[0048] (2) Take 1 mL of cell suspension from the enrichment medium and transfer it into 100 mL of sterile high-salt nitrification medium with a salinity of 50 g NaCl / L and an initial ammonia nitrogen concentration of 100 mg / L. The high-salt nitrification medium formula is: sodium pyruvate 4.28 g / L, (NH4)2SO4 0.47 g / L, K2HPO4·3H2O 0.75 g / L, NaH2PO4·2H2O 0.25 g / L, MgSO4·7H2O 0.05 g / L, NaCl 50 g / L, MnSO4·4H2O 0.01 g / L, FeSO4·7H2O 0.01 g / L, pH=7.0. Selectively culture salt-tolerant nitrifying and denitrifying bacteria at 30℃ and 120 rpm for 2 days. This acclimatization process needs to be repeated 3 times.

[0049] (3) The mixed bacterial solution from step (2) is subjected to 10 −1 -10 −9 Gradient dilution, take 10 −5 10 −6 and 10 −7 100 μL of serially diluted solution was added to solid nitrification medium with an initial ammonia nitrogen concentration of 100 mg / L. The solid nitrification medium formulation consisted of sodium pyruvate 4.28 g / L, (NH4)2SO4 0.47 g / L, K2HPO4·3H2O 0.75 g / L, NaH2PO4·2H2O 0.25 g / L, MgSO4·7H2O 0.05 g / L, MnSO4·4H2O 0.01 g / L, and FeSO4·7H2O 0.01 g / L. The pH of the medium was adjusted to 7.0 with NaOH or HCl, and then 20 g / L of agar powder was added. The solid plates were then incubated at 30°C for 3–5 days. Single colonies of different morphologies were extracted and cultured, and this process was repeated three times to obtain 100 salt-tolerant denitrifying bacteria.

[0050] (4) The 100 purified salt-tolerant denitrifying bacteria strains were first tested using a biosensor method, with the reporter bacteria themselves as a negative control. The 100 salt-tolerant denitrifying bacteria strains were then compared with the quorum sensing reporter strains. Agrobacterium tumefaciens A136 and Agrobacterium tumefaciens KYC55 was used for parallel streaking, and finally, based on the report of plate color development results, nine strains of AHLs signal molecule producing bacteria were preliminarily screened.

[0051] (5) The nine AHLs-type signal molecule producing bacteria from step (4) were inoculated into 100 mL of nitrification and denitrification medium with a salinity of 30 g NaCl / L, respectively. The nitrification medium consisted of 4.28 g / L sodium pyruvate, 0.47 g / L (NH4)2SO4, 0.75 g / L K2HPO4·3H2O, 0.25 g / L NaH2PO4·2H2O, 0.05 g / L MgSO4·7H2O, 0.01 g / L MnSO4·4H2O, and 0.01 g / L FeSO4·7H2O, with a pH of 7.0. The denitrification medium consisted of 4.28 g / L sodium pyruvate, 0.61 g / L NaNO3, 0.75 g / L K2HPO4·3H2O, and 0.01 g / L NaH2PO4·2H2O. The bacterial strain was cultured at 0.25 g / L, MgSO4·7H2O 0.05 g / L, MnSO4·4H2O 0.01 g / L, and FeSO4·7H2O 0.01 g / L, pH=7.0, in a shaker at 30℃ and 120 rpm. The growth and nitrification / denitrification performance of the strain were periodically monitored. The experimental results are shown in the appendix. Figure 1 and 2 Under high-salinity conditions (30 g NaCl / L), the ammonia nitrogen removal rates of the nine strains within 24 hours were 98.29%, 98.26%, 98.42%, 97.86%, 30.22%, 23.56%, 24.33%, 28.69%, and 6.22%, respectively, while the nitrate nitrogen removal rates were 58.29%, 55.26%, 55.42%, 49.86%, 18.31%, 19.35%, 17.62%, 17.89%, and 4.68%, respectively. These results indicate that within the study range, the four signal molecule-producing strains ZY1, ZY2, Q1, and YN2 exhibited good salt tolerance for nitrification and denitrification.

[0052] (6) Finally, LC / MS was used to further characterize the signal molecule types in the crude extracts of the four salt-tolerant bacteria ZY1, ZY2, Q1, and YN2 from step (5). The experimental results are shown in the appendix. Figure 3 Within the scope of this study, strains ZY1, ZY2, Q1, and YN2 can all produce acyl-homoserine lactones. Among them, strain Q1 exhibits the highest signal molecule production capacity, primarily producing two signal molecules, C10-HSL and 3-OC10-HSL, during high-salt nitrification. Considering the nitrification and denitrification performance, as well as the signal molecule production capacity, of strains ZY1, ZY2, Q1, and YN2 under high-salt conditions, strain Q1 was selected as the target strain and named LV-Q1. Example 2

[0053] The identification of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain LV-Q1, which produces acyl homoserine lactone, is as follows: Strain strain LV-Q1 was inoculated onto a solid agar high-salt nitrification medium. The solid high-salt nitrification medium formulation was: sodium pyruvate 4.28 g / L, (NH4)2SO4 0.47 g / L, K2HPO4·3H2O 0.75 g / L, NaH2PO4·2H2O 0.25 g / L, MgSO4·7H2O 0.05 g / L, NaCl 50 g / L, MnSO4·4H2O 0.01 g / L, FeSO4·7H2O 0.01 g / L. The pH of the medium was adjusted to 7.0 with NaOH or HCl, and then 20 g / L of agar powder was added. The culture was incubated at 30℃ for 3–5 days. The colony morphology of strain LV3 is shown below. Figure 4 As shown, the colonies are milky white, round, raised, with neat edges, smooth spots, slightly raised bodies, smooth surfaces, moist texture, opaque, and regular edges.

[0054] Gram staining of strain LV-Q1 revealed that the stained cells turned red, indicating that strain LV-Q1 is a Gram-negative bacterium.

[0055] The 16S rDNA sequencing base sequence of strain LV-Q1 is shown in the attached sequence listing. The sequence was submitted to the GenBank database on the NCBI website for analysis. Blast homology analysis showed that ( Figure 5 Strain LV-Q1 is closely related to the genus Vibrio and is also related to strains Vibrio fluvialis strain BBS 2087 and Vibrio fluvialis The strain EP27-13-11 showed a sequence homology of up to 99%, therefore strain LV-Q1 was identified as a Vibrio species. Vibrio This strain was deposited on October 20, 2023, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the collection center registration number CGMCC NO. 28696. Example 3

[0056] The growth and aerobic nitrification experiments of strain LV-Q1 under different salinities are as follows: The working solution was prepared by extracting purified strain LV-Q1 from preserved strains and activating it in a 250 mL Erlenmeyer flask containing 100 mL of high-salt nitrification medium (same as in Example 1). The flask was then cultured at 30°C and 120 rpm in a constant-temperature shaker until the logarithmic growth phase (OD50) of the strain. 600 ≈1.3).

[0057] Using sodium pyruvate and ammonia nitrogen as the sole carbon and nitrogen sources, respectively, nitrification media with NaCl concentrations of 0, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, and 80 g / L were prepared (same as in Example 1), corresponding to salinities of 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%, respectively. 1 mL of the working solution was inoculated into 100 mL of the above-mentioned media, sealed with sealing film, and placed on a shaker at 30°C and 120 rpm to study the growth and ammonia nitrogen degradation of strain LV-Q1 under different salinities. Figure 6 As shown, when the salinity is not higher than 5%, strain LV-Q1 almost does not have a lag phase and enters the rapid growth logarithmic phase. Figure 6 However, as salinity continues to rise, the lag phase of strain LV-Q1 prolongs with increasing salinity, during which almost no ammonia nitrogen is degraded. Once strain LV-Q1 enters the rapid growth logarithmic phase, the ammonia nitrogen content decreases rapidly. Figure 7 The results indicate that the degradation of ammonia nitrogen by strain LV-Q1 is closely related to its growth. In summary, strain LV-Q1 can perform aerobic nitrification using ammonia nitrogen as the sole nitrogen source under both salt-free and high-salt conditions (10 g NaCl / L–80 g NaCl / L). Furthermore, when the salinity is not higher than 50 g NaCl / L, strain LV-Q1 achieves optimal ammonia nitrogen removal performance within 24 hours, with a removal rate exceeding 98%. Therefore, a salinity of 50 g NaCl / L is selected as the preferred salinity for strain LV-Q1. Example 4

[0058] The heterotrophic nitrification experiment of strain LV-Q1 under high-salt conditions with ammonia nitrogen as the sole nitrogen source is as follows: The experiment was conducted in a nitrification medium (same as in Example 1) with sodium pyruvate and ammonia nitrogen as the sole carbon and nitrogen sources, respectively, a carbon-to-nitrogen ratio of 16, an ammonia nitrogen concentration of 100 mg / L, and a salinity of 50 g NaCl / L. 1 mL of the working solution (same as in Example 3) was inoculated into 100 mL of the above medium, sealed with sealing film, and placed on a shaker at 30°C and 120 rpm to study the aerobic nitrification performance of strain LV-Q1 under high-salt conditions with ammonia nitrogen as the sole nitrogen source. Figure 8 As shown, under high-salt conditions with ammonia nitrogen as the sole nitrogen source, strain LV-Q1 exhibited almost no lag phase during growth, with an OD value of 6 h. 600The value reached 0.12, at which point the ammonia nitrogen concentration decreased from the initial 97.09 mg / L to 91.76 mg / L after 6 hours. Subsequently, from 6 to 24 hours, strain LV-Q1 entered the logarithmic growth phase, during which the ammonia nitrogen content showed a rapid decreasing trend, indicating that ammonia nitrogen removal was closely related to the growth of strain LV-Q1. The removal rates of ammonia nitrogen and total nitrogen at 24 hours reached 97.97% and 97.72%, respectively, with a remaining total nitrogen concentration of 2.23 mg / L. Subsequently, the growth of strain LV-Q1 entered the decline phase, and the OD... 600 The values ​​showed a decreasing trend, while the total nitrogen and ammonia nitrogen contents in the solution increased slightly, possibly due to the release of ammonia nitrogen caused by the death of the strain. Nitrate nitrogen accumulation was detected throughout the experiment, initially increasing with the growth of strain LV-Q1, reaching its maximum at 12 hours (16.25 mg / L). Subsequently, the nitrate nitrogen content rapidly decreased, stabilizing at around 2.23 mg / L at 24 hours. It is speculated that strain LV-Q1 produces nitrate nitrogen during nitrification, and this intermediate nitrate nitrogen undergoes aerobic denitrification to convert ammonia nitrogen into a gaseous nitrogen source (nitrogen generation was detected in later gas chromatography). TOC removal and total nitrogen removal occurred simultaneously, with maximum removal rates of 70.00% and 97.72%, respectively, indicating that strain LV-Q1 can utilize organic carbon sources for heterotrophic nitrification to remove ammonia nitrogen. Example 5

[0059] The following is an aerobic denitrification experiment of strain LV-Q1 under high-salt conditions with nitrate nitrogen as the sole nitrogen source: The experiment was conducted in a denitrification medium (same as in Example 1) with sodium pyruvate and nitrate nitrogen as the sole carbon and nitrogen sources, respectively, a carbon-to-nitrogen ratio of 16, a nitrate nitrogen concentration of 100 mg / L, and a salinity of 50 g NaCl / L. 1 mL of working solution (same as in Example 3) was inoculated into 100 mL of the above medium, sealed with sealing film, and placed on a shaker at 30°C and 120 rpm to study the aerobic denitrification performance of strain LV-Q1 under high-salt conditions with nitrate nitrogen as the sole nitrogen source. Figure 9 As shown, under high-salt conditions with sodium nitrate as the sole nitrogen source, the lag phase of strain LV-Q1 was prolonged compared to when ammonia nitrogen was the sole nitrogen source, and the OD at 12 h was [not specified]. 600 The value only reached 0.13, at which point the nitrate nitrogen concentration decreased from an initial 101.34 mg / L to 93.92 mg / L after 12 hours. Subsequently, from 12 to 36 hours, strain LV-Q1 entered the logarithmic growth phase, during which the nitrate nitrogen content showed a rapid decreasing trend, indicating that nitrate nitrogen removal was closely related to the growth of strain LV-Q1. The removal rates of nitrate nitrogen and total nitrogen reached 100% and 87.65% at 36 hours, respectively, with a remaining total nitrogen concentration of 13.18 mg / L. Subsequently, the growth of strain LV-Q1 entered the decline phase, and the OD...600 The values ​​showed a decreasing trend, while the ammonia nitrogen and total nitrogen contents in the solution increased slightly, possibly due to the release of ammonia nitrogen caused by the death of the strain. During the nitrate nitrogen removal process, strain LV-Q1 detected the accumulation of nitrite nitrogen, an intermediate denitrification product. The accumulation of nitrite nitrogen reached its maximum at 24 hours, with a maximum content of 6.70 mg / L, indicating that strain LV-Q1 can effectively remove nitrate nitrogen through aerobic denitrification using nitrite nitrogen as the sole nitrogen source. TOC removal occurred simultaneously with nitrate nitrogen removal, with maximum removal rates of 57.01% and 87.65% for TOC and total nitrogen, respectively, indicating that strain LV-Q1 can simultaneously perform nitrogen and carbon removal. Example 6

[0060] The simultaneous nitrification and denitrification experiment of strain LV-Q1 under high-salt conditions using ammonia nitrogen and nitrate nitrogen as a mixed nitrogen source is as follows: The experiment was conducted in a simultaneous nitrification and denitrification medium with ammonia nitrogen and nitrate nitrogen as mixed nitrogen sources, sodium pyruvate as the sole carbon source, a carbon-to-nitrogen ratio of 1:6, an ammonia nitrogen concentration of 50 mg / L, a nitrate nitrogen concentration of 50 mg / L, and a salinity of 50 g NaCl / L. The formulation of the simultaneous nitrification and denitrification medium was as follows: sodium pyruvate 4.28 g / L, (NH4)2SO4 0.24 g / L, NaNO3 0.31 g / L, K2HPO4·3H2O 0.75 g / L, NaH2PO4·2H2O 0.25 g / L, MgSO4·7H2O 0.05 g / L, NaCl 50 g / L, MnSO4·4H2O 0.01 g / L, FeSO4·7H2O 0.01 g / L, pH=7.0. Take 1 mL of working solution (same as in Example 3) and inoculate it into 100 mL of the above-mentioned culture medium. Seal the container with sealing film and place it on a shaker at 30 °C and 120 rpm to study the simultaneous nitrification and denitrification performance of strain LV-Q1 under high-salt conditions with ammonia nitrogen and nitrate nitrogen as mixed nitrogen sources. Figure 10 As shown, under high-salt conditions with ammonia nitrogen and nitrate nitrogen as a mixed nitrogen source, the lag phase of strain LV-Q1 was shorter than that with nitrate nitrogen as the sole nitrogen source, and the OD at 6 h was [not specified]. 600The value reached 0.13, at which point the concentrations of ammonia nitrogen and nitrate nitrogen decreased slightly. Subsequently, strain LV-Q1 entered the logarithmic growth phase, reaching a maximum bacterial density of 1.64 at 24 hours. During the logarithmic phase, the concentrations of ammonia nitrogen and nitrate nitrogen decreased rapidly, reaching their lowest levels at 24 hours and 18 hours, respectively, corresponding to maximum removal rates of 96.04% and 100%. This indicates that strain LV-Q1 can utilize ammonia nitrogen and nitrate nitrogen for growth under high-salt conditions, and the addition of ammonia nitrogen promotes the removal of nitrate nitrogen, demonstrating simultaneous nitrification and denitrification capabilities. TOC removal and total nitrogen removal occurred simultaneously, with maximum removal rates of 64.72% and 91.90%, respectively. Almost no intermediate products, nitrite nitrogen and hydroxylamine nitrogen, accumulated throughout the experiment. These results further indicate that strain LV-Q1 can utilize organic carbon sources for simultaneous nitrification and denitrification under a single aerobic condition, demonstrating that strain LV3 possesses simultaneous nitrification and denitrification capabilities under high-salt conditions. Example 7

[0061] The following is an experiment on the secretion of AHL-type signaling molecules by strain LV-Q1 during high-salt denitrification: The experiment was conducted in a nitrification medium with sodium pyruvate and ammonia nitrogen as the sole carbon and nitrogen sources, respectively, a carbon-to-nitrogen ratio of 1:2, an ammonia nitrogen concentration of 100 mg / L, and a salinity of 50 g NaCl / L. The nitrification medium formula was as follows: sodium pyruvate 3.67 g / L, (NH4)2SO4 0.47 g / L, K2HPO4·3H2O 0.75 g / L, NaH2PO4·2H2O 0.25 g / L, MgSO4·7H2O 0.05 g / L, NaCl 50 g / L, MnSO4·4H2O 0.01 g / L, FeSO4·7H2O 0.01 g / L, pH=7.0. Take 1 mL of working solution (same as in Example 3) and inoculate it into 100 mL of the above culture medium. Seal the container with sealing film and place it on a shaker at 30 °C and 120 rpm to study the AHLs of strain LV-Q1 during high-salt denitrification. The secretion of signaling molecules. For example... Figure 11 As shown, under a salinity of 50 g NaCl / L, the degradation of ammonia nitrogen by strain LV-Q1 was closely related to its growth: strain LV-Q1 exhibited almost no lag phase, reaching a bacterial density of 0.16 after 6 hours, at which point the ammonia nitrogen concentration decreased from the initial 96.92 mg / L to 92.11 mg / L. It then entered the logarithmic growth phase, reaching its maximum bacterial density of 1.07 at 24 hours; simultaneously, the ammonia nitrogen concentration rapidly decreased, reaching its lowest point of 1.48 mg / L at 24 hours, at which point the ammonia nitrogen removal rate was highest at 98.47%. Afterward, the bacterial density gradually decreased, and strain LV-Q1 entered its growth decline phase. Figure 12As shown, the signal molecule secretion capacity of strain LV-Q1 during high-salt denitrification increases with the growth of strain LV-Q1. The secretion capacity gradually increases during the 6-24 h logarithmic growth phase, and significantly decreases once strain LV-Q1 enters its decline phase. This indicates that strain LV-Q1 has the strongest AHLs secretion capacity during the logarithmic growth phase. In summary, strain LV-Q1 secretes AHLs-like signal molecules during high-salt denitrification, especially during the logarithmic growth phase. Within the scope of signal molecule research, the strain can secrete C8-HSL, C10-HSL, 3OC6-HSL, 3OC8-HSL, 3OC10-HSL, and 3OC12-HSL, with 3OC10-HSL and C10-HSL being the predominantly secreted signal molecules. Example 8

[0062] The effects of exogenous signaling molecules and their inhibitors on the high-salt denitrification performance of strain LV-Q1 were investigated as follows: The experiment was conducted in a nitrification medium (same as in Example 6) with sodium pyruvate and ammonia nitrogen as the sole carbon and nitrogen sources, respectively, a carbon-to-nitrogen ratio of 1:2, an ammonia nitrogen concentration of 100 mg / L, and a salinity of 50 g NaCl / L. 1 mL of working solution (same as in Example 3) was inoculated into 100 mL of the above medium, sealed with sealing film, and placed on a shaker at 30°C and 120 rpm to study the effects of exogenously applied signaling molecules C10-HSL and 3OC10-HSL on the high-salt denitrification and biofilm formation performance of strain LV-Q1. Figure 13 As shown, within the study range, external application of C10-HSL at concentrations ranging from 50 to 200 nmol / L promoted the high-salt nitrogen removal performance of strain LV-Q1. The most significant promoting effect was observed at a C10-HSL concentration of 50 nmol / L (P<0.05), at which point the ammonia nitrogen removal rate reached 89.95% (after 18 hours of cultivation), which was 4.41% higher than that of the control group. Similarly, as... Figure 14 As shown, within the study range, the application of 3OC10-HSL at concentrations ranging from 10 to 200 nmol / L promoted the high-salt denitrification performance of strain LV-Q1. The most significant promoting effect was observed at a 3OC10-HSL concentration of 100 nmol / L (P<0.05), at which point the ammonia nitrogen removal rate reached 92.73% (after 18 h of culture), exceeding the control group's ammonia nitrogen removal rate by 6.47%. These results indicate that the application of a certain concentration of signaling molecules can promote the denitrification performance of strain LV-Q1 under high-salt conditions. Vanillin strongly inhibits the secretion of AHL-type signaling molecules; therefore, this patent investigates its effect on the high-salt denitrification performance of strain LV-Q1 by applying the inhibitor vanillin. Figure 15As shown, within the study range, the concentration of vanillin applied externally at 50 mg / L had almost no effect on the growth of the strain and the ammonia nitrogen removal performance (P>0.05). However, when the vanillin concentration ranged from 100 to 500 mg / L, the growth of strain LV-Q1 and the ammonia nitrogen removal performance decreased significantly with the increase of the inhibitor concentration (P<0.05). This indicates that the application of inhibitors can significantly inhibit the growth of strain LV-Q1 and its high-salt denitrification performance. Example 9

[0063] The effects of exogenous signaling molecules and their inhibitors on the biofilm formation performance of strain LV-Q1 were investigated as follows: The experiment was conducted in a nitrification medium (same as in Example 6) with sodium pyruvate and ammonia nitrogen as the sole carbon and nitrogen sources, respectively, a carbon-to-nitrogen ratio of 1:2, an ammonia nitrogen concentration of 100 mg / L, and a salinity of 50 g NaCl / L. 1 mL of the working solution (same as in Example 3) was inoculated into 100 mL of the above medium, sealed with sealing film, and incubated overnight for 12 h at 30°C and 120 rpm on a shaker. The overnight cultured LV-Q1 bacterial suspension was adjusted to an initial bacterial density of OD600 ≈ 1.00 using nitrified medium (same as in Example 6). 1 mL of the bacterial suspension was added to 100 mL of nitrified medium (same as in Example 6) containing different concentrations of signaling molecules (C10-HSL and 3OC10-HSL) or the inhibitor vanillin for a second dilution. The diluted bacterial suspension was then inoculated into 12-well cell culture plates (BKMAM, China), with 4 mL of bacterial suspension added to each well. Six wells were inoculated per plate for parallel experiments. After inoculation, the plates were sealed with sealing film and incubated at 30°C for 18 hours. After incubation, 3 mL of bacterial suspension was aspirated from each well. After treating the biofilm with crystal violet staining and ethanol destaining, an appropriate amount of the liquid was taken and measured at OD600 ≈ 1.00. 570 The amount of biofilm formed is characterized under certain conditions. For example... Figure 16 As shown, both exogenous application of signaling molecules C10-HSL and 3OC10-HSL significantly promoted biofilm formation in strain LV-Q1 (P<0.05), and under the same concentration gradient, 3OC10-HSL had a more significant promoting effect on biofilm formation in strain LV-Q1. Figure 17 The study investigated the effect of exogenous vanillin, a signaling molecule inhibitor, on biofilm formation in strain LV-Q1 during high-salt denitrification. Results showed that exogenous inhibition significantly suppressed biofilm formation in strain LV-Q1 during high-salt denitrification (P<0.05). These findings indicate that exogenous enhancement or inhibition of AHL-type signaling molecule secretion affects the biofilm formation performance of strain LV-Q1, which is significant for regulating biofilm formation in functional bacteria and for the bioaugmented treatment of high-salt nitrogenous wastewater.

[0064] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any modifications or equivalent substitutions made to the technical solutions of the present invention based on the description and drawings of the present invention shall also be included within the patent protection scope of the present invention.

Claims

1. A salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain producing acylhomoserine lactone, characterized in that, The species is classified as Vibrio ( ). Vibrio sp.) LV-Q1, deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.28696.

2. The application of a salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain producing acyl homoserine lactone as described in claim 1 in the treatment of saline and nitrogenous wastewater.

3. The application of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain producing acylhomoserine lactone according to claim 2 in the treatment of saline and nitrogenous wastewater, characterized in that, Wastewater salinity is calculated in terms of NaCl, and the salinity range is 10 g / L–80 g / L.

4. The application of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain producing acylhomoserine lactone according to claim 2 in the treatment of saline and nitrogenous wastewater, characterized in that, The Vibrio LV-Q1 strain grows using ammonia nitrogen as the sole nitrogen source under a salinity of 50 g NaCl / L and utilizes an organic carbon source for aerobic heterotrophic nitrification.

5. The application of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain producing acylhomoserine lactone according to claim 2 in the treatment of saline and nitrogenous wastewater, characterized in that, The Vibrio LV-Q1 strain described herein grows using nitrate nitrogen as the sole nitrogen source under a salinity of 50 g NaCl / L and utilizes organic carbon sources for aerobic denitrification.

6. The application of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain producing acylhomoserine lactone according to claim 2 in the treatment of saline and nitrogenous wastewater, characterized in that, Vibrio LV-Q1 grows using a mixture of ammonia and nitrate nitrogen as nitrogen sources under a salinity of 50 g NaCl / L, and simultaneously nitrifies and denitrifies using organic carbon sources under aerobic conditions.

7. The application of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain producing acylhomoserine lactone according to claim 2 in the treatment of saline and nitrogenous wastewater, characterized in that, During the high-salt denitrification process with a salinity of 50 g NaCl / L, during the logarithmic growth phase, the Vibrio LV-Q1 secretes AHLs-like signaling molecules, namely C8-HSL, C10-HSL, 3OC6-HSL, 3OC8-HSL, 3OC10-HSL, and 3OC12-HSL signaling molecules.

8. The application of a salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain producing acyl homoserine lactone as described in claim 1 in regulating biofilm denitrification.

9. The application of the salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain producing acylhomoserine lactone according to claim 8 in controlled biofilm denitrification, characterized in that, Biofilm formation and denitrification performance of Vibrio LV-Q1 in a high-salt denitrification process at a salinity of 50 g NaCl / L can be promoted or inhibited by exogenously applying signaling molecules C10-HSL and 3OC10-HSL or the inhibitor vanillin; wherein the concentration range of signaling molecules C10-HSL or 3OC10-HSL is 10–200 nmol / L; or the concentration range of signaling molecule inhibitor vanillin is 10–500 mg / L.

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