A strain of Alcaligenes HC-1 and its application

Abstract

This invention discloses an Alcaligenes HC-1 strain and its applications. This invention belongs to the field of microbiology, specifically relating to an Alcaligenes HC-1 strain and its applications. The Alcaligenes provided in this invention is Alcaligenes nematophilus CGMCC No. 27571, which is registered with the China General Microbiological Culture Collection Center (CGMCC) under the number CGMCC No. 27571. The Alcaligenes HC-1 strain provided in this invention possesses highly efficient denitrification capabilities, effectively removing or reducing the ammonia nitrogen and total nitrogen content in wastewater. Using Alcaligenes HC-1 for direct ammonia oxidation denitrification in wastewater greatly simplifies traditional denitrification steps, improves denitrification efficiency, and has broad application value.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to an Alcaligenes HC-1 strain and its applications. Background Technology

[0002] Nitrogen is a key limiting nutrient element, controlling the growth, reproduction, and primary production of organisms in most Earth's ecosystems. The nitrogen cycle forms an extremely complex system, supporting diverse life forms. With rapid societal development, human activities have gradually led to widespread and excessive nitrogen pollution in aquatic environments. Excessive nitrogen emissions cause eutrophication in aquatic ecosystems, resulting in algal blooms and red tides; increased ammonia concentrations (enhanced toxicity) cause the death of aquatic organisms, among other environmental problems. Exploring microbial denitrification mechanisms and isolating and developing highly efficient denitrifying microbial strains is of great significance for the efficient management of aquatic environments.

[0003] Currently, wastewater treatment processes in China are mainly biological, including nitrification-denitrification (SND), anaerobic ammonia oxidation (ANAMMOX), and heterotrophic nitrification-aerobic denitrification (HN-AD). The current mainstream nitrogen removal process is based on traditional nitrification-denitrification, which separates the nitrification and denitrification processes according to the adaptation conditions and aerobic requirements of nitrifying and denitrifying bacteria. This leads to a series of disadvantages, including high energy consumption, the need to add organic matter as a carbon source for denitrification, large equipment scale, and the generation of large amounts of N2O greenhouse gases. Although anaerobic ammonia oxidation has high nitrogen removal efficiency, the microorganisms involved are autotrophic, grow extremely slowly, and have high requirements for their growth environment. Heterotrophic nitrification combined with aerobic denitrification can achieve simultaneous nitrification-denitrification (SND) and COD removal in the same reactor. Heterotrophic nitrifying bacteria have become a hot topic in current biological nitrogen removal research due to their advantages such as rapid growth, low oxygen requirements, and wide applicability. However, the HN-AD simultaneous nitrogen removal process is still in the laboratory research stage, and there are few reports of industrial applications.

[0004] Direct ammonia oxidation (Dirammox) is a novel denitrification pathway discovered in heterotrophic bacteria. Mediated by the dnf gene cluster, ammonia is oxidized to hydroxylamine, directly producing nitrogen gas. This significantly simplifies traditional denitrification steps and improves denitrification efficiency. Heterotrophic denitrifying bacteria have enormous application potential in the field of biological denitrification. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to improve and simplify the traditional denitrification steps in wastewater treatment processes and increase the denitrification efficiency of wastewater treatment.

[0006] To solve the above-mentioned technical problems, the present invention first provides an alkaloid bacterium.

[0007] The Alcaligenes nematophilus provided by this invention is CGMCC No. 27571, which is registered with the China General Microbiological Culture Collection Center under the number CGMCC No. 27571.

[0008] The Alcaligenes HC-1 strain possesses a complete direct ammonia oxidation gene cluster, including ten genes: dnfI, dnfH (dnfT1), dnfR, dnfG (dnfT2), dnfA, dnfB, dnfC, and dnfD. Among them, dnfA is 954 bp in length, with the nucleotide sequence being sequence 2 in the sequence listing, and is predicted to encode diferrooxygenase; dnfB is 1053 bp in length, with the nucleotide sequence being sequence 3 in the sequence listing, and is predicted to encode a 2Fe-2S iron-sulfur cluster-binding domain protein; dnfC is 723 bp in length, with the nucleotide sequence being sequence 4 in the sequence listing, and is predicted to encode glutamine aminotransferase. At the genomic level, this demonstrates that strain HC-1 has the ability to complete denitrification via the direct ammonia oxidation (Dirammox) pathway.

[0009] The present invention also provides a microbial agent.

[0010] The bacterial agent provided by this invention contains the Alcaligenes bacillus described above and / or the metabolites or cultures of the Alcaligenes bacillus.

[0011] The culture of Alcaligenes described above is a substance obtained by culturing the Alcaligenes mentioned above in a bacterial culture medium.

[0012] In addition to the active ingredients, the above-mentioned bacterial agent also contains a carrier. The carrier can be a commonly used biological carrier that is biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, plant material, or polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material can be at least one of corn flour, soybean flour, and starch; the polymer compound can be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent can be decane and / or dodecane.

[0013] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0014] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the bacterial agent.

[0015] In a specific embodiment of the present invention, the bacterial agent is 10 8 The fermentation broth of *Alcaligenes nematophilus* HC-1 at CFU / mL was further prepared by a method including the following steps: inoculating *Alcaligenes nematophilus* HC-1 into a bacterial culture medium and culturing it to obtain a concentration of 10 CFU / mL. 8 The bacterial solution containing CFU / mL is the bacterial agent.

[0016] The term "metabolite" refers to the primary and / or secondary metabolites produced during microbial metabolism. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the external environment and, through catabolism and anabolism, generate substances and energy to sustain life activities. The products of primary metabolism are called primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, and lipids. Secondary metabolism refers to the process by which microorganisms, at a certain growth stage, use primary metabolites as precursors to synthesize substances that have no clearly defined function for their life activities. The products of secondary metabolism are called secondary metabolites, and are mostly compounds with relatively complex molecular structures. Based on their functions, they can be classified into types such as antibiotics, hormones, alkaloids, and toxins.

[0017] In the above text, the metabolites can be obtained from the fermentation broth of the *Alcaligenes*. The metabolites can be sterile metabolites of the *Alcaligenes* or bacterial metabolites of the *Alcaligenes*. Specifically, the sterile metabolites of the *Alcaligenes* (sterile fermentation filtrate) can be prepared by culturing the *Alcaligenes* in a liquid culture medium and filtering to remove the *Alcaligenes* from the liquid culture (fermentation broth). Specifically, the bacterial metabolites of the *Alcaligenes* can be prepared by culturing the *Alcaligenes* in a liquid fermentation medium and collecting the fermentation broth, which is the bacterial metabolite of the *Alcaligenes*.

[0018] Furthermore, the bacterial culture medium is LB liquid medium.

[0019] Furthermore, the culture medium may be a heterotrophic nitrification medium (HNM).

[0020] The heterotrophic nitrification medium consists of the following components: 6.74 g sodium succinate hexahydrate, 0.66 g ammonium sulfate, 0.5 g potassium dihydrogen phosphate, 1.25 g magnesium sulfate heptahydrate, 1.25 g disodium hydrogen phosphate dodecahydrate, 2 ml trace elements (Trace), 1 L ddH2O, and pH 7.0.

[0021] Trace elements included: EDTA·Na2 57.1g, FeSO4·7H2O 5g, ZnSO4·7H2O 3.9g, MnCl2·4H2O 1g, CoCl2·6H2O 1.6g, CuSO4·5H2O 1.6g, (NH4)6Mo7O24·4H2O 1.1g, CaCl2·2H2O 7g, ddH2O 1L, pH 6.0.

[0022] The term "culture" refers to a liquid or solid product (all substances within the culture container) that has grown a microbial community after artificial inoculation and cultivation. It is a product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

[0023] The present invention also provides the use of the Alcaligenes bacillus, the bacterial agent, or the culture described above in at least one of the following:

[0024] (a1) Biological denitrification;

[0025] (a2) Preparation of biological denitrification products;

[0026] (a3) Wastewater treatment;

[0027] (a4) Prepare wastewater treatment products.

[0028] In the above applications, biological denitrification is manifested in all or part of the following:

[0029] (b1) Remove or reduce ammonia nitrogen under normal salinity conditions;

[0030] (b2) Remove or reduce ammonia nitrogen under salt-tolerant conditions;

[0031] (b3) Remove or reduce total nitrogen under normal salinity conditions;

[0032] (b4) Remove or reduce total nitrogen under salt-tolerant conditions;

[0033] (b5) Remove or reduce ammonia nitrogen under normal ammonia nitrogen conditions;

[0034] (b6) Remove or reduce ammonia nitrogen under high concentration conditions;

[0035] (b7) Remove or reduce total nitrogen under normal ammonia nitrogen conditions;

[0036] (b8) Remove or reduce total nitrogen under high concentrations of ammonia nitrogen;

[0037] (b9) Remove or reduce ammonia nitrogen under low temperature conditions;

[0038] (b10) Remove or reduce ammonia nitrogen under normal temperature conditions;

[0039] (b11) Remove or reduce total nitrogen under low temperature conditions;

[0040] (b12) Remove or reduce total nitrogen under normal temperature conditions;

[0041] (b13) Remove or reduce total nitrogen under conditions where organic nitrogen is used as the nitrogen source.

[0042] The present invention also provides a method for preparing the above-mentioned bacterial agent, comprising the following steps: using the Alcaligenes bacillus described above as the active ingredient to obtain the bacterial agent.

[0043] The present invention also provides a biological denitrification product, wherein the biological denitrification product contains the Alcaligenes described above or the bacterial agent or the culture described above.

[0044] The present invention also provides a method for denitrification in wastewater treatment, comprising the following steps: under high ammonia nitrogen concentration conditions, applying the aforementioned Alcaligenes bacillus, or the aforementioned bacterial agent, or the aforementioned culture to the wastewater, thereby removing or reducing the ammonia nitrogen and total nitrogen content in the wastewater.

[0045] The present invention also provides the application of the above-described application or method in at least one of the following:

[0046] (c1) Biological denitrification;

[0047] (c2) Improve nitrogen removal efficiency;

[0048] (c3) Wastewater treatment;

[0049] (c4) Improve wastewater treatment efficiency.

[0050] The Alcaligenes HC-1 strain provided by this invention is a Gram-negative bacterium isolated and purified from activated sludge samples from urban wastewater treatment plants. This strain exhibits excellent performance in wastewater treatment, including wastewater containing ammonia nitrogen and organic nitrogen; environmental remediation, restoration of black and odorous water bodies and sediment; and microbial treatment in horticultural ponds, aquaculture, home fish tanks, ornamental fish farming, and other aquaculture applications. The HC-1 strain of this invention possesses a complete dnf gene cluster, and its denitrification mechanism primarily involves the conversion to gas, mainly nitrogen. It oxidizes ammonia to hydroxylamine via the Dirammox pathway, directly producing gaseous nitrogen products. This significantly simplifies traditional denitrification steps, improves denitrification efficiency, and saves space and costs.

[0051] The strain HC-1 of this invention, as a heterotrophic bacterium, possesses the ability to aerobically convert ammonia into nitrogen gas, and exhibits excellent removal capabilities for both ammonia nitrogen and total nitrogen. When grown using ammonia nitrogen (140 mg / L) as the sole nitrogen source, it can convert 55.61% into nitrogen gas, achieving a 100% ammonia nitrogen removal rate and a 72.62% removal rate for total nitrogen in the water. Strain HC-1 demonstrates good pH adaptability, growing well within a pH range of 6-10 and possessing direct ammonia oxidation capabilities, efficiently removing 100% of ammonia nitrogen and over 62% of total nitrogen. Strain HC-1 exhibits salt tolerance, still achieving a 100% ammonia nitrogen removal rate and a 66% total nitrogen removal rate even at a NaCl concentration of 60 g / L. Strain HC-1 also exhibits broad C / N adaptability, growing well within a C / N ratio range of 3-20, and efficiently removing both ammonia nitrogen and total nitrogen. Even under low C / N ratio (C / N = 3 or 5), it still maintains a high ammonia nitrogen removal rate (100%) and a total nitrogen removal rate (over 56%). Strain HC-1 can maintain good growth and efficient nitrogen removal even under high ammonia nitrogen concentrations. At an ammonia nitrogen concentration of 5600 g / L, it still grows well, achieving an ammonia nitrogen removal rate of 69.4% and a total nitrogen removal rate of 52.2%. Strain HC-1 can also grow well and efficiently remove nitrogen using organic nitrogen sources (such as corn steep liquor) as the sole nitrogen source, achieving a total nitrogen removal rate of 67.50 ± 2.68%.

[0052] Preservation Instructions

[0053] Strain name: Alcaligenes

[0054] Latin name: Alcaligenes nematophilus

[0055] Strain number: HC-1

[0056] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0057] Collection institution abbreviation: CGMCC

[0058] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0059] Deposit date: June 7, 2023

[0060] Registered with the China National Collection Center (CGMCC) No. 27571. Attached Figure Description

[0061] Figure 1 This is a phylogenetic tree of strain HC-1 based on its 16S rRNA gene sequence and whole genome sequence. Left: 16S rRNA gene sequence; Right: whole genome sequence.

[0062] Figure 2 This is a diagram of the complete dnf gene cluster of strain HC-1.

[0063] Figure 3 The graph shows the growth and direct ammonia oxidation capacity of strain HC-1 using ammonia nitrogen as the sole nitrogen source.

[0064] Figure 4 This is a nitrogen balance analysis diagram of nitrogen-containing gaseous products of strain HC-1 using ammonia nitrogen as the sole nitrogen source.

[0065] Figure 5 The total nitrogen removal rate of strain HC-1 was determined using ammonia nitrogen as the sole nitrogen source.

[0066] Figure 6 The effects of different pH conditions on the growth and direct ammonia oxidation capacity of strain HC-1.

[0067] Figure 7 The effects of different NaCl concentrations on the growth and direct ammonia oxidation capacity of strain HC-1 were investigated.

[0068] Figure 8 The effects of different C / N ratios on the growth and direct ammonia oxidation capacity of strain HC-1 were investigated.

[0069] Figure 9 For different NH4 + Effects of -N concentration on the growth and direct ammonia oxidation capacity of strain HC-1.

[0070] Figure 10 The effects of different dissolved oxygen conditions on the growth and direct ammonia oxidation capacity of strain HC-1 were investigated.

[0071] Figure 11 The effects of different temperatures on the growth and direct ammonia oxidation capacity of strain HC-1.

[0072] Figure 12 To detect the growth of strain HC-1 and the total nitrogen removal rate under conditions where organic nitrogen is the sole nitrogen source. Detailed Implementation

[0073] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0074] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0075] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0076] The culture medium preparation methods involved in the following examples are as follows:

[0077] LB medium: 10g tryptone, 5g yeast extract, 10g NaCl, 1L ddH2O, pH 7.0.

[0078] Heterotrophic Nitrification Media (HNM): Sodium succinate hexahydrate 6.74g, ammonium sulfate 0.66g, potassium dihydrogen phosphate 0.5g, magnesium sulfate heptahydrate 1.25g, disodium hydrogen phosphate dodecahydrate 1.25g, trace elements (Trace) 2mL, to be made up to 1L with ddH2O, pH 7.0.

[0079] Trace elements included: EDTA·Na₂ 57.1g, FeSO₄·7H₂O 5g, ZnSO₄·7H₂O 3.9g, MnCl₂·4H₂O 1g, CoCl₂·6H₂O 1.6g, CuSO₄·5H₂O 1.6g, (NH₄)₆Mo₇O 24 ·4H2O 1.1g, CaCl2·2H2O 7g, add ddH2O to make up to 1L, pH 6.0.

[0080] The following examples used Origin 2023 GraphPad Prism 9 and SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0081] Example 1. Screening, isolation, identification and preservation of strain HC-1

[0082] 1. Screening and isolation of strains

[0083] Enrichment Culture: Activated sludge samples were collected from the Changsha Municipal Wastewater Treatment Plant in Hunan Province. (Collector: Chen Han chenhan@ihb.ac.cn) The collected activated sludge samples were mixed and added to a conical flask containing 100 mL of HNM medium at a ratio of 1:500. Since hydroxylamine is highly toxic to most microorganisms, 1 mol / L hydroxylamine stock solution was added to the HNM medium to achieve a final concentration of 2 mmol / L as a pressure stress for selective enrichment. The flasks were then placed in a constant temperature shaking incubator at 28°C and 180 rpm.

[0084] Strain isolation: For the samples enriched above, pipette 1 mL daily and add it to 9 mL of sterile physiological saline (0.9% NaCl), mixing thoroughly by inverting the tube at each step. Finally, dilute 10-fold serially to a final concentration of 10-fold. -1 10 -2 10 -3 10 -4 10 -5 10 -6 200 μL of each dilution was spread onto HNM agar plates containing 2 mM hydroxylamine and incubated at 28°C for 24–72 h until single colonies appeared. The bacterial growth morphology was observed and preliminary classification was performed. After continuous selective enrichment and screening, a bacterium with direct ammonia oxidation ability was finally obtained and named strain HC-1.

[0085] 2. Molecular biological identification and morphological analysis of the strain

[0086] A single colony of strain HC-1 obtained from the preliminary classification in step 1 was picked and placed in 3 mL of HNM liquid medium containing 2 mM hydroxylamine, and cultured at 28°C and 180 rpm until OD500. 600 Approximately 0.6. DNA was extracted from strain HC-1, and molecular biological identification of the strain was completed using PCR amplification.

[0087] The 16S rRNA gene sequence fragment (nucleotide sequence is sequence 1 in the sequence listing) of strain HC-1 was obtained using universal primers 27F / 1492R (specific primer sequences: 1492R: 5'-TACGGCTACCTTGTTACGACTT-3', 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'). PCR system: 25 μL 2×Es Taq master Mi, 1 μL 27F, 1 μL 1492R, 1 μL bacterial culture, 22 μL ddH2O. PCR reaction conditions: 1) 94℃ pre-denaturation for 5 min; 2) 94℃ denaturation for 30 sec; 3) 55℃ annealing for 30 sec; 4) 72℃ extension for 1 min; 5) 72℃ extension for 10 min. 30 cycles were performed for 2) to 5).

[0088] The PCR products were sent to a sequencing company for first-generation sequencing, and the sequencing results were analyzed using the NCBI BLASTN database. http: / / www.ncbi.nlm.nih.gov / BLAST The analysis was compared with standard strains selected from the genus *Alcaligenes*. Analysis of its 16S rRNA genome and whole genome phylogenetic tree, collinearity, and ANI / AAI all revealed... Figure 1 HC-1 is more closely related to *Alcaligenes nematophilus*, followed by *Alcaligenes aquatilis* and *Alcaligenes ammonioxydans*. Ultimately, it was named *Alcaligenes nematophilus* HC-1, or simply strain HC-1.

[0089] Strain HC-1 is a Gram-negative bacterium. Colonies are milky white, round, smooth and moist, opaque, with relatively regular edges. After culturing on LB agar at 30°C for 2 days, the colony diameter is approximately 2-3 mm. The cells are short rods, with a cell size of approximately (0.5-0.6) μm × (0.7-0.9) μm.

[0090] 3. Preservation of strain HC-1

[0091] Alcaligenes nematophilus HC-1 was deposited on June 7, 2023, at the China General Microbiological Culture Collection Center (CGMCC, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC 27571. It will be referred to as Alcaligenes CGMCC No. 27571, Alcaligenes HC-1, or strain HC-1.

[0092] Example 2: Denitrification capacity analysis of Alcaligenes HC-1

[0093] Strain HC-1 was pre-cultured in LB medium to complete strain activation. The activated strain HC-1 was then transferred to HNM medium at an inoculum of 1% and cultured at 30℃ and 160rpm for 16h to finally obtain Alcaligenes HC-1 bacterial agent.

[0094] 1. Identification of the dnf gene cluster of Alcaligenes HC-1

[0095] Whole-genome sequencing of strain HC-1 revealed the presence of a complete direct ammonia oxidation gene cluster (dnf gene cluster). Figure 2 As shown in the figure, the strain HC-1 contains ten genes: dnfI, dnfH (dnfT1), dnfR, dnfG (dnfT2), dnfA, dnfB, dnfC, and dnfD. Among them, dnfA is 954 bp in length, with the nucleotide sequence being sequence 2 in the sequence listing, and is predicted to encode diferrooxygenase; dnfB is 1053 bp in length, with the nucleotide sequence being sequence 3 in the sequence listing, and is predicted to encode a 2Fe-2S iron-sulfur cluster-binding domain protein; and dnfC is 723 bp in length, with the nucleotide sequence being sequence 4 in the sequence listing, and is predicted to encode glutamine aminotransferase. This demonstrates at the genomic level that strain HC-1 possesses the ability to denitrify via the direct ammonia oxidation (Dirammox) pathway.

[0096] 2. Determination of the growth ability and direct ammonia oxidation ability of strain HC-1 using ammonia nitrogen as the sole nitrogen source.

[0097] Strain HC-1 was pre-cultured in LB medium to activate the strain. Then, 1 mL of the HC-1 seed culture was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of HNM medium (C / N = 10) with 5 mM (NH4)2SO4. The flask was incubated on a rotating shaker at 30°C and 160 rpm. Bacterial growth (OD) was measured every 4 hours during the 60-hour incubation period. 600 ), NH4 - -N, NH2OH-N, NO2 —N content. The determination method for ammonia nitrogen is as follows: Determination of ammonia nitrogen in water quality by salicylic acid spectrophotometry HJ 536-2009; the determination method for nitrite nitrogen is as follows: Determination of nitrite nitrogen in water quality by spectrophotometry GB7493-87; the determination method for hydroxylamine nitrogen is as follows: the paper "Frear, DS, & Burrel l, RC (1955). SPECTROPHOTOMETRIC METHOD FOR DETERMINING HYDROXYLAMINE REDUCTASE ACTIVITY IN HIGHER PLANTS. Analytical Chemistry, 27(10), 1664-1665." The determination principle is that in the presence of ethanol and sodium carbonate, hydroxylamine and excess 8-hydroxyquinoline react quantitatively to form a stable green condensate 5,8-quinolinequinone-5-(8-hydroxy-5-quinolyl imide). The hydroxylamine content in the sample can be quantified by measuring the photometric value of its absorption peak at a wavelength of 705 nm.

[0098] Tests on its ammonia oxidation and denitrification capabilities in HNM medium revealed that ammonia consumption accompanied cell growth during ammonia conversion. Figure 3 As shown, hydroxylamine accumulates in large quantities during this process, eventually being completely consumed, accompanied by the production of small amounts of nitrite and nitrate.

[0099] After 4 hours of growth, strain HC-1 cells entered the logarithmic growth phase, and after 40 hours of culture, OD... 600 It reached a peak of 0.328, then decreased slightly. With NH4... - As -N decreases, NH2OH-N gradually accumulates, reaching a maximum concentration of 48.42 mg / L at 30 hours, and then gradually decreases to almost undetectable levels. The accumulation of nitrite (9.50 mg / L) and nitrate (5.33 mg / L) is relatively small throughout the reaction process.

[0100] 3. Detection of nitrogen-containing gaseous products of strain HC-1 using ammonia nitrogen as the sole nitrogen source

[0101] The HC-1 strain was pre-cultured in LB medium, and then 40 μL of the HC-1 culture was inoculated into a medium containing 4 mL of HNM (containing 5 mM... 15 The culture medium (NH4)2SO4 was placed in a 50 mL anaerobic flask. The headspace of the flask was filled with He / O2 at a ratio of 4:1 as described above. The flask was incubated on a rotating shaker at 30 °C and 160 rpm for 120 h. N2-N and N2O-N were measured, followed by bacterial growth (OD). 600 ), NH4 - -N, NH2OH-N, NO2 --N, NO3 — N.

[0102] The results showed that HC-1 produced nitrogen gas during the aerobic conversion of ammonia. Based on nitrogen balance analysis, strain HC-1 could ultimately produce 10 mM nitrogen gas. 15 NH4-N is completely consumed, and 55.61% of it can be consumed. 15 NH4 + Transform into 15 N2, simultaneously producing 10.51% 15 N2O ( Figure 4 Meanwhile, trace amounts of nitrite (6.52%) and nitrate (0.96%) were detected. In addition, most of the consumed ammonia was converted into cellular nitrogen and organic nitrogen, with a total nitrogen removal rate of 75.81%.

[0103] 4. Detection of total nitrogen removal rate of strain HC-1 using ammonia nitrogen as the sole nitrogen source

[0104] The removal rate of total nitrogen in water by strain HC-1 was determined by the following method: the supernatant of strain HC-1 was treated with a total nitrogen reagent (Test). Nitrogen persulfate (NH4N, HACH) was digested with alkaline potassium persulfate using a COD heater (DRB200, HACH), and then measured using a spectrophotometer (DR890, HACH). The biomass of strain HC-1 was determined using the same method as total nitrogen, with biomass nitrogen calculated by differential method.

[0105] The results showed that the total nitrogen removal rate in the water was 72.62%. Figure 5 As shown, the result is basically consistent with the nitrogen balance analysis result (75.81%) in step 3.

[0106] Example 3: Effects of different HNM culture medium conditions on the growth and direct ammonia oxidation capacity of strain HC-1

[0107] 1. Optimization of pH conditions in HNM medium

[0108] The initial pH of the HNM medium was adjusted to 3, 4, 5, 6, 7, 8, 9, and 10. Sodium citrate was used as the sole carbon source, and the initial ammonia nitrogen concentration was set at 140 mg / L with a C / N ratio of 10. The culture was carried out with shaking at 30°C and 160 rpm. The growth and direct ammonia oxidation capacity of strain HC-1 were measured. The growth assay, direct ammonia oxidation capacity assay, and total nitrogen (TN) removal rate assay were performed using the same methods as in Example 2.

[0109] The results are as follows Figure 6As shown, strain HC-1 grows well in the pH range of 6-10 and exhibits good direct ammonia oxidation capacity. It shows a high growth rate at pH 7-9; growth decreases at pH 6, but hydroxylamine accumulation is very high, approximately 1600 μM; similarly, nitrite accumulation is highest at pH 6, approximately 800 μM.

[0110] The ammonia nitrogen and total nitrogen removal capabilities are shown in Table 1 below. Within the pH range of 6-10, strain HC-1 achieved a 100% ammonia nitrogen removal rate and a total nitrogen removal rate between 62.62% and 68.81%.

[0111] Table 1. Removal capacity of ammonia nitrogen and total nitrogen from fermentation broth under different pH conditions

[0112] pH Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) 3 0 0 4 0 0 5 0 0 6 100 62.62±1.19 7 100 65.00±2.58 8 100 66.67±4.06 9 100 68.81±2.88 10 100 63.10±5.46

[0113] 2. Optimization of different NaCl concentrations in HNM medium

[0114] The NaCl concentration in HNM medium was adjusted to 0 g / L, 20 g / L, 40 g / L, 60 g / L, and 80 g / L, respectively. Sodium citrate was used as the sole carbon source. The initial ammonia nitrogen concentration was 140 mg / L, the C / N ratio was 10, and the initial pH was 7.0. The culture was carried out with shaking at 30°C and 160 rpm. The growth and direct ammonia oxidation capacity of strain HC-1 were measured. The growth assay, direct ammonia oxidation capacity assay, and total nitrogen (TN) removal rate assay were performed using the same methods as in Example 2.

[0115] The results are as follows Figure 7 As shown, strain HC-1 grows well in the NaCl concentration range of 0-60 g / L and exhibits good direct ammonia oxidation capacity. At NaCl concentrations of 20-60 g / L, it shows higher growth compared to the control group (NaCl concentration of 0 g / L); the accumulation of hydroxylamine and nitrite nitrogen decreases with increasing NaCl concentration.

[0116] The removal capacities of ammonia nitrogen and total nitrogen are shown in Table 2 below. Within the NaCl concentration range of 0-60 g / L, strain HC-1 achieved a 100% ammonia nitrogen removal rate and a total nitrogen removal rate between 65.00% and 68.33%.

[0117] Table 2. Removal capacity of ammonia nitrogen and total nitrogen in fermentation broth under different NaCl concentrations

[0118] NaCl (g / L) Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) 0 100 65.00±2.58 20 100 68.33±0.83 40 100 67.38±2.89 60 100 66.19±1.80 80 0 0

[0119] 3. Optimization of different C / N conditions in HNM culture medium

[0120] Sodium citrate was used as the sole carbon source, and the C / N ratios were adjusted to 3, 5, 10, 15, and 20, respectively. The initial ammonia nitrogen concentration was set to 140 mg / L, and the initial pH of the culture medium was adjusted to 7.0. The culture was carried out with shaking at 30°C and 160 rpm. The growth and direct ammonia oxidation capacity of strain HC-1 were determined. The growth assay, direct ammonia oxidation capacity assay, and total nitrogen (TN) removal rate assay were performed using the same methods as in Example 2.

[0121] The results are as follows Figure 8 As shown, strain HC-1 grows well and exhibits good direct ammonia oxidation capacity within a C / N ratio range of 3-20. Within this range, the growth rate and amount of HC-1 increase with increasing C / N ratio, but show no significant increase when C / N = 10; hydroxylamine accumulation shows no significant difference within the C / N range of 3-20.

[0122] The removal capacities of ammonia nitrogen and total nitrogen are shown in Table 3 below. Within the C / N ratio range of 3-20, strain HC-1 achieved a 100% ammonia nitrogen removal rate and a total nitrogen removal rate between 56.19% and 69.05%.

[0123] Table 3. Removal capacity of ammonia nitrogen and total nitrogen in fermentation broth under different C / N ratios

[0124] C / N Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) 3 100 56.19±2.30 5 100 65.24±2.30 10 100 65.00±2.58 15 100 67.38±1.48 20 100 69.05±2.18

[0125] 4. Different NH4+ in HNM culture medium + -N concentration conditions optimization

[0126] NH4 in HNM medium + The -N concentration was adjusted to 700, 1400, 2800, and 5600 mg / L, respectively. Sodium citrate was used as the sole carbon source with an initial carbon source concentration of 7000 mg / L and an initial pH of 7.0. The culture was carried out with shaking at 30°C and 160 rpm. The growth and direct ammonia oxidation capacity of strain HC-1 were measured. The growth assay, direct ammonia oxidation capacity assay, and total nitrogen (TN) removal rate assay were performed using the same methods as in Example 2.

[0127] The results are as follows Figure 9 As shown, strain HC-1 grows well in the ammonia nitrogen concentration range of 700-5600 mg / L and has good direct ammonia oxidation capacity. Growth is generally consistent across the ammonia nitrogen concentration range of 700-5600 mg / L; hydroxylamine accumulation decreases with increasing ammonia nitrogen concentration after 6 hours; nitrite nitrogen accumulates only in small amounts after the growth enters the decline phase.

[0128] The removal capacities of ammonia nitrogen and total nitrogen are shown in Table 4 below. Within the ammonia nitrogen concentration range of 700-5600 mg / L, the ammonia nitrogen removal rate of strain HC-1 is 69.41-99.02%, and the total nitrogen removal rate is between 52.19-71.43%.

[0129] Table 4. Different NH4 4+ Ammonia and total nitrogen removal capacity in fermentation broth under -N concentration conditions

[0130] Ammonia nitrogen concentration (mg / L) Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) 700 99.02±0.21 71.43±0.71 1400 81.42±0.53 75.71±1.43 2800 81.39±0.06 66.91±2.68 5600 69.41±0.12 52.19±2.68

[0131] 5. Optimization of different dissolved oxygen conditions in HNM medium

[0132] Sodium citrate was used as the sole carbon source. The shaking speed was adjusted to 0, 80, 160, and 200 rpm, the initial ammonia nitrogen concentration was set to 140 mg / L, the initial pH of the culture medium was adjusted to 7.0, C / N = 10, and the culture was carried out at 30℃. The growth of strain HC-1, its direct ammonia oxidation capacity, and the total nitrogen (TN) removal rate were measured.

[0133] The results are as follows Figure 10 As shown, strain HC-1 grows well within a rotation speed range of 0-200 rpm and exhibits good direct ammonia oxidation capacity. Within a C / N ratio range of 0-200 rpm, the growth rate of strain HC-1 increases with increasing dissolved oxygen content (rotation speed), but shows no significant increase when the rotation speed reaches 160 rpm; hydroxylamine accumulation increases with increasing dissolved oxygen content (rotation speed).

[0134] The ammonia nitrogen and total nitrogen removal capabilities are shown in Table 5. Within the rotation speed range of 0-200 rpm, strain HC-1 achieved ammonia nitrogen removal rate of 100% and a total nitrogen removal rate of 45.24-65.00%.

[0135] Table 5. Removal capacity of ammonia nitrogen and total nitrogen in fermentation broth under different dissolved oxygen conditions

[0136] Rotational speed (rpm) Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) 0 100 45.24±3.79 80 100 63.57±3.78 160 100 65.00±2.58 200 100 59.29±2.08

[0137] 6. Optimization of different temperature conditions for HNM culture medium

[0138] Sodium citrate was used as the sole carbon source. The temperature was adjusted to 10, 15, 20, 30 and 40℃ respectively. The initial ammonia nitrogen concentration was set to 140 mg / L. The initial pH of the culture medium was adjusted to 7.0 and C / N = 10. The culture was shaken at 160 rpm. The growth of strain HC-1, its direct ammonia oxidation capacity and the total nitrogen (TN) removal rate were measured.

[0139] The results are as follows Figure 11As shown, strain HC-1 grows well in the temperature range of 10-40℃ and exhibits good direct ammonia oxidation capacity. Within the 10-40℃ range, the growth rate and yield of strain HC-1 increase with increasing temperature. Whether under low-temperature conditions of 10℃ or high-temperature conditions of 40℃, strain HC-1 maintains excellent ammonia nitrogen and total nitrogen degradation capabilities.

[0140] The ammonia nitrogen and total nitrogen removal capabilities are shown in Table 6. Within the temperature range of 10-40℃, strain HC-1 achieved an ammonia nitrogen removal rate of 92.5-100% and a total nitrogen removal rate of 49.05-69.29%.

[0141] Table 6. Removal capacity of ammonia nitrogen and total nitrogen in fermentation broth under different culture temperatures

[0142] Temperature (°C) Ammonia nitrogen removal rate (%) Total nitrogen removal rate (%) 10 92.5±1.37 64.52±2.89 15 100 64.76±2.18 20 100 69.29±1.00 30 100 65.00±2.58 40 100 49.05±5.02

[0143] 7. Optimization of different nitrogen source conditions in HNM medium

[0144] The inorganic nitrogen source (ammonium sulfate) in HNM medium was adjusted to an organic nitrogen source (corn steep liquor), with sodium citrate as the sole carbon source. The initial pH was 7.0, and the culture was carried out with shaking at 30°C and 160 rpm. The growth and total nitrogen removal capacity of strain HC-1 were then measured. The growth assay, direct ammonia oxidation capacity assay, and total nitrogen (TN) removal rate assay were performed using the same methods as in Example 2.

[0145] The results are as follows Figure 12 As shown, the strain grew well under conditions where corn steep liquor was the sole nitrogen source, with a total nitrogen removal rate of 67.50 ± 2.68%.

[0146] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Alcaligenes, characterized by: The alkaloid bacillus is Alcaligenes. Alcaligenes nematophilus CGMCC No. 27571 is registered with the China General Microbiological Culture Collection Center (CGMCC).

2. A microbial agent, characterized in that: The bacterial agent contains the Alcaligenes bacillus as described in claim 1.

3. The culture of Alcaligenes as described in claim 1 is a substance obtained by culturing Alcaligenes as described in claim 1 in a bacterial culture medium.

4. The use of the Alcaligenes bacillus of claim 1, the bacterial agent of claim 2, or the culture of claim 3 in at least one of the following: (a1) Biological denitrification; (a2) Preparation of biological denitrification products; (a3) Wastewater treatment; (a4) Prepare wastewater treatment products.

5. The application according to claim 4, characterized in that: The biological denitrification is manifested in all or part of the following: (b1) Remove or reduce ammonia nitrogen under normal salinity conditions; (b2) Remove or reduce ammonia nitrogen under salt-tolerant conditions; (b3) Remove or reduce total nitrogen under normal salinity conditions; (b4) Remove or reduce total nitrogen under salt-tolerant conditions; (b5) Remove or reduce ammonia nitrogen under normal ammonia nitrogen conditions; (b6) Removing or reducing ammonia nitrogen under high concentration conditions; (b7) Remove or reduce total nitrogen under normal ammonia nitrogen conditions; (b8) Removing or reducing total nitrogen under high concentration ammonia nitrogen conditions; (b9) Removing or reducing ammonia nitrogen under low temperature conditions; (b10) Remove or reduce ammonia nitrogen under normal temperature conditions; (b11) Removing or reducing total nitrogen under low temperature conditions; (b12) Remove or reduce total nitrogen under normal temperature conditions; (b13) Remove or reduce total nitrogen under conditions where organic nitrogen is used as the nitrogen source.

6. The method for preparing the bacterial agent according to claim 2 comprises the following steps: using the Alcaligenes bacillus according to claim 1 as the active ingredient to obtain the bacterial agent.

7. A biological denitrification product, characterized in that: The biological denitrification product contains the Alcaligenes bacillus of claim 1, the bacterial agent of claim 2, or the culture of claim 3.

8. A method for denitrification in wastewater treatment, comprising the following steps: applying the Alcaligenes bacillus of claim 1, the bacterial agent of claim 2, or the culture of claim 3 to the wastewater, thereby removing or reducing the ammonia nitrogen and total nitrogen content in the wastewater.

9. The use of the product of claim 7 or the method of claim 8 in at least one of the following: (c1) Biological denitrification; (c2) Improve nitrogen removal efficiency; (c3) Wastewater treatment; (c4) Improve wastewater treatment efficiency.

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