Alcaligenes HC-1 and application thereof

Abstract

The invention discloses an alcaligenes sp. HC-1 and an application thereof. The invention belongs to the field of microorganisms, and particularly relates to alcaligenes HC-1 and application thereof. The alcaligenes provided by the invention is the alcaligenes nematphilus with the accession number of CGMCC (China General Microbiological Culture Collection Center) No. 27571, and the registration number of the alcaligenes nematphilus in the China General Microbiological Culture Collection Center is CGMCC No. 27571. The alcaligenes HC-1 provided by the invention has efficient denitrification capability, and can effectively remove or reduce the content of ammonia nitrogen and total nitrogen in sewage. The alcaligenes HC-1 is used for carrying out direct ammonia oxidation denitrification on the sewage, so that the traditional denitrification steps are greatly simplified, the denitrification efficiency is improved, and the alcaligenes HC-1 has wide application value.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to a strain of Alcaligenes HC-1 and an application thereof. Background Art

[0002] Nitrogen is a key limiting nutrient that controls the growth, reproduction and primary production of organisms in most of the earth's ecosystems. The cycle of nitrogen forms an extremely complex circulation system that supports diverse life forms. With the rapid development of society, human activities have gradually caused nitrogen pollution in aquatic environments. For example, excessive nitrogen emissions cause eutrophication of aquatic ecosystems, resulting in algal blooms and red tides, and increased ammonia concentrations (increased toxicity) cause the death of aquatic organisms and other environmental problems. Exploring the mechanism of microbial denitrification and isolating and developing efficient denitrification microbial strain resources are of great significance to our efficient management of the water environment.

[0003] At present, the domestic sewage treatment process is mainly based on biological methods, including nitrification-denitrification (Autotrophicnitrification and denitrification), anaerobic ammonia oxidation (ANAMMOX), heterotrophic nitrification-aerobic denitrification (Heterotrophic nitrifying and aerobicdenitrifying bacteria, HN-AD) and other processes. The current mainstream denitrification process is based on traditional nitrification-denitrification, which separates the nitrification and denitrification processes according to the adaptation conditions and oxygen demand of nitrifying bacteria and denitrifying bacteria. This brings 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 production of a large amount of N2O greenhouse gas. Although the anaerobic ammonium oxidation process has a high denitrification efficiency, the anaerobic ammonium oxidation microorganisms are autotrophic bacteria, grow extremely slowly and have high requirements for the growth environment. The heterotrophic nitrification and aerobic denitrification denitrification process can achieve simultaneous nitrification and denitrification (SND) and COD removal in the same reactor. Heterotrophic nitrifying bacteria have the advantages of rapid growth, low oxygen demand and wide application range, and have become a hot topic in current biological denitrification research. However, the HN-AD simultaneous denitrification process is still in the laboratory research stage, and there are few reports on its industrial application.

[0004] Direct ammonia oxidation (Dirammox) is a new denitrification pathway discovered in heterotrophic bacteria. Under the mediation of the dnf gene cluster, ammonia can be oxidized to hydroxylamine, and then nitrogen gas can be directly produced, which greatly simplifies the traditional denitrification steps and improves the denitrification efficiency. Heterotrophic denitrification bacteria have great application potential in the field of biological denitrification. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to improve and simplify the traditional denitrification steps of sewage treatment process and improve the denitrification efficiency of sewage treatment.

[0006] In order to solve the above technical problems, the present invention first provides a strain of Alcaligenes.

[0007] The alcaligenes nematophilus provided by the present invention is Alcaligenes nematophilus CGMCC No. 27571, and its registration number in the General Microbiological Center of China National Committee for the Preservation and Administration of Microbiological Cultures is CGMCC No. 27571.

[0008] The alcaligenes HC-1 has 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 954bp long, the nucleotide sequence is sequence 2 in the sequence list, and it is predicted to encode diiron oxygenase; dnfB is 1053bp long, the nucleotide sequence is sequence 3 in the sequence list, and it is predicted to encode 2Fe-2S iron-sulfur cluster binding domain protein; dnfC is 723bp long, the nucleotide sequence is sequence 4 in the sequence list, and it is predicted to encode glutamine aminotransferase. At the genome level, it is proved that strain HC-1 has the ability to complete denitrification through the direct ammonia oxidation (Dirammox) pathway.

[0009] The invention also provides a bacterial agent.

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

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

[0012] In the above-mentioned bacterial agent, in addition to the active ingredient, the bacterial agent also contains a carrier. The carrier can be a carrier commonly used in the biological field and biologically inert. The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material or a 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 polyglycol; 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 bacterial agents may be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0014] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

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

[0016] The term "metabolite" refers to the primary metabolites and / or secondary metabolites produced during the metabolism of microorganisms. Primary metabolism refers to the process in which microorganisms absorb various nutrients from the outside world and generate substances and energy to maintain life activities through catabolism and anabolism. The products of primary metabolism are primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids and other monomers, as well as various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the process in which microorganisms use primary metabolites as precursors to synthesize some substances that have no clear function in the life activities of microorganisms during a certain growth period. The products of secondary metabolism are secondary metabolites, most of which are compounds with relatively complex molecular structures. According to their functions, they can be divided into types such as antibiotics, hormones, alkaloids, toxins, etc.

[0017] In the above, the metabolite can be obtained from the fermentation broth of the Alcaligenes Bacillus. The metabolite can be a sterile metabolite of the Alcaligenes Bacillus or a bacteria-containing metabolite of the Alcaligenes Bacillus. The sterile metabolite of the Alcaligenes Bacillus (sterile fermentation filtrate) can be prepared specifically according to the following method: the Alcaligenes Bacillus is cultured in a liquid culture medium, and the Alcaligenes Bacillus in the liquid culture (fermentation broth) is filtered out to obtain the sterile metabolite of the Alcaligenes Bacillus. The bacteria-containing metabolite of the Alcaligenes Bacillus can be prepared specifically according to the following method: the Alcaligenes Bacillus is cultured in a liquid fermentation medium, and the fermentation broth is collected, and the fermentation broth is the bacteria-containing metabolite of the Alcaligenes Bacillus.

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

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

[0020] The components of the heterotrophic nitrification medium are specifically: 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, pH 7.0.

[0021] Trace elements: 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 general term for liquid or solid products (all materials in the culture container) that have a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which 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 culture process. The term "culture" also includes a subculture obtained by subculturing a microorganism, which can be a culture of a certain generation or a mixture of several generations.

[0023] The present invention also provides the use of the aforementioned alcaligenes, the aforementioned bacterial agent, or the aforementioned culture in at least one of the following:

[0024] (a1) Biological denitrification;

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

[0026] (a3) sewage treatment;

[0027] (a4) Preparing products for sewage treatment.

[0028] In the above application, the biological denitrification is embodied in whole or in part as follows:

[0029] (b1) Removal or reduction of ammonia nitrogen under normal salinity conditions;

[0030] (b2) Removal or reduction of ammonia nitrogen under salt-tolerant conditions;

[0031] (b3) Removal or reduction of total nitrogen under normal salinity conditions;

[0032] (b4) Removal or reduction of total nitrogen under salt-tolerant conditions;

[0033] (b5) Removing or reducing ammonia nitrogen under normal ammonia nitrogen conditions;

[0034] (b6) Removing or reducing ammonia nitrogen under high ammonia nitrogen concentration conditions;

[0035] (b7) Removal or reduction of total nitrogen under normal ammonia nitrogen conditions;

[0036] (b8) Removal or reduction of total nitrogen under conditions of high ammonia nitrogen concentration;

[0037] (b9) Removing or reducing ammonia nitrogen under low temperature conditions;

[0038] (b10) Removal or reduction of ammonia nitrogen under normal temperature conditions;

[0039] (b11) Removal or reduction of total nitrogen under low temperature conditions;

[0040] (b12) Removal or reduction of total nitrogen under normal temperature conditions;

[0041] (b13) Removal or reduction of total nitrogen using organic nitrogen 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 above-mentioned Alcaligenes bacillus as an active ingredient to obtain the bacterial agent.

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

[0044] The present invention also provides a method for sewage treatment and denitrification, comprising the following steps: under conditions of high ammonia nitrogen concentration, using the above-mentioned Alcaligenes or the above-mentioned bacterial agent or the above-mentioned culture on the sewage to remove or reduce the ammonia nitrogen and total nitrogen content in the sewage.

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

[0046] (c1) Biological denitrification;

[0047] (c2) Improve denitrification efficiency;

[0048] (c3) sewage treatment;

[0049] (c4) Improve sewage treatment efficiency.

[0050] The alcaligenes HC-1 provided by the present invention is a Gram-negative bacterium isolated and purified from an activated sludge sample of an urban sewage treatment plant. The strain has good effects in sewage treatment, wastewater containing ammonia nitrogen and organic nitrogen; environmental remediation, black and odorous water body and bottom mud remediation; horticultural pond live aquaculture, family fish tanks, ornamental fish farming, aquaculture and other microbial treatments. The strain HC-1 of the present invention has a complete dnf gene cluster, and the denitrification method is mainly converted into gas, and the gas is mainly nitrogen. Ammonia is oxidized to hydroxylamine through the Dirammox pathway, and then a gaseous nitrogen product is directly produced. The traditional denitrification steps are greatly simplified, the denitrification efficiency is improved, and space and cost are saved.

[0051] The strain HC-1 of the present invention has the ability of aerobically converting ammonia into nitrogen as a heterotrophic bacterium, and has good ammonia nitrogen and total nitrogen removal capabilities. When grown with ammonia nitrogen (140 mg / L) as the only nitrogen source, 55.61% can be converted into nitrogen, the ammonia nitrogen removal rate is 100%, and the total nitrogen removal rate of the water body is 72.62%; the strain HC-1 has good pH adaptability, grows well in the pH range of 6-10 and has direct ammonia oxidation ability, can efficiently remove 100% of ammonia nitrogen and more than 62% of total nitrogen; the strain HC-1 has salt tolerance, and can still achieve 100% ammonia nitrogen removal rate and 66% total nitrogen removal rate at a NaCl concentration of 60 g / L; the strain HC-1 has a wide C / N adaptability, grows well in the C / N range of 3-20, and can efficiently remove ammonia nitrogen and total nitrogen. Even under low carbon-nitrogen ratio (C / N=3 or 5), the strain HC-1 can still maintain high ammonia nitrogen removal rate (100%) and total nitrogen removal rate (more than 56%); the strain HC-1 can maintain good growth and efficient nitrogen removal under high ammonia nitrogen concentration conditions. Under the condition of ammonia nitrogen concentration of 5600g / L, it still grows well, with an ammonia nitrogen removal rate of 69.4% and a total nitrogen removal rate of 52.2%; the strain HC-1 can grow well and efficiently remove nitrogen under the condition of organic nitrogen source (such as corn pulp) as the only nitrogen source, and the total nitrogen removal rate reaches 67.50±2.68%.

[0052] Collection Instructions

[0053] Bacteria name: Alcaligenes

[0054] Latin name: Alcaligenes nematophilus

[0055] Strain ID: HC-1

[0056] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0057] Abbreviation of depository institution: CGMCC

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

[0059] Deposit date: June 7, 2023

[0060] The registration number of the Collection Center is: CGMCC No.27571. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The phylogenetic tree of strain HC-1 based on 16S rRNA gene sequence and whole genome sequence. Left: 16S rRNA gene sequence, right: whole genome sequence.

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

[0063] Figure 3 This is a graph showing the growth and direct ammonia oxidation ability of strain HC-1 using ammonia nitrogen as the sole nitrogen source.

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

[0065] Figure 5 This is a test of the total nitrogen removal rate of strain HC-1 using ammonia nitrogen as the only nitrogen source.

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

[0067] Figure 7 Effects of different NaCl concentrations on the growth and direct ammonia oxidation ability of strain HC-1.

[0068] Figure 8 Effects of different C / N conditions on the growth and direct ammonia oxidation ability of strain HC-1.

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

[0070] Fig.10 Effects of different dissolved oxygen conditions on the growth and direct ammonia oxidation ability of strain HC-1.

[0071] Fig.11 The effect of different temperatures on the growth and direct ammonia oxidation ability of strain HC-1.

[0072] Fig.12 To detect the growth and total nitrogen removal rate of strain HC-1 under the condition of organic nitrogen as the only nitrogen source. DETAILED DESCRIPTION

[0073] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0074] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0075] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0076] The culture medium preparation method involved in the embodiment of the following embodiment is as follows:

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

[0078] Heterotrophic Nitrification Media (HNM): 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), make up to 1 L with ddH2O, pH 7.0.

[0079] Trace elements: 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, make up to 1L with ddH2O, pH 6.0.

[0080] The following examples used Origin 2023GraphPad Prism 9 and SPSS11.5 statistical software to process the data. The experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated a very 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 municipal sewage treatment plant in Changsha, Hunan Province. (Collector: Chen Han chenhan@ihb.ac.cn) The collected activated sludge samples were mixed and added to a conical flask containing 100mL 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 make the final concentration of hydroxylamine 2mmol / L as a pressure stress for selective enrichment, and placed in a constant temperature shaking incubator at 28°C and 180rpm for culture.

[0084] Strain isolation: For the above enrichment culture samples, use a pipette to take 1 mL each day and add it to 9 mL of sterile saline (0.9% NaCl). Invert each step to mix evenly. Finally, dilute 10 times to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 200 μL of each dilution was applied to a HNM medium plate containing 2 mM hydroxylamine, and cultured at 28°C for 24-72 hours until a single colony grew, and the bacterial growth morphology was observed and preliminarily classified. After continuous selective enrichment screening, a strain of bacteria with direct ammonia oxidation ability was finally obtained, which was named strain HC-1.

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

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

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

[0088] The PCR product was sent to a sequencing company for first-generation sequencing, and the sequencing results were compared with the NCBI BLASTN database ( http: / / www.ncbi.nlm.nih.gov / BLAST ) and the selected standard strains of Alcaligenes. The analysis of 16S rRNA genome and whole genome evolutionary tree, colinearity, ANI / AAI showed that ( Figure 1 ), HC-1 has a higher affinity with Alcaligenes nematophilus. It is followed by Alcaligenes aquatilis and Alcaligenes ammonioxydans. Finally, it was named Alcaligenes nematophilus HC-1, or strain HC-1 for short.

[0089] The strain HC-1 is a Gram-negative bacterium. The colonies are milky white, round, smooth and moist, opaque, and have relatively neat edges. After culturing on an LB plate at 30°C for 2 days, the colony diameter is about 2-3 mm. The cells are short rod-shaped, and the cell size is about (0.5-0.6) μm×(0.7-0.9) μm.

[0090] 3. Storage of strain HC-1

[0091] Alcaligenes nematophilus HC-1 was deposited in China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​June 7, 2023, with the deposit number CGMCC 27571. Hereinafter referred to as Alcaligenes nematophilus CGMCC No. 27571 or Alcaligenes nematophilus HC-1 or strain HC-1.

[0092] Example 2: Analysis of denitrification capacity of Alcaligenes HC-1

[0093] The strain HC-1 was pre-cultured in LB medium to complete strain activation. The activated strain HC-1 was transferred to HNM medium at a 1% inoculation amount and cultured at 30°C and 160rpm for 16 hours to finally obtain the Alcaligenes HC-1 bacterial agent.

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

[0095] By sequencing the whole genome of strain HC-1, it was found that HC-1 had a complete direct ammonia oxidation gene cluster (dnf gene cluster Figure 2 The dnfA gene is 954 bp long, and the nucleotide sequence is sequence 2 in the sequence list, which is predicted to encode di-iron oxygenase; the dnfB gene is 1053 bp long, and the nucleotide sequence is sequence 3 in the sequence list, which is predicted to encode 2Fe-2S iron-sulfur cluster binding domain protein; the dnfC gene is 723 bp long, and the nucleotide sequence is sequence 4 in the sequence list, which is predicted to encode glutamine aminotransferase. This proves at the genome level that strain HC-1 has the ability to complete denitrification through the direct ammonia oxidation (Dirammox) pathway.

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

[0097] The strain HC-1 was pre-cultured in LB medium to complete the strain activation. Then, 1 mL of the strain HC-1 seed solution was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of HNM medium (C / N=10), which contained 5 mM (NH4)2SO4. The Erlenmeyer flask was incubated on a rotary shaker at 30°C and 160 rpm. During the 60 h incubation period, the bacterial growth (OD 600 ), NH4 - -N, NH2OH-N, NO2 —N content. The determination method of ammonia nitrogen refers to: Determination of ammonia nitrogen in water by salicylic acid spectrophotometry HJ 536-2009; the determination method of nitrite nitrogen refers to: Determination of nitrite nitrogen in water by spectrophotometry GB7493-87; the determination method of hydroxylamine nitrogen refers to: the paper "Frear, DS, & Burrel, 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 generate a stable green condensation product 5,8-quinol inequinone-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 705nm.

[0098] The ammonia oxidation and denitrification abilities of the cells were tested in HNM medium and it was found that during the ammonia conversion process, ammonia consumption was accompanied by cell growth. Figure 3 Hydroxylamine accumulates in large quantities during this process and is eventually consumed completely, accompanied by the production of small amounts of nitrite and nitrate.

[0099] After 4 h of growth, the HC-1 cells entered the logarithmic phase, and the OD 600 It reaches the highest value (0.328) and then decreases slightly. - -N decreased, NH2OH-N gradually accumulated, reaching a maximum concentration of 48.42 mg / L at 30 h, and then gradually decreased to almost undetectable. The accumulation of nitrite (9.50 mg / L) and nitrate (5.33 mg / L) was relatively small during the entire reaction process.

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

[0101] The strain HC-1 was pre-cultured in LB medium, and then 40 μL of HC-1 bacterial solution was inoculated into 4 mL of HNM (containing 5 mM ( 15 The top space of the bottle was filled with He / O2 at 4:1 as above, and the flask was incubated on a rotary shaker at 30°C and 160 rpm. The N2-N and N2O-N were measured for 120 h, and then the bacterial growth (OD 600 ), NH4 - -N, NH2OH-N, NO2 --N、NO3 — N.

[0102] The results showed that HC-1 produced nitrogen during the aerobic conversion of ammonia. According to nitrogen balance analysis, strain HC-1 could eventually convert 10mM 15 NH4-N is completely consumed and 55.61% of the consumed 15 NH4 + Convert to 15 N2, while generating 10.51% 15 N2O( Figure 4 ). Meanwhile, small 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, and the total nitrogen removal rate reached 75.81%.

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

[0104] The removal rate of total nitrogen in water by strain HC-1 was tested by using a total nitrogen reagent (Test ˉN Tub, HACH), digested with alkaline potassium persulfate by COD heater (DRB200, HACH), and then measured by spectrophotometer (DR890, HACH). The method for determining the biomass of strain HC-1 was the same as that for determining total nitrogen, and the biomass nitrogen was calculated by the difference method.

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

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

[0107] 1. Optimization of pH conditions of 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, the initial ammonia nitrogen concentration was set to 140 mg / L, C / N was 10, and the culture was shaken at 30°C and 160 rpm to determine the growth and direct ammonia oxidation capacity of the strain HC-1. The growth determination, direct ammonia oxidation capacity determination, and total nitrogen (TN) removal rate detection were the same as those 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 has good direct ammonia oxidation ability. At pH 7-9, it has a very high growth rate; at pH 6, the growth rate is reduced, but the accumulation of hydroxylamine is very high, about 1600μM; similarly, the accumulation of nitrite nitrogen is the highest at pH 6, about 800μM.

[0110] The removal capacity of ammonia nitrogen and total nitrogen is shown in Table 1 below. In the pH range of 6-10, the ammonia nitrogen removal rate of strain HC-1 is 100%, and the total nitrogen removal rate is between 62.62-68.81%.

[0111] Table 1. Ammonia nitrogen and total nitrogen removal capacity in 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 concentration conditions of HNM medium

[0114] The NaCl concentration of the 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, C / N was 10, the initial pH was 7.0, and the culture was shaken at 30°C and 160 rpm to determine the growth and direct ammonia oxidation capacity of the strain HC-1. The growth determination, direct ammonia oxidation capacity determination, and total nitrogen (TN) removal rate detection were the same as those in Example 2.

[0115] The results are as follows Figure 7 As shown, strain HC-1 grew well in the range of NaCl concentration of 0-60 g / L and had good direct ammonia oxidation ability. When the NaCl concentration was 20-60 g / L, it had a higher growth rate than the control group (NaCl concentration was 0 g / L); the accumulation of hydroxylamine and nitrite nitrogen decreased with the increase of NaCl concentration.

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

[0117] Table 2. Ammonia nitrogen and total nitrogen removal capacity in fermentation broth under different NaCl concentration conditions

[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 of HNM medium

[0120] Sodium citrate was used as the sole carbon source, and the C / N ratio was 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 shaken at 30°C and 160 rpm. The growth and direct ammonia oxidation capacity of strain HC-1 were determined. The growth determination, direct ammonia oxidation capacity determination, and total nitrogen (TN) removal rate detection were the same as those in Example 2.

[0121] The results are as follows Figure 8 As shown, strain HC-1 grows well in the range of C / N of 3-20 and has good direct ammonia oxidation ability. In the range of C / N of 3-20, the growth rate and growth amount of strain HC-1 increase with the increase of C / N, and there is no obvious increase when C / N=10; there is no obvious difference in the accumulation of hydroxylamine in the range of C / N of 3-20.

[0122] The removal capacity of ammonia nitrogen and total nitrogen is shown in Table 3 below. In the range of C / N of 3-20, the ammonia nitrogen removal rate of strain HC-1 is 100%, and the total nitrogen removal rate is between 56.19-69.05%.

[0123] Table 3. Ammonia nitrogen and total nitrogen removal capacity in fermentation broth under different C / N conditions

[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 medium + -N concentration optimization

[0126] Add NH4 + -N concentration was adjusted to 700, 1400, 2800 and 5600 mg / L, respectively, sodium citrate was used as the sole carbon source, the initial carbon source concentration was 7000 mg / L, the initial pH was 7.0, and the culture was shaken at 30°C and 160 rpm to determine the growth and direct ammonia oxidation capacity of strain HC-1. The growth determination, direct ammonia oxidation capacity determination, and total nitrogen (TN) removal rate detection were the same as those in Example 2.

[0127] The results are as follows Fig. 9 As shown, strain HC-1 grows well in the range of ammonia nitrogen concentration of 700-5600 mg / L and has good direct ammonia oxidation ability. When the ammonia nitrogen concentration is 700-5600 mg / L, the growth situation is basically the same; the accumulation of hydroxylamine decreases with the increase of ammonia nitrogen concentration at 6 hours; nitrite nitrogen only accumulates a small amount after the growth enters the decay stage.

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

[0129] Table 4. Different NH 4+ -N concentration under the condition of ammonia nitrogen and total nitrogen removal capacity in fermentation broth

[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 culture medium

[0132] Sodium citrate was used as the sole carbon source. The shaking speed was adjusted to 0, 80, 160, and 200 rpm, respectively. 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 cultured at 30°C. The growth, direct ammonia oxidation ability, and total nitrogen (TN) removal rate of strain HC-1 were determined.

[0133] The results are as follows Fig.10 As shown, strain HC-1 grows well in the range of 0-200rpm and has good direct ammonia oxidation ability. In the range of C / N of 0-200rpm, the growth rate of strain HC-1 increases with the increase of dissolved oxygen (rotation speed), and there is no obvious increase when the rotation speed reaches 160rpm; the accumulation of hydroxylamine increases with the increase of dissolved oxygen (rotation speed).

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

[0135] Table 5. Ammonia nitrogen and total nitrogen removal capacity in fermentation broth under different dissolved oxygen conditions

[0136] 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 of HNM culture medium

[0138] Sodium citrate was used as the sole carbon source, and the temperature was adjusted to 10, 15, 20, 30, and 40°C, respectively. 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 shaken at 160 rpm to determine the growth, direct ammonia oxidation ability, and total nitrogen (TN) removal rate of strain HC-1.

[0139] The results are as follows Fig.11As shown, strain HC-1 grows well in the temperature range of 10-40℃ and has good direct ammonia oxidation ability. In the range of 10-40℃, the growth rate and growth amount of strain HC-1 increase with the increase of temperature. Whether under low temperature conditions of 10℃ or high temperature conditions of 40℃, strain HC-1 can ensure excellent ammonia nitrogen and total nitrogen degradation ability.

[0140] The ammonia nitrogen and total nitrogen capabilities are shown in Table 6. In the temperature range of 10-40°C, the ammonia nitrogen removal rate of strain HC-1 was 92.5-100%, and the total nitrogen removal rate was between 49.05-69.29%.

[0141] Table 6. Ammonia nitrogen and total nitrogen removal capacity in fermentation broth under different culture temperature conditions

[0142] Temperature(℃) 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 for HNM medium

[0144] The inorganic nitrogen source (ammonium sulfate) of the HNM medium was adjusted to an organic nitrogen source (corn steep liquor), sodium citrate was used as the sole carbon source, the initial pH was 7.0, and the culture was shaken at 30°C and 160rpm to determine the growth and total nitrogen removal capacity of the strain HC-1. The growth determination, direct ammonia oxidation capacity determination, and total nitrogen (TN) removal rate detection were the same as those in Example 2.

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

[0146] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Alcaligenes, characterized in that: The alcaligenes nematophilus is Alcaligenes nematophilus CGMCC No. 27571, and its registration number in the General Microbiological Center of China National Culture Collection Administration is CGMCC No. 27571.

2. A bacterial agent, characterized in that: The bacterial agent contains the Alcaligenes Bacillus according to claim 1 and / or a metabolite of the Alcaligenes Bacillus.

3. The culture of Alcaligenes according to claim 1, which is obtained by culturing the Alcaligenes according to claim 1 in a bacterial culture medium.

4. Use of the alcaligenes bacillus according to claim 1, the bacterial agent according to claim 2, or the culture according to claim 3 in at least one of the following: (a1) Biological denitrification; (a2) preparing products of biological denitrification; (a3) sewage treatment; (a4) Preparing products for sewage treatment.

5. The use according to claim 4, characterized in that: The biological denitrification is embodied in whole or in part as follows: (b1) Removal or reduction of ammonia nitrogen under normal salinity conditions; (b2) Removal or reduction of ammonia nitrogen under salt-tolerant conditions; (b3) Removal or reduction of total nitrogen under normal salinity conditions; (b4) Removal or reduction of total nitrogen under salt-tolerant conditions; (b5) Removing or reducing ammonia nitrogen under normal ammonia nitrogen conditions; (b6) Removing or reducing ammonia nitrogen under high ammonia nitrogen concentration conditions; (b7) Removal or reduction of total nitrogen under normal ammonia nitrogen conditions; (b8) Removal or reduction of total nitrogen under conditions of high ammonia nitrogen concentration; (b9) Removing or reducing ammonia nitrogen under low temperature conditions; (b10) Removal or reduction of ammonia nitrogen under normal temperature conditions; (b11) Removal or reduction of total nitrogen under low temperature conditions; (b12) Removal or reduction of total nitrogen under normal temperature conditions; (b13) Removal or reduction of total nitrogen using organic nitrogen as the nitrogen source.

6. A method for preparing the bacterial agent according to claim 2, comprising the following steps: using the Alcaligenes bacillus according to claim 1 as an active ingredient to obtain the bacterial agent.

7. Biological denitrification product, characterized by: The biological denitrification product contains the Alcaligenes described in claim 1 or the bacterial agent described in claim 2 or the culture described in claim 3.

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

9. 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 denitrification efficiency; (c3) sewage treatment; (c4) Improve sewage treatment efficiency.

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