Application of a salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacterium in wastewater treatment and plant growth promotion

Abstract

The present invention discloses an application of a salt-tolerant heterotrophic nitrification-aerobic denitrifying bacteria in wastewater treatment and plant growth promotion. The present invention provides an application of Glutamicibacter nicotianae XH-220 or a bacterial agent containing the Glutamicibacter nicotianae XH-220 in nitrogen-containing wastewater and cucumber seedling growth promotion treatment; the Glutamicibacter nicotianae XH-220 is numbered ACCC 62208 in the Agricultural Microbiology Center of the China Microbiological Culture Collection Administration. The strain has acid resistance, salt resistance and heavy metal resistance, and also has a plant growth promotion effect. The present invention has important application prospects in the fields of sewage treatment and plant growth promotion.

Description

Technical Field

[0001] The invention relates to the technical field of environmental microorganisms, and in particular to application of a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium in wastewater treatment and plant growth promotion. Background Art

[0002] Nitrogen is a major component of nature. It is constantly transformed and circulated between -5-valent to +3-valent compounds and simple substances in the natural environment. + -N, NO2 - -N、NO3 - -N four forms exist stably in the water environment. In the steady-state environment of nitrogen cycle, the accumulation of certain types of nitrogen will not occur, but in recent years, more and more human production and life activities have caused damage to this natural cycle to a certain extent. With the progress of industry, agricultural development and the improvement of residents' living standards, the problem of water resource pollution has become increasingly serious and high, posing a threat to the ecological environment and human health. A large amount of nitrogen discharged from different sources cannot be effectively treated, and the discharge into the water will cause serious harm to human health and the natural environment. When the nitrate nitrogen and nitrite nitrogen in the water exceed the "Drinking Water Standard DB4403 / T 60-2020"> 10mg / L, long-term accumulation can easily lead to intolerance in infants and young children, causing "blue baby disease" and hemoglobinemia, and in severe cases, it can cause cancer or death. For the natural ecological environment, the continuous discharge and enrichment of high nitrogen will lead to water pollution and eutrophication, which will greatly reduce the dissolved oxygen in the water, and a large number of autotrophic photosynthetic bacteria and algae will multiply, and eventually the vicious cycle will become "dead water". Similarly, when nitrite nitrogen accumulates in water, it is easy for aquatic animals to absorb it, which will cause the iron valence in their hemoglobin to change, damage the ability of red blood cells to bind oxygen, and eventually cause the aquatic animals to suffocate and die. Moreover, polluted water enters plants through agricultural irrigation, and eventually accumulates in the human body through human drinking, causing great harm to the human body.

[0003] Biological denitrification mainly utilizes the nitrification and denitrification of denitrification-related microorganisms. Through the action of the enzyme pathway of microorganisms, various nitrogen-containing substances in the water are finally converted into nitrogen gas to escape, thereby reducing or removing nitrogen-containing substances in sewage. At present, in conjunction with related processes such as immobilization, biological denitrification technology is widely used in sewage denitrification treatment. The reaction process mainly converts organic nitrogen into ammonia nitrogen, nitrite nitrogen and nitrate nitrogen through ammonification and nitrification under aerobic conditions, and then uses denitrification to reduce nitrate nitrogen to gaseous nitrogen overflowing from sewage under anaerobic conditions, thereby achieving the purpose of denitrification. However, it has the following disadvantages: (1) Nitrifying bacteria are inorganic chemoautotrophic bacteria, and their nutritional metabolism type determines their slow growth, long generation cycle, low biomass concentration and poor environmental adaptability. (2) Traditional nitrifying bacteria are strictly aerobic bacteria, and traditional denitrifying bacteria are anaerobic or facultative anaerobic bacteria. Different conditions limit the rate of water denitrification under the same environment.

[0004] So people began to explore bacterial strains with nitrification and denitrification functions. In 1983, Robertson et al. successfully isolated a strain that can use organic carbon to oxidize ammonia nitrogen into hydroxylamine, then convert it into nitrite nitrogen and nitrate nitrogen under the condition of oxygen participation, and finally generate nitrogen gas (NH4 + →NH2OH→NO2 - →NO3 - →NO2 - →NO→N2O→N2), which was later revised to Paracoccus pantotrophus, and then started the screening and research of nitrification and denitrification co-functional bacteria. And this bacterium reacts in a different way under aerobic conditions. Therefore, Robertson proposed the heterotrophic nitrification-aerobic denitrification HN-AD (Heterotrophic Nitrification-Aerobic Denitrification) theory. Heterotrophic nitrification-aerobic denitrification bacteria have a faster growth rate and shorter cycle than traditional microbial denitrification bacteria, a simple denitrification procedure, a faster denitrification rate, low requirements for the living environment, and strong adaptability to temperature, pH, and DO. And this strain of bacteria has the ability to remove nitrogen sources of different valence types in wastewater (organic nitrogen, NH4 + -N, NO2 - -N、NO3 --N). Low energy consumption, in the process of denitrification reaction, only the organic matter supply of nitrification reaction needs to be started at the same time as the initial addition of bacteria, and no carbon source needs to be added in the middle. The acid and alkali produced by itself can be neutralized during the whole reaction process, and the pH of the system can be stabilized. However, most of these related reports are reports on the denitrification characteristics of bacterial strains, and there is no report on the heterotrophic nitrifying aerobic denitrifying bacteria Glutamicibacter nicotianae that has acid resistance, heavy metal resistance, salt resistance and can simultaneously remove ammonia nitrogen, nitrate nitrogen and nitrite nitrogen from environmental wastewater. Summary of the invention

[0005] The invention aims to provide a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium for use in wastewater treatment and plant growth promotion.

[0006] In the first aspect, the present invention claims the use of Glutamicibacter nicotianae XH-220 or a bacterial agent containing the Glutamicibacter nicotianae XH-220 in the treatment of nitrogen-containing wastewater. Wherein, the number of the Glutamicibacter nicotianae XH-220 in the Agricultural Microbiology Center of the China Microbiological Culture Collection Administration is ACCC 62208.

[0007] Furthermore, the wastewater treatment may be biological denitrification of the water body.

[0008] Furthermore, the nitrogen in the wastewater may be ammonia nitrogen, nitrite nitrogen and / or nitrate nitrogen.

[0009] Further, the pH of the wastewater is not less than 3. For example, the pH is not less than 5, and further, the pH is 5-11, and further, the pH is 7-11.

[0010] Furthermore, the total salinity of the wastewater is not higher than 10% by mass (ie, the mass fraction of NaCl in the wastewater is not higher than 10%).

[0011] Furthermore, the Mn in the wastewater 2+ Not higher than 10mmol / L, and / or Pb 2+ , Fe 3+ , Cu 2+ Not higher than 5mmol / L, and / or Zn 2+ 、Co 2+ 、Ni 2+ Cr 6+ Not higher than 1mmol / L.

[0012] Furthermore, the C / N ratio of the wastewater is 10±0.2.

[0013] In a second aspect, the present invention claims a method for treating wastewater.

[0014] The wastewater treatment method claimed in the present invention may include the following steps: inoculating tobacco glutamicum bacillus (Glutamicibacter nicotianae) XH-220 or a bacterial agent containing the tobacco glutamicum (Glutamicibacter nicotianae) XH-220 into the wastewater to be treated for cultivation. Wherein, the tobacco glutamicum (Glutamicibacter nicotianae) XH-220 has a deposit number of ACCC 62208 in the Agricultural Microbiology Center of the China Microbiological Culture Collection Administration.

[0015] Furthermore, after inoculation, the content of Glutamicibacter nicotianae XH-220 in the wastewater system was 1×10 6 CFU / ml-3.0×10 7 CFU / ml, such as 1×10 7 CFU / ml.

[0016] Furthermore, the culture is carried out at a temperature of 28-32°C, such as 30°C.

[0017] Furthermore, the culturing time is 36-60h, such as 48h.

[0018] Furthermore, the culture may be shaken at 180 r / min.

[0019] Furthermore, the wastewater is nitrogen-containing wastewater. Furthermore, the nitrogen in the wastewater may be ammonia nitrogen, nitrite nitrogen and / or nitrate nitrogen.

[0020] Furthermore, the pH of the wastewater is not less than 5. For example, the pH is 5-11, and for example, the pH is 7-11.

[0021] Furthermore, the total salinity of the wastewater is not higher than 10% by mass (ie, the mass fraction of NaCl in the wastewater is not higher than 10%).

[0022] Furthermore, the Mn in the wastewater 2+ Not higher than 10mmol / L, Pb 2+ , Fe 3+ Not higher than 5mmol / L, Zn 2+ 、Co 2+ 、Ni 2+ Cr 6+ Not higher than 1mmol / L.

[0023] Furthermore, the C / N ratio of the wastewater is 10±0.2.

[0024] In the first and second aspects above, the initial relevant nitrogen concentration in the wastewater is preferably not higher than 100 mg / L.

[0025] In a third aspect, the present invention claims to protect the use of Glutamicibacter nicotianae XH-220 or a bacterial agent containing the Glutamicibacter nicotianae XH-220 in biological denitrification and / or promoting plant growth. Wherein, the deposit number of the Glutamicibacter nicotianae XH-220 in the Agricultural Microbiology Center of the China Microbiological Culture Collection Administration is ACCC 62208.

[0026] Further, the nitrogen is ammonia nitrogen, nitrite nitrogen and / or nitrate nitrogen;

[0027] Furthermore, the promoting of plant growth is manifested as promoting an increase in plant height and / or promoting an increase in plant leaf length.

[0028] In a specific embodiment of the present invention, the biological denitrification is to inoculate the tobacco glutamicum bacteria (Glutamicibacter nicotianae) XH-220 into a heterotrophic nitrification-aerobic denitrification medium for cultivation, thereby completing denitrification; the solvent of the heterotrophic nitrification-aerobic denitrification medium is water, and the solutes and concentrations are as follows: CH3COONa3.42g / L, NH4Cl 0.0.1284g / L, NaNO2 0.1643g / L, KNO3 0.241g / L, K2HPO4 0.2g / L, MgCl20.05g / L, CaCl2 0.05g / L, ZnSO4 0.05g / L, 0.1% (V / V) trace element solution; pH 7.2. The solvent of the trace element solution is water, and the solutes and concentrations are as follows: EDTA 50g / L, CaCl2 5.5g / L, ZnSO4 2.2g / L, MnCl2·4H2O5.06g / L, FeSO4·7H2O 5.0g / L, (NH4)6Mo7O 24 ·4H2O 1.1g / L, CuSO4·5H2O 1.57g / L, CoCl2·6H2O 1.61g / L; pH7.0.

[0029] Furthermore, after inoculation, the content of Glutamicibacter nicotianae XH-220 in the culture system can be 1×106 CFU / ml-3.0×10 7 CFU / ml, such as 1×10 7 CFU / ml.

[0030] Furthermore, the culture may be carried out at a temperature of 28-32°C, such as 30°C.

[0031] Furthermore, the culturing time may be 36-60 hours, such as 48 hours.

[0032] Furthermore, the culture may be shaken at 180 rpm.

[0033] Furthermore, before carrying out the biological denitrification, the method further includes the steps of activating the tobacco glutamicum bacillus (Glutamicibacternicotianae) XH-220 and / or preparing a bacterial suspension. Specifically, the Glutamicibacternicotianae XH-220 is inoculated on an LB slant culture medium and cultured at 35° C. for 1-2 days to obtain activated bacterial cells. Then, the activated bacterial cells are inoculated into 100 ml of LB liquid seed culture medium according to a conventional amount and cultured at 35° C. for 12 hours to obtain a bacterial suspension of the tobacco glutamicum bacillus (Glutamicibacter nicotianae) XH-220.

[0034] In a fourth aspect, the present invention claims a method for promoting plant growth.

[0035] The method for promoting plant growth claimed in the present invention may include the following steps: inoculating tobacco glutamicum bacillus (Glutamicibacter nicotianae) XH-220 or a bacterial agent containing the tobacco glutamicum (Glutamicibacternicotianae) XH-220 into the rhizosphere soil of the plant to be treated. For example, the plant to be treated is irrigated with a bacterial suspension of the tobacco glutamicum (Glutamicibacter nicotianae) XH-220). Wherein, the preservation number of the tobacco glutamicum (Glutamicibacter nicotianae) XH-220 in the Agricultural Microbiology Center of the China Microbiological Culture Collection Administration is ACCC 62208.

[0036] In the third aspect or the fourth aspect, the plant may be a plant of the Cucurbitaceae family.

[0037] Furthermore, the Cucurbitaceae plant may be a Cucumber plant.

[0038] Furthermore, the Cucumis plant may be cucumber.

[0039] In a specific embodiment of the present invention, the cucumber is specifically the cucumber variety Jinyan No. 4.

[0040] Experiments have shown that Glutamicibacter nicotianae XH-220 (ACCC 62208) can grow under aerobic conditions with the addition of sodium acetate as the carbon source, using ammonia nitrogen, nitrate nitrogen or nitrite nitrogen as the sole nitrogen source, and simultaneously carry out heterotrophic nitrification and aerobic denitrification, and has the ability to remove ammonia nitrogen, nitrate nitrogen and nitrite nitrogen. Within 48 hours, 1×10 7 CFU / ml concentration of bacteria in the ammonium salt ammoniation performance test medium to 100mg / L ammonia nitrogen (NH4 + -N) removal rate was 35.14%; the nitrite denitrification performance was determined in the culture medium for 100 mg / L nitrite nitrogen (NO2 - -N) removal rate was 18.53%; the denitrification performance was tested in the culture medium for 100 mg / L nitrate nitrogen (NO3 - -N) removal rate was 56.84%; in heterotrophic nitrification-aerobic denitrification medium, 100 mg / L of three nitrogen mixed nitrogen sources (the three nitrogen sources were 33.33 mg / L) nitrate nitrogen (NO3 - -N), nitrite nitrogen (NO2 - -N), ammonia nitrogen (NH4 + -N) removal rates were 46.56%, 26.48%, and 83.62% respectively. It can tolerate a salt concentration of 10% and a pH of 3, and can remove 10 mmol / L of metal ions Mn 2+ ,5mmol / L metal ion Pb 2+ , Fe 3+ , Cu 2+ ,1mmol / L metal ion Zn 2+ 、Ni 2+ 、Co 2+ Cr 6+ It has tolerance. It has a certain growth-promoting effect on cucumber seedlings. Therefore, Glutamicibacter nicotianae XH-220 has important application prospects in the fields of sewage treatment and / or plant growth promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The following is the analysis result of the genetic evolution tree (neighbor-joining method) of the strain Glutamicibacter nicotianae XH-220 of the present invention.

[0042] Figure 2The figure is a morphological diagram of a single colony streaked on a XH-220LB solid plate of the tobacco glutamicum bacillus (Glutamicibacter nicotianae) of the present invention.

[0043] Figure 3 This is a 100-fold optical microscope morphology diagram of the tobacco glutamicum bacillus (Glutamicibacter nicotianae) XH-220LB liquid culture of the present invention.

[0044] Figure 4 The effect of different carbon sources on the 48h growth of Glutamicibacter nicotianae XH-220.

[0045] Figure 5 The effect of different carbon-nitrogen ratios on the 48h growth of Glutamicibacter nicotianae XH-220.

[0046] Figure 6 The effect of different pH on the growth of Glutamicibacter nicotianae XH-220 for 48 hours.

[0047] Figure 7 The effect of different temperatures on the 48h growth of Glutamicibacter nicotianae XH-220.

[0048] Figure 8 The effect of different salinities on the growth of Glutamicibacter nicotianae XH-220 for 48 hours.

[0049] Fig. 9 The effects of different heavy metal concentrations on the growth of Glutamicibacter nicotianae XH-220 48.

[0050] Fig.10 This is the removal effect of ammonia nitrogen by Glutamicibacter nicotianae XH-220 for 48 hours.

[0051] Fig.11 This is the removal effect of nitrite nitrogen by Glutamicibacter nicotianae XH-220 for 48 hours.

[0052] Fig.12This is the removal effect of nitrate nitrogen by Glutamicibacter nicotianae XH-220 in 48 hours.

[0053] Fig.13 This is the removal effect of mixed nitrogen sources by Glutamicibacter nicotianae XH-220 for 48 hours.

[0054] Fig.14 This is the growth-promoting effect of Glutamicibacter nicotianae XH-220 on cucumber seedlings for 48 hours.

[0055] Fig.15 The data show that Glutamicibacter nicotianae XH-220 can promote the growth of cucumber seedlings for 14 days after 48 hours of exposure. DETAILED DESCRIPTION

[0056] 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.

[0057] 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.

[0058] The Glutamicibacter nicotianae XH-220 in the following embodiments has been collected by the Agricultural Microorganism Center of the China Microorganism Culture Collection Administration Committee before the filing date of this application, also known as the Agricultural Culture Collection of China (ACCC for short, address: No. 12, Zhongguancun South Street, Haidian District, Beijing, Institute of Agricultural Resources and Agricultural Zoning, Chinese Academy of Agricultural Sciences, Postal Code 100081), the strain number is ACCC 62208, the collection date is June 28, 2022, from the date of collection, the public can obtain the strain from the Agricultural Microorganism Center of the China Microorganism Culture Collection Administration Committee. ACCC has a special website, the website address is: http: / / www.accc.org.cn, the public can directly order strains online. The website address of Glutamicibacter nicotianae XH-220 is http: / / www.accc.org.cn / Column.asp? Column_ID=34929&Model=product_detail&P_ID=1817434873.

[0059] Example 1. Isolation and Identification of Glutamicibacter nicotianae XH-220

[0060] A sludge sample of a fish pond in Caofeidian District, Tangshan City, Hebei Province was taken, and after being acclimated and enriched with carbon dioxide carbon source and nitrate nitrogen source, it was spread and separated to obtain the Glutamicibacter nicotianae XH-220 of the present invention.

[0061] 1. Acclimatization, enrichment, separation and expansion culture media

[0062] Acclimation enrichment medium: The solvent is water, and the solutes and concentrations are as follows: KNO3 0.722g / L, CO2 is continuously introduced, K2HPO4 0.2g / L, MgCl2 0.05g / L, CaCl2 0.05g / L, ZnSO4 0.05g / L, 0.1% (V / V) trace element solution; pH 7.2. Trace element solution: The solvent is water, and the solutes and concentrations are as follows: EDTA 50g / L, CaCl2 5.5g / L, ZnSO42.2g / L, MnCl2·4H2O 5.06g / L, FeSO4·7H2O 5.0g / L, (NH4)6Mo7O 24·4H2O 1.1g / L, CuSO4·5H2O 1.57g / L, CoCl2·6H2O 1.61g / L; pH7.0.

[0063] Separation medium: The solvent is water, and the solutes and concentrations are as follows: CH3COONa 1.0g / L, KNO3 0.2g / L, K2HPO40.2g / L, MgCl2 0.05g / L, CaCl2 0.05g / L, ZnSO4 0.05g / L, 0.1% (V / V) trace element solution; pH 7.2. The solid medium is supplemented with 2% agar (20g / L) and 0.1% (V / V) bromo-musk thymol indicator solution.

[0064] Expansion culture medium: The solvent is water, and the solutes and concentrations are as follows: trypsin 10 g / L, yeast extract 5 g / L, NaCl 10 g / L; pH 7.2.

[0065] 2. Isolation and purification of strain XH-220

[0066] The sterile water of the sample acclimated in the high nitrate environment was prepared into 100 mL solution at a ratio of 1:9. At this time, the solution was diluted by 10 -1 , add 1 mL of the solution into a 9 mL test tube filled with sterile water, and repeat this step to dilute to 10 -2 , 10 -3 , 10 -4 , 10 -5 There are five gradients in total. In a sterile clean bench, take 20 μL of the bacterial suspension of 5 gradient concentrations on the surface of the prepared solid separation medium, and spread it evenly with a sterile glass coating rod. After it is completely infiltrated, cover the culture dish lid and seal the culture dish. Each concentration gradient is three parallel. Invert the plate to be cultured in an incubator with a culture temperature of 30°C for 2-4 days. After a clear single colony grows, screen and mark the single strain to be selected with a marker and count it. In a sterile environment, open the culture dish, dip a small amount of single colony with a sterile inoculation loop in the pre-prepared LB culture medium and gently draw lines to avoid scratching. After an area is drawn, slowly rotate the plate, place the inoculation loop on the alcohol lamp to burn, remove the microorganisms on it, wait until the inoculation loop temperature cools down, connect the drawn area with the blank area and continue to draw lines. A plate is divided into 4 areas and drawn, and the plate is sealed with a sealing film. Invert the plate and culture it in an incubator at 30°C for 2-3 days. If the streaked strain is not a single strain, it needs to be streaked again until the streaked pure culture is a single strain. Each strain is purified more than twice. One of the strains obtained is numbered as strain XH-220.

[0067] 3. PCR Amplification and Sequencing of 16S rRNA

[0068] The DNA of the obtained XH-220 strain was extracted and purified using the Solebold Bacterial DNA Extraction D1600 Kit (Beijing Solebold Technology Co., Ltd.). 5 μL of DNA sample was subjected to electrophoresis at 110 V for 20 min and quality inspection was performed under a gel electrophoresis instrument.

[0069] The bacterial universal primers 16S-27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 16S-1492R (5'-CGGTTACCTTGTTACGACTTC-3') were used for PCR amplification. The PCR reaction conditions were: 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 90s, 35 cycles; then 72℃ extension for 10min; finally 4℃ storage.

[0070] After PCR product purification and quality inspection, the recovered PCR amplification products were entrusted to Sangon Biotechnology Co., Ltd. for sequencing.

[0071] After sequencing, the strain obtained a 1438 bp 16S rDNA sequence (SEQ ID No. 1). Using the BLASTN program of the National Center for Biotechnology Information (NCBI), it was found that the gene sequence of the 16S rRNA of the XH-220 strain of the present invention had 99.79% homology with the gene sequences of the 16S rRNA of multiple Glutamicibacter nicotianae strains registered by NCBI. The phylogenetic tree was constructed using the Neighbour Joining method using the Systematic Analysis (MEGA11) software. Figure 1 ), indicating that the XH-220 strain of the present invention is closely related to the known Glutamicibacter nicotianae NBRC 14234.

[0072] 4. Observation of morphological characteristics of XH-220 strain

[0073] The experimental method for observing morphological characteristics refers to the "Handbook of Systematic Identification of Common Bacteria" compiled by Dong Xiuzhu, Cai Miaoying, etc., Science Press, 2001, first edition, p353-363.

[0074] XH-220 strain morphological characteristics: solid culture colonies are round, convex, with neat edges, smooth surface, yellow, opaque, such as Figure 2The strain is observed under a microscope as a bacillus, does not form spores, and has a straight or slightly curved body shape with irregular rod-like shapes at both ends. Figure 3 .

[0075] 5. Identification of Physiological and Biochemical Characteristics of XH-220 Strain

[0076] The culture medium and experimental methods of physiological and biochemical tests refer to the biolog GenIII plate of the American BIOLOG company Biolog, Inc., US Patent #5,627,045.

[0077] Physiological and biochemical characteristics of XH-220 strain: Gram staining positive, aerobic, optimal growth temperature 30-40℃, at 30℃, it can utilize glucose, sucrose, fructose, glycerol, salt tolerance and acid tolerance; the results of physiological and biochemical experiments are detailed in Table 1.

[0078] Table 1. Some physiological and biochemical characteristics of strain XH-220 Biolog GenIII plate

[0079]

[0080]

[0081] Note: “(+)” means good growth or positive; “(-)” means no growth or negative; “(W)” means growth close to the blank control or false positive.

[0082] In view of the above-mentioned morphological characteristics, physiological and biochemical characteristics and 16S sequencing results of the XH-220 strain, the XH-220 strain was identified as Glutamicibacter nicotianae, and was collected in the Agricultural Microbiology Center of the China Culture Collection of Microorganisms, also known as the Agricultural Culture Collection of China (ACCC, address: No. 12, Zhongguancun South Street, Haidian District, Beijing, Institute of Agricultural Resources and Agricultural Zoning, Chinese Academy of Agricultural Sciences, Postal Code 100081) on June 28, 2022, with the number ACCC 62208.

[0083] VI. Effect of Carbon Source on the Growth of Glutamicibacter nicotianae XH-220

[0084] The suspension of Glutamicibacter nicotianae XH-220 cultured in LB medium was resuspended in PBS, and glucose, sucrose, sodium acetate, sodium citrate, and sodium succinate were added to the basal universal medium as the sole carbon source, and 100 mg / L ammonia nitrogen (NH4 + -N), 100mg / L nitrite nitrogen (NO2 - -N), 100mg / L nitrate nitrogen (NO3 - -N) and 100 mg / L three kinds of nitrogen mixed nitrogen sources (three nitrogen sources are 33.33 mg / L) respectively, and the carbon-nitrogen ratio in the control medium is C / N=10 (basal universal medium: K2HPO4 0.2 g / L, MgCl2 0.05 g / L, CaCl2 0.05 g / L, ZnSO4 0.05 g / L, 0.1% (V / V) trace element solution; pH 7.2. Trace element solution: EDTA 50 g / L, CaCl2 5.5 g / L, ZnSO4 2.2 g / L, MnCl2·4H2O 5.06 g / L, FeSO4·7H2O 5.0 g / L, (NH4)6Mo7O 24 ·4H2O 1.1g / L, CuSO4·5H2O 1.57g / L, CoCl2·6H2O 1.61g / L; pH7.0). After culturing at 30℃ and 180rpm for 48h, the OD600 value of Glutamicibacter nicotianae XH-220 in the system was monitored. The results showed that the growth rate of Glutamicibacter nicotianae XH-220 using sodium acetate was the fastest, so sodium acetate was selected as the carbon source for the subsequent experimental culture medium. For details, see Figure 4 .

[0085] VII. Effect of Carbon-Nitrogen Ratio on the Growth of Glutamicibacter nicotianae XH-220

[0086] The suspension of Glutamicibacter nicotianae XH-220 cultured in LB was resuspended in PBS, and sodium acetate was selected as the sole carbon source, and 100 mg / L ammonia nitrogen (NH4 + -N), 100mg / L nitrite nitrogen (NO2 - -N), 100mg / L nitrate nitrogen (NO3 --N) and 100 mg / L of three nitrogen mixed nitrogen sources (33.33 mg / L for each nitrogen source) were used as nitrogen sources, and different amounts of C / N of 5, 10, 15, 20, and 25 were added to the basal universal medium. (Basic universal medium: K2HPO4 0.2 g / L, MgCl2 0.05 g / L, CaCl2 0.05 g / L, ZnSO4 0.05 g / L, 0.1% (V / V) trace element solution; pH 7.2. Trace element solution: EDTA 50 g / L, CaCl2 5.5 g / L, ZnSO4 2.2 g / L, MnCl2·4H2O 5.06 g / L, FeSO4·7H2O 5.0 g / L, (NH4)6Mo7O 24 ·4H2O 1.1g / L, CuSO4·5H2O 1.57g / L, CoCl2·6H2O1.61g / L; pH7.0), 30℃, 180rpm, after 48h of culture, the OD600 value of Glutamicibacternicotianae XH-220 was monitored. The results showed that the growth rate of Glutamicibacternicotianae XH-220 was the fastest when C / N was 10, so C / N was selected as the carbon-nitrogen ratio of the subsequent experimental culture medium. For details, see Figure 5 .

[0087] 8. Effect of pH on the Growth of Glutamicibacter nicotianae XH-220

[0088] Glutamicibacter nicotianae XH-220 was inoculated into LB medium with pH values ​​of 3, 5, 7, 9, and 11, respectively, and cultured at 30°C and 180rpm for 48 hours, and the OD600 value of Glutamicibacter nicotianae XH-220 was monitored. The results showed that Glutamicibacter nicotianae XH-220 could tolerate pH 3 and had a good growth rate at pH 5-11. The subsequent experiments were conducted at pH 7. For details, see Figure 6 .

[0089] IX. Effect of Temperature on the Growth of Glutamicibacter nicotianae XH-220

[0090] Glutamicibacter nicotianae XH-220 was inoculated into LB medium with temperature values ​​of 10℃, 20℃, 30℃, 40℃, and 50℃, respectively. After culturing at 30℃ and 180rpm for 48h, the OD600 value of Glutamicibacter nicotianae XH-220 was monitored. The results showed that Glutamicibacter nicotianae XH-220 had a good growth rate at a temperature of 30-40℃. The subsequent experiments were conducted at a temperature of 30℃. For details, see Figure 7 .

[0091] 10. Effect of salinity on the growth of Glutamicibacter nicotianae XH-220

[0092] Glutamicibacter nicotianae XH-220 was inoculated into LB medium with salinity of 0%, 1%, 3%, 5%, and 10% (mass fraction of NaCl) respectively, and cultured at 30℃ and 180rpm for 48h, and then the OD600 value of Glutamicibacter nicotianae XH-220 was monitored. The results showed that Glutamicibacter nicotianae XH-220 had a good growth rate at a salinity of 5% and could tolerate a salinity of 10%. For details, see Figure 8 .

[0093] 11. Tolerance of Glutamicibacter nicotianae XH-220 to Heavy Metals

[0094] Glutamicibacter nicotianae XH-220 were inoculated with Mn containing heavy metal concentrations of 1mM, 5mM, and 10mM, respectively. 2+ , Pb 2+ , Fe 3+ 、Zn 2+ , Cu 2+ 、Co 2+ 、Cd 2+ , Hg 2+ 、Ni 2+ Cr 6+LB medium was used, and the OD600 value of Glutamicibacter nicotianae XH-220 was monitored after culturing at 30°C and 180rpm for 48h. The results showed that Glutamicibacter nicotianae XH-220 could effectively inhibit the growth of 10mmol / L metal ions Mn 2+ ,5mmol / L metal ion Pb 2+ , Fe 3+ , Cu 2+ ,1mmol / L metal ion Zn 2+ 、Co 2+ 、Ni 2+ Cr 6+ Tolerable. Fig. 9 .

[0095] 12. Removal capacity of different types of nitrogen sources by Glutamicibacter nicotianae XH-220

[0096] The following methods for determining ammonia nitrogen, nitrite nitrogen and nitrate nitrogen are as follows: Ammonia nitrogen (NH4 + -N) was determined by the "Determination of Ammonia Nitrogen in Water - Nessler's Reagent Spectrophotometric Method" (HJ535-2009); Nitrite Nitrogen (NO2 - -N) was determined by the "Determination of Nitrite Nitrogen in Water by Diazo Coupling Spectrophotometry" (GB7493-87); Nitrate Nitrogen (NO3 - -N) UV spectrophotometric method for the determination of nitrate nitrogen in water (HJ 346-2007).

[0097] The culture medium formula used below is as follows:

[0098] Basic expansion medium: The solvent is water, and the solutes and concentrations are as follows: trypsin 10.0 g / L; yeast powder 5.0 g / L; NaCl 10.0 g / L.

[0099] Denitrification performance determination culture medium: CH3COONa 3.42g / L, KNO3 0.722g / L, K2HPO4 0.2g / L, MgCl20.05g / L, CaCl2 0.05g / L, ZnSO4 0.05g / L, 0.1% (V / V) trace element solution; pH 7.2.

[0100] Nitrite denitrification performance determination culture medium: CH3COONa 3.42g / L, NaNO2 0.4928g / L, K2HPO40.2g / L, MgCl2 0.05g / L, CaCl2 0.05g / L, ZnSO4 0.05g / L, 0.1% (V / V) trace element solution; pH 7.2.

[0101] Ammonium salt ammoniation performance determination culture medium: CH3COONa 3.42g / L, NH4Cl 0.3853g / L, K2HPO4 0.2g / L, MgCl2 0.05g / L, CaCl2 0.05g / L, ZnSO4 0.05g / L, 0.1% (V / V) trace element solution; pH 7.2.

[0102] Heterotrophic nitrification-aerobic denitrification medium: CH3COONa 3.42g / L, NH4Cl 0.1284g / L, NaNO20.1643g / L, KNO3 0.241g / L, K2HPO4 0.2g / L, MgCl2 0.05g / L, CaCl2 0.05g / L, ZnSO4 0.05g / L, 0.1% (V / V) trace element solution; pH 7.2.

[0103] Trace element solution: EDTA 50g / L, CaCl2 5.5g / L, ZnSO4 2.2g / L, MnCl2·4H2O 5.06g / L, FeSO4·7H2O 5.0g / L, (NH4)6Mo7O 24 ·4H2O 1.1g / L, CuSO4·5H2O 1.57g / L, CoCl2·6H2O1.61g / L; pH7.0.

[0104] Glutamicibacter nicotianae XH-220 was inoculated on LB plate culture medium and cultured at 35°C for 24 hours to activate the bacteria.

[0105] The activated bacterial cells prepared were inoculated into 100 ml of LB liquid seed culture medium at a ratio of 5% according to a conventional amount, and cultured at 35° C. for 12 hours to obtain a seed suspension of Glutamicibacter nicotianae XH-220.

[0106] The prepared seed suspension was resuspended with PBS and inoculated into the corresponding ammonia nitrogen removal capacity determination medium so that the bacterial count in the system was 1×10 7 CFU / ml. Cultured at 30°C, 180 rpm for 48 h, and the ammonia nitrogen (NH4 +-N), nitrite nitrogen (NO2 - -N), nitrate nitrogen (NO3 - -N) index. Denitrification performance within 48 hours was measured by nitrate nitrogen (NO3 - -N) removal rate was 56.84%; nitrite denitrification performance was determined by nitrite nitrogen (NO2 - -N) removal rate was 18.53%; ammonium salt ammoniation performance test medium ammonia nitrogen (NH4 + -N) removal rate was 35.14%; nitrate nitrogen (NO3 - -N), nitrite nitrogen (NO2 - -N), ammonia nitrogen (NH4 + -N) removal rates were 46.53%, 26.48%, and 83.62% respectively. Figures 10 to 13 .

[0107] 13. Growth-promoting effect of Glutamicibacter nicotianae XH-220 on cucumber seedlings

[0108] Glutamicibacter nicotianae XH-220 was inoculated on LB slant medium and cultured at 35°C for 1-2 days to activate the bacteria.

[0109] The activated bacteria prepared were inoculated into 100 ml of LB liquid seed culture medium according to the conventional amount, and cultured at 35° C. for 12 hours to obtain a seed suspension of Glutamicibacter nicotianae XH-220.

[0110] The prepared Glutamicibacter nicotianae XH-220 seed solution was resuspended in PBS solution to make the bacterial count in the system 1×10 7 CFU / ml, aspirate 10ml of the resuspended bacterial solution and irrigate the roots until the 4-leaf budding stage of cucumber seedlings (variety: Jinyan No. 4). The untreated group CK used sterile water instead of root irrigation. After growing for 14 days, 10ml of sterile water was added every day to ensure soil moisture.

[0111] The results showed that the plant height of the tobacco Glutamicibacter nicotianae XH-220 treated group increased by an average of 0.4 cm, the length of the 3rd leaf increased by an average of 0.24 cm, and the length of the 4th leaf increased by an average of 0.16 cm. Fig.14 and Fig.15 shown.

[0112] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Nicotiana glutamicum Glutamicibacter nicotianae ) XH-220 or containing the tobacco glutamicum bacteria ( Glutamicibacter nicotianae )Application of XH-220 bacterial agent in the treatment of nitrogen-containing wastewater; The tobacco glutamicum bacteria ( Glutamicibacter nicotianae )XH-220 is registered in the Agricultural Microbiology Center of China Microbiological Culture Collection Committee as ACCC 62208; The wastewater treatment is to carry out biological denitrification on the water body; The nitrogen in the wastewater is ammonia nitrogen, nitrite nitrogen and / or nitrate nitrogen.

2. The use according to claim 1, characterized in that: The pH of the wastewater is not less than 3.

3. The use according to claim 1, characterized in that: The total salinity in the wastewater is not higher than 10% by mass.

4. The use according to claim 1, characterized in that: The Mn in the wastewater 2+ Not higher than 10 mmol / L, and / or Pb 2+ , Fe 3+ , Cu 2+ Not higher than 5 mmol / L, and / or Zn 2+ 、Co 2+ 、Ni 2+ Cr 6+ Not higher than 1 mmol / L.

5. A wastewater treatment method comprising the following steps: treating Nicotiana glutamicum ( Glutamicibacter nicotianae ) XH-220 or containing the tobacco glutamicum bacteria ( Glutamicibacter nicotianae )The XH-220 bacterial agent is inoculated into the wastewater to be treated for cultivation; The tobacco glutamicum bacteria ( Glutamicibacter nicotianae )The deposit number of XH-220 in the Agricultural Microbiology Center of China Microbiological Culture Collection Committee is ACCC 62208; The nitrogen in the wastewater is ammonia nitrogen, nitrite nitrogen and / or nitrate nitrogen; After inoculation, the Nicotiana glutamicum Glutamicibacter nicotianae )The content of XH-220 in the wastewater system is 1×10 6 CFU / ml-3.0×10 7 CFU / ml; The culture temperature is 28-32°C. The culture is carried out for 36-60 hours.

6. The method according to claim 5, characterized in that: The culture is performed by shaking at 180 r / min.

7. Nicotiana glutamicum Glutamicibacter nicotianae ) XH-220 or containing the tobacco glutamicum bacteria ( Glutamicibacter nicotianae )Application of XH-220 microbial agent in promoting plant growth; The tobacco glutamicum bacteria ( Glutamicibacter nicotianae )The deposit number of XH-220 in the Agricultural Microbiology Center of China Microbiological Culture Collection Committee is ACCC 62208; The plant is cucumber.

8. The use according to claim 7, characterized in that: The promoting of plant growth is manifested in promoting an increase in plant height and / or promoting an increase in plant leaf length.

9. A method for promoting plant growth, comprising the steps of: introducing Nicotiana glutamicum ( Glutamicibacter nicotianae ) XH-220 or containing the tobacco glutamicum bacteria ( Glutamicibacter nicotianae ) XH-220 bacterial agent is inoculated into the rhizosphere soil of the plants to be treated; The tobacco glutamicum bacteria ( Glutamicibacter nicotianae )The deposit number of XH-220 in the Agricultural Microbiology Center of China Microbiological Culture Collection Committee is ACCC 62208; The plant is cucumber.

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