Stenotrophomonas geniculatum TA2-8, microbial inoculum thereof and application of microbial inoculum in ammonia removal of stenotrophomonas geniculatum TA2-8

Abstract

The invention relates to stenotrophomonas geniculata TA2-8, a microbial agent thereof and application of the microbial agent in ammonia removal, and belongs to the technical field of microorganisms. The stenotrophomonas geniculata TA2-8 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.32385, the preservation date is October 28, 2024, and the address of the preservation institution is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. In the decomposition process, the stenotrophomonas geniculata TA2-8 provided by the invention is resistant to higher temperature and higher in ammonia removal and deodorization efficiency, ammonia gas volatilization is reduced, nitrogen elements are reserved in a decomposition system, and pollution to the environment is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Oligotrophomonas geniculate TA2-8 and its bacterial agent and application in ammonia removal. Background Art

[0002] Crop straw is an important by-product of agricultural production, containing rich organic matter, nitrogen, phosphorus, potassium and other nutrients. Through composting treatment, straw can be converted into organic fertilizer, realizing the recycling of resources, reducing dependence on chemical fertilizers, and reducing agricultural production costs. After being decomposed, straw can be converted into high-quality organic fertilizer, rich in organic matter and nutrients, which can improve soil structure, increase soil fertility, promote crop growth, and realize the recycling of resources. Through composting treatment, straw can be prevented from being directly exposed to the natural environment, reducing the stench caused by straw rotting and the pollution of water and soil. Composted straw will produce a bad smell, which is due to the decomposition of microorganisms, which will produce some smelly gases, such as hydrogen sulfide, ammonia, volatile organic compounds, etc. These odors will not only affect the environment and the quality of life of residents, but also cause harm to human health. Straw contains a certain amount of nitrogen, but during the composting process, nitrogen loss will occur due to the action of microorganisms and ammonia volatilization. This will not only reduce the fertilizer efficiency of organic fertilizers after decomposition, but also increase the demand for chemical nitrogen fertilizers in agricultural production, increasing production costs and environmental pressure.

[0003] At present, there are physical methods for treating the odor of straw decomposition, such as using adsorbents such as activated carbon to absorb odorous substances; chemical methods, such as spraying chemical deodorants; biological methods, such as using specific microorganisms to degrade odorous substances. No relevant reports on the use of Oligotrophomonas geniculate in the field of ammonia removal have been retrieved in the prior art. Summary of the invention

[0004] In view of the problem that the application of Stenotrophomonas geniculate is not found in the existing biological methods for removing ammonia, the present invention provides a strain of Stenotrophomonas geniculate TA2-8 and its bacterial agent and application for removing ammonia to solve the above problem.

[0005] In a first aspect, the present invention provides a strain of St. genu TA2-8, wherein the St. genu TA2-8 Stenotrophomonas geniculata )TA2-8 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32385 and the deposit date October 28, 2024. The address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] In a second aspect, the present invention provides a bacterial agent containing Stenotrophomonas geniculate TA2-8, wherein the bacterial agent is a solid bacterial agent.

[0007] A method for preparing the above solid bacterial agent comprises the following steps: A ring of activated St. Geniculate was inoculated into LB liquid culture medium and cultured at a constant temperature of 200 rpm and 30°C for 24 hours; then it was transferred to a primary fermentation tank for expansion culture and cultured at a constant temperature of 200 rpm and 30°C for 10-12 hours, and then transferred to a secondary fermentation tank; the secondary fermentation tank was expanded and cultured at a constant temperature of 200 rpm and 30°C for 24 hours; the bacterial liquid was vacuum dried to prepare a powder, which is a solid bacterial agent containing St. Geniculate TA2-8.

[0008] The LB liquid culture medium had the following components: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and pH=7.0.

[0009] The culture medium components in the primary fermentation tank and the secondary fermentation tank are: sodium citrate 3.5 g / L, ammonium sulfate 0.35 g / L, potassium dihydrogen phosphate 0.25 g / L, disodium hydrogen phosphate 0.3 g / L, sodium chloride 0.12 g / L, magnesium sulfate 1 g / L, pH=7.0~7.5.

[0010] In a third aspect, the present invention provides an application of Stenotrophomonas geniculate TA2-8 in removing ammonia.

[0011] Furthermore, the St. Geniculate TA2-8 is used to remove ammonia generated during the straw composting process.

[0012] Furthermore, the straw composting temperature is 30℃~45℃.

[0013] The beneficial effects of the present invention are: The oligotrophic mononas TA2-8 provided by the present invention can tolerate higher temperatures during the decomposition process, has higher efficiency in removing ammonia and deodorizing, reduces ammonia volatilization, retains nitrogen in the decomposition system, and reduces pollution to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 This is a trend chart of ammonia degradation by the four bacterial strains in Example 1 of the present invention.

[0016] Figure 2 This is the colony map of Stenotrophomonas geniculate TA2-8 in Example 1 of the present invention.

[0017] Figure 3This is the Gram staining image of Stenotrophomonas geniculate TA2-8 in Example 1 of the present invention.

[0018] Figure 4 This is a trend diagram of ammonia degradation by Oligotrophomonas geniculate TA2-8 at 35°C in Example 2 of the present invention.

[0019] Figure 5 This is a trend diagram of ammonia degradation by Oligotrophomonas geniculate TA2-8 at 40°C in Example 2 of the present invention.

[0020] Figure 6 This is a trend diagram of ammonia degradation by Oligotrophomonas geniculate TA2-8 at 45°C in Example 2 of the present invention.

[0021] Figure 7 This is a trend diagram of ammonia degradation by Oligotrophomonas geniculate TA2-8 at 50°C in Example 2 of the present invention. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0023] Example 1 Isolation, Screening and Identification of Stenotrophomonas geniculate TA2-8 1. Sampling On March 23, 2024, sludge was taken from the sewage aerobic tank of Sinopec Shanghai Gaoqiao Petrochemical Co., Ltd.

[0024] 2. Separation Take out 500g of sludge, add 1kg of sterile water to soak, use a mixer to mix the sludge for 20min, and filter through an 80-mesh sieve to obtain a filtrate. Centrifuge the filtrate at 8000rpm for 5min to obtain a small amount of bacterial sludge. Wash the bacterial sludge with sterilized sterile water and centrifuge it 3 times. Collect the bacterial sludge and inoculate all of it into 200ml of sterilized NH 3 Selective medium (NH 3 The selective culture medium composition is as follows: sodium citrate 0.35 g / L, 25% ammonia water 0.35 g / L, potassium dihydrogen phosphate 0.25 g / L, disodium hydrogen phosphate 0.3 g / L, sodium chloride 0.12 g / L, magnesium sulfate 1 g / L, pH = 7.0~7.5) in a shake flask, the initial temperature of the shaker is set to 30°C, the speed is 180 rpm, and the enrichment culture is carried out for 48 hours.

[0025] During the enrichment culture, the turbidity of the culture medium was observed, and the nitrate nitrogen content and ammonia nitrogen content were detected every 12 hours (equipment model: GNST-900, manufacturer: Henan Suijing Environmental Protection Technology Co., Ltd.). At the end of 48 hours of culture, the ammonia nitrogen in the culture medium dropped from the initial 88 ppm to 0 ppm, and the nitrate nitrogen increased from 0 ppm to 40 ppm, indicating that some strains utilized ammonia water and converted it into nitrate nitrogen.

[0026] The bacterial suspension after enrichment culture was spread on the plate by the doubling dilution method to isolate single colonies: 10 ml of the bacterial suspension was taken and added to 90 ml of sterile water with glass beads, and the shaker was fully shaken at 200 r / min for 30 min. 5 ml of the above mother liquid bacterial suspension was taken with a sterile pipette and added to 45 ml of sterile water, and serial dilutions were performed from 1 to 10 to obtain 1×10 -1 , 1×10 -2 , 1×10 -3 , 1×10 -4 , 1×10 -5 and 1×10 -6 times diluted bacterial suspension.

[0027] Take 3 appropriate dilutions of 10 -4 , 10 -5 and 10 -6 Use a sterile pipette to take 0.1 ml of the bacterial suspension diluted 2 times, add it to the pre-prepared nutrient agar solid medium plate, and use a sterile spreader to evenly spread the bacterial suspension of different dilutions on the surface of the medium, and culture it in a 30°C constant temperature incubator. After 24 hours of culture, colonies grow, and smear, stain, and microscopic examination are performed. A total of 4 strains were selected and stored in 50% sterile glycerol tubes and stored in a -80°C refrigerator. The four strains were marked as TA2-5, TA2-6, TA2-7, and TA2-8.

[0028] 3. Screening (1) Strain activation: Streak the four preserved strains on sterile plates of nutrient agar solid medium, place them in a 30°C constant temperature incubator and culture for 24 h. After the bacteria have grown well, scrape a loopful of the bacteria and inoculate them into LB liquid medium (tryptone 10 g, yeast extract 5 g, NaCl 10 g, pH = 7.0, constant volume 1 L, 121°C, 30 min), culture them at 30°C, shake at 200 r / min for 24 h, and place them in a 4°C refrigerator for later use.

[0029] (2) The four isolated and purified strains of bacteria have the function of degrading ammonia water and deodorizing it. The four strains of bacteria were verified separately. 2.5 kg of straw samples from the straw composting base in Qihe County, Dezhou City were taken and divided into 5 equal portions, each 500 g, and placed in 3L stoppered conical flasks. One end of the glass tube was inserted into the conical flask, and the other end was inserted into a stoppered conical flask containing 100 ml of 2% boric acid solution, with the port inserted below the liquid surface (methyl red-methylene blue indicator was added to the boric acid solution).

[0030] The suspension of the four strains was centrifuged, washed twice with sterile water, and then restored to the volume before centrifugation with sterile water. The four strains were inoculated into the straw at an inoculation rate of 10%, and the same amount of sterile water was added to the blank control. The culture was carried out at 30°C for 12 days, and the ammonia nitrogen concentration in the boric acid absorption solution was measured every 2 days (titration method). The NH 4 + The concentration reached 522 ppm, while the NH 4 + The concentration was 135 ppm, and the NH 4 + The concentration was 466ppm, and the NH 4 + The concentration was 369 ppm, and the NH 4 + The concentration ppm was 425ppm. It was finally determined that strain TA2-8 had a good ammonia degradation ability, and the ammonia nitrogen growth was the slowest, only 25.8% of the blank control, that is, the ammonia degradation rate reached 74.2%. For details, see Figure 1 .

[0031] 4. Identification The colony morphology of strain TA2-8 is as follows Figure 2 As shown, the colonies are light yellow, with neat edges, raised and rounded, and the surface of the bacteria is moist, smooth and viscous. The TA2-8 strain was stained with Gram stain using a staining kit, and the staining of the strain was observed under a microscope (equipment model: Nikon Ei100, manufacturer: Nanjing Jiangnan Yongxin Optical Co., Ltd.). The Gram staining of the TA2-8 strain is shown in Figure 3 As shown. It can be seen that the strain is red, rod-shaped, and is a Gram-negative bacterium. After the strain TA2-8 was activated, the plate was sent to Beijing Qingke Biotechnology Co., Ltd. for gene identification, and the gene sequence of the strain TA2-8 was obtained as shown in SEQ ID NO.1. The identification result is Stenotrophomonas geniculate Stenotrophomonas geniculata, Stenotrophomonas geniculate TA2-8 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.32385, the deposit date is October 28, 2024, and the address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0032] Example 2 Verification of ammonia removal performance of Oligotrophomonas geniculate TA2-8 1. Effect of straw maturity temperature on ammonia removal performance of Oligotrophomonas geniculate TA2-8 During the straw composting process, high temperature can quickly complete the decomposition and composting of organic matter, but too high temperature may also cause the death of some beneficial microorganisms, affecting the composting effect. At the same time, improper temperature control may also lead to the death of bacteria. In view of the composting characteristics, the ammonia removal performance of Oligotrophomonas geniculate TA2-8 was verified at 30℃, 35℃, 40℃, 45℃ and 50℃.

[0033] Streak a glycerol tube of the genuformis TA2-8 strain on a nutrient agar solid culture medium plate and culture it at 30°C for 24 h. Scrape a loopful of the bacteria and inoculate it into 300 ml LB liquid culture medium (10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, pH=7.0, constant volume 1 L, 121°C, 30 min) and culture it at 30°C with a shaker at 200 r / min for 24 h.

[0034] At 30℃, the data of ammonia degradation rate of 74.2% was obtained when screening strains, so the test was not repeated. The experiment was carried out according to the test method for screening strains, with 10% inoculation, and the experiments were carried out at 35℃, 40℃, 45℃, and 50℃ respectively (detected according to the titration method in the screening step), and blank controls were made at each temperature point. The results are shown in Table 1 and Figure 4~Figure 7 As shown: Table 1 - Ammonia degradation rate of Oligotrophomonas geniculate TA2-8 at different temperatures

[0035] Note: NH in Table 1 4 + The concentrations are all detected data at 12 days.

[0036] according to Figure 4~Figure 7 As can be seen from the data in Table 1, at 30℃~45℃, as the temperature increases, the amount of ammonia released during the composting process also increases, but the degradation efficiency of ammonia by genu shaped oligotrophic mononas TA2-8 is not greatly affected. At 50℃, the ammonia degradation rate decreases significantly, which is due to the death of genu shaped oligotrophic mononas TA2-8 caused by the high temperature. The experiment proves that the genu shaped oligotrophic mononas TA2-8 strain has a wide tolerance to temperature and is suitable for the current composting process.

[0037] Example 3 Preparation of solid bacterial agent containing Stenotrophomonas geniculate TA2-8 A ring of activated St. Geniculate was inoculated into LB liquid medium. The LB liquid medium composition is as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH = 7.0. Cultured at 30°C for about 24 hours, transferred to a primary fermenter (30L fermenter) for expansion, 200 rpm, 30°C constant temperature culture for 10-12 hours, and then transferred to a secondary fermenter (300L). The secondary fermenter was 30°C, 200 r / min, 24 hours, and the fermentation medium was 3.5 g / L sodium citrate, 0.35 g / L ammonium sulfate, 0.25 g / L potassium dihydrogen phosphate, 0.3 g / L disodium hydrogen phosphate, 0.12 g / L sodium chloride, 1 g / L magnesium sulfate, pH = 7.0-7.5, and a total of 200 kg of bacterial liquid was obtained, with a colony count of 2.2×10 9 CFU / ml. The solid inoculum containing St. Geniculate TA2-8 was obtained by vacuum drying and powder preparation. The colony count of the solid inoculum was 2×10 10 CFU / ml.

[0038] Example 4 On-site ammonia removal experiment The experimental site is the straw composting base in Qihe County, Dezhou City. The detection equipment includes the gas detector SW-7500A for detecting ammonia concentration and the thermometer DLX-HC2510. First, the experimental area is divided: the site is divided into the experimental group and the control group, each with 1000kg of straw (pay attention to heat preservation). In the original composting process, the experimental group added 10kg of solid bacterial agent containing oligotrophic monocytogenes TA2-8 and mixed it evenly with the straw, with a dosage of 10kg / ton of straw. The control group followed the composting process without adding bacterial agent treatment.

[0039] Before the experiment, the ammonia concentration of the experimental group and the control group was tested using a gas detector, and the initial ammonia concentration and temperature data were recorded. After the experiment started, the ammonia concentration was tested and the temperature was recorded for the experimental group and the control group every 2 days. The experimental results are shown in Table 2: Table 2 - Ammonia removal rate of Oligotrophomonas geniculate TA2-8 at different times

[0040] As can be seen from the results in Table 2, the on-site ammonia removal test cycle is 12 days. As time goes by, the ammonia concentration in the control group increases, and the peak value reaches 285ppm. By comparing the ammonia concentration data of the experimental group and the control group at each time point, the ammonia removal effect is significant, indicating that the genuformis has a good ammonia removal effect, and the deodorization effect is stable over time.

[0041] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A strain of Stenotrophomonas geniculate TA2-8, characterized in that: The Stenotrophomonas geniculate Stenotrophomonas geniculata )TA2-8 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32385 and the deposit date October 28, 2024. The address of the deposit institution is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The oligotrophomonas geniculate TA2-8 according to claim 1, characterized in that The 16S rDNA sequence of Stenotrophomonas geniculate TA2-8 is shown in SEQ ID NO.

1.

3. A bacterial agent containing the oligotrophomonas geniculate TA2-8 according to claim 1, characterized in that: The bacterial agent is a solid bacterial agent.

4. The bacterial agent according to claim 3, characterized in that The preparation of the solid bacterial agent comprises the following steps: A ring of activated St. Geniculate was inoculated into LB liquid culture medium and cultured at 200 rpm and 30°C for 24 h; then it was transferred to a primary fermenter for expansion culture and cultured at 200 rpm and 30°C for 10-12 h, and then transferred to a secondary fermenter; the culture was expanded in the secondary fermenter at 200 rpm and 30°C for 24 h, and the bacterial liquid was vacuum dried to obtain a solid bacterial agent containing St. Geniculate TA2-8.

5. The bacterial agent according to claim 4, characterized in that The composition of the LB liquid culture medium is as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH=7.

0.

6. The bacterial agent according to claim 4, characterized in that The culture medium components in the primary fermentation tank and the secondary fermentation tank are: sodium citrate 3.5 g / L, ammonium sulfate 0.35 g / L, potassium dihydrogen phosphate 0.25 g / L, disodium hydrogen phosphate 0.3 g / L, sodium chloride 0.12 g / L, magnesium sulfate 1 g / L, pH=7.0~7.

5.

7. Use of the oligotrophomonas geniculate TA2-8 as described in claim 1 in removing ammonia.

8. The use according to claim 7, characterized in that The oligotrophic monocytogenes TA2-8 is used to remove ammonia generated during the straw composting process.

9. The use according to claim 8, characterized in that The straw composting temperature is 30℃~45℃.

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