A high-concentration ammonia nitrogen-degrading bacterium with low carbon-nitrogen ratio and its application

Abstract

The present invention discloses a high-concentration ammonia nitrogen degrading bacteria with a low carbon-nitrogen ratio and its application. The strain is Bacillus spizizenii QY01, with a deposit number of GDMCC No.63869, which was deposited in Guangdong Province Microbial Culture Collection Center on October 11, 2023; the strain QY01 can efficiently reduce ammonia nitrogen pollutants in a low carbon-nitrogen ratio and high ammonia nitrogen environment. Compared with the prior art, the strain QY01 of the present invention can tolerate high-concentration ammonia nitrogen and can achieve an ideal degradation effect on ammonia nitrogen with a concentration of 530 mg / L under conditions of relatively scarce nutrition and low C / N ratio, and can achieve a maximum degradation rate of about 60% when the C / N ratio is 1:1.

Description

Technical Field

[0001] The invention relates to the technical field of ammonia nitrogen pollutant degradation, in particular to a high-concentration ammonia nitrogen degradation bacterium with a low carbon-nitrogen ratio and application thereof. Background Art

[0002] Bacillus subtilis subsp.spizizenii, as a subspecies of Bacillus subtilis, was previously classified as B. subtilis subsp.spizizenii due to the difficulty in distinguishing phenotypes. It was not until comparative genomics and sequencing technologies such as housekeeping genes such as gyrA that it was promoted to species status (Bacillus spizizenii) in 2020. Since the bacterium has broad-spectrum antimicrobial agents and can produce lipopeptide metabolites such as biosurfactants and bioemulsifiers, it shows significant plasticity in synthesizing antibiotics. Therefore, it has great utilization value in agriculture, especially in the biological control of crop diseases. In addition, as a species of Bacillus, B.spizizenii has a powerful enzyme production system and secretes a large amount of extracellular enzymes to decompose pollutants. Therefore, it can degrade, dilute, and transfer pollutants in environmental pollution, thereby achieving ultimate environmental purification.

[0003] In my country, the removal of ammonia nitrogen by microbial methods is through traditional nitrification and denitrification. In the denitrification process, carbon sources are used as electron donors to achieve the denitrification process and thus achieve the purpose of denitrification. However, the biggest problem faced by biological denitrification of nitrogen-containing wastewater or soil (especially wastewater with a low C / N ratio) is insufficient carbon sources, resulting in poor denitrification effects. Therefore, under the premise of successful denitrification by microorganisms, it is often necessary to add a large amount of additional carbon sources and even consider the content of other nutrients to achieve effective removal of ammonia nitrogen, resulting in high costs. Therefore, it is urgent to find a way to achieve efficient degradation of ammonia nitrogen in the case of low or even no carbon source and relatively nutrient-deficient conditions. As a biological control agent and bioremediation agent, the research on B.spizizenii is mostly focused on how to prevent and control crop diseases and pests and produce antibiotics. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a high-concentration ammonia nitrogen-degrading bacteria with a low carbon-nitrogen ratio and application thereof.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] The first object of the present invention is to provide a high-concentration ammonia nitrogen degrading bacteria with a low carbon-nitrogen ratio, the strain is Bacillus spizizenii QY01, the preservation number is: GDMCC No.63869, and it was preserved in Guangdong Provincial Microbiological Culture Collection Center on October 11, 2023, and the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City.

[0007] The second object of the present invention is to provide a kind of high-concentration ammonia nitrogen degrading bacteria with low carbon-nitrogen ratio for use in efficiently reducing ammonia nitrogen pollutants in low carbon-nitrogen ratio and high ammonia nitrogen environment.

[0008] Preferably, the carbon-nitrogen ratio is 1:1.

[0009] Compared with the prior art, the strain QY01 of the present invention can tolerate high concentrations of ammonia nitrogen and can achieve an ideal degradation effect on ammonia nitrogen concentration of 530 mg / L under conditions of relative nutrient scarcity and low C / N ratio, and can achieve a maximum degradation rate of about 60% when the C / N ratio is 1:1. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the colony morphology of strain QY01 in broth culture medium.

[0011] Figure 2 Effects of different initial ammonia nitrogen concentrations (a), C / N ratio (b) and fermentation temperature (c) on the degradation of ammonia nitrogen by strain QY01. DETAILED DESCRIPTION

[0012] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0013] Example 1 Isolation and purification of strain QY01

[0014] (I) Source of soil samples and culture medium used in this example

[0015] The source of soil samples: Yuhelong, Mendong Village, Baisha Town, Yingde City, Qingyuan City, Guangdong Province (center coordinates: N24°5′20.00″, E113°40′48.00″), a historical ionic rare earth mine.

[0016] Enrichment medium: (NH4)2SO4 as the only nitrogen source, glucose as the carbon source, Na + Mg 2+ , Fe 2+, K+ is the mineral element required for microbial growth to prepare the culture medium for microbial growth. Among them, (NH4)2SO45.0g, glucose5.0g, NaCl2.0g, FeSO4·7H2O0.4g, K2HPO41.0g, MgSO4·7H2O0.5g, ultrapure water is fixed to 1000mL, pH7.0-7.4, initial ammonia nitrogen concentration is 1060mg / L, C / N=1:1, and sterilized at 121℃ for 30min.

[0017] Isolation medium: Add 20 g of agar to the enrichment medium.

[0018] LB liquid culture medium: peptone 10 g / mL, yeast extract 5 g / mL, NaCl 10 g / mL, pH 7.5, sterilization at 121°C for 30 min.

[0019] Slant culture medium: (LB solid culture medium): Add 20 g / mL agar to LB liquid culture medium.

[0020] Fermentation medium: (NH4)2SO42.5g, sodium citrate 5.0g, NaCl1.0g, K2HPO40.5g, MgSO4·7H2O0.25g, dilute to 1000mL with ultrapure water, sterilize at 121℃ for 30min. At this time, the initial ammonia nitrogen concentration is 530mg / L, and the initial theoretical value of ammonia nitrogen and the carbon-nitrogen ratio are adjusted by adjusting the amount of (NH4)2SO4 added.

[0021] (II) High-concentration ammonia nitrogen acclimation culture: Considering the barren environment of the mine, the types and number of microorganisms are relatively small, so the microbial groups that can utilize ammonia nitrogen are obtained by targeted enrichment culture. That is, take 200mL of enrichment culture medium with an initial ammonia nitrogen concentration of 1060mg / L and C / N=1:1 after sterilization, mix the collected soil samples in equal amounts, weigh 40g of the mixed soil samples and inoculate them in a conical flask, and carry out enrichment culture at 28℃ and 120r / min for 20h. Then, the enrichment culture solution is connected to the fresh enrichment culture solution for the second round of enrichment culture. Repeat the enrichment culture 5 times to eliminate the bacterial groups that cannot utilize (NH4)2SO4 and obtain the microbial groups that can utilize ammonia nitrogen.

[0022] (III) Gradient dilution of bacterial suspension: Take the supernatant (bacterial suspension) of the last enrichment culture and dilute it with physiological saline (0.9% NaCl) under aseptic conditions. The specific operation steps are as follows: 9 test tubes are numbered in advance: 1, 2, 3, 4, 5, 6, 7, 8, 9, take 9mL of physiological saline in each test tube, sterilize at 121℃ for 20min, take 1mL of the supernatant (bacterial suspension) of the last enrichment culture and add it to the test tube numbered 1 to make the first gradient bacterial suspension (usually called 10－1 Gradient), after mixing on oscillator, pipette 1mL10 －1 Gradient bacterial suspension to test tube No. 2 to obtain 10 －2 Gradient bacterial suspension, and so on can get 10 －3 -10 －9 Gradient bacterial suspension.

[0023] (IV) Spreading separation: Discard 10% of the bacterial solution with too high or too low concentration -1 -10 -2 and 10 -8 -10 -9 , take 10 respectively -3 -10 -7 0.1mL (100μL) of bacterial solution of each dilution gradient concentration was spread on a separation medium plate containing 1060mg / L, C / N=1:1 ammonia nitrogen, and inverted cultured at 28°C for 72h. Three replicates were set for each gradient. After a single colony grew on the plate, the single colony was picked up by an inoculation needle and placed on a separation medium containing 1060mg / L, C / N=1:1 ammonia nitrogen for plate streak separation, and inverted cultured at 28°C for 48h. After repeating the plate separation 3 times, 15 single colonies were selected and inoculated into 30mL fermentation medium containing 1060mg / L, C / N=1:1, and cultured at 32°C, 160r / min for 72h. The ammonia nitrogen degradation ability of the 15 single colonies was determined, and the 5 single colonies with the best degradation ability were selected and inoculated into 30mL fermentation culture medium containing 1060mg / L ammonia nitrogen at an inoculation amount of 5%, and continuously subcultured at 32°C, 160r / min for 72h. After continuous high-concentration ammonia nitrogen acclimation and degradation ability testing, a strain with the strongest ammonia nitrogen degradation ability was obtained.

[0024] Example 2 Identification of QY01 strain

[0025] (I) Observation of QY01’s morphology

[0026] A strain with the highest ammonia nitrogen degradation rate was selected and named strain QY01.

[0027] like Figure 1 As shown, the colony of strain QY01 is irregular in shape, with smooth and rounded edges, opaque, milky white, can reduce nitrates, is aerobic, and has rod-shaped cells, sometimes in chains. It can form heat-resistant spores, so it can survive and grow normally in a harsh environment. B.spizizenii has flocculent edges in broth culture medium, forms a biofilm, and has a dark color in the middle.

[0028] (II) Molecular identification of QY01

[0029] After strain QY01 was cultured in LB liquid shake flasks, it was centrifuged at 5000r / min for 2min at 4℃, and the bacteria were collected. The bacterial liquid was sent to Guangdong Meige Gene Technology Co., Ltd. for molecular biological identification. After genome extraction, 16S rDNA was amplified using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), and the obtained sequence was subjected to BLAST comparison analysis through the nt library of the NCBI website to screen out homologous sequences. In order to more accurately determine the species of QY01 and avoid the error judgment caused by many homologous sequences with high similarity, the housekeeping gene gyrA of the bacteria was detected by the Guangdong Provincial Microbiological Culture Collection Center on the basis of 16s rDNA detection. The gene sequence of QY01 gene GyrA is:

[0030] .

[0031] It was finally determined that strain QY01 was Bacillus spizizenii. Bacillus spizizenii QY01 was sent to Guangdong Microbiological Culture Collection Center for preservation on October 11, 2023, with the preservation number: GDMCC No.63869.

[0032] Example 3 Ammonia nitrogen degradation ability of QY01 in different environments

[0033] (I) Preparation of seed solution: Inoculate QY01 into LB liquid medium to activate the strain, activate at 30℃, 160r / min for 24h, centrifuge the cultured bacterial solution at 4℃, 5000r / min for 2min, collect the bacterial cells, resuspend and wash the bacterial cells three times with sterile physiological saline, and prepare a bacterial suspension containing a certain cell concentration (OD=1) as seed solution for later use.

[0034] (II) Ammonia nitrogen degradation ability at gradient concentration: The seed solution QY01 with OD=1 was cultured in fermentation medium with C / N=10:1 and initial ammonia nitrogen concentrations of 53 mg / L, 106 mg / L, 212 mg / L, 530 mg / L, and 848 mg / L at 32°C and 160 r / min. After 72 h, the national standard Nessler's reagent spectrophotometry (HJ535-2009) was used to determine the ammonia nitrogen degradation effect of QY01 under different initial ammonia nitrogen concentrations. Figure 2 (a) By Figure 2 (a) B.spizizenii has a certain tolerance to high concentrations of ammonia nitrogen, and has an ideal degradation effect at a low initial ammonia nitrogen concentration (ammonia nitrogen concentration <200 mg / L). When the ammonia nitrogen concentration is >200 mg / L and as the ammonia nitrogen concentration continues to increase, its degradation rate decreases significantly but still has a certain degradation rate.

[0035] (III) Ammonia degradation capacity at different C / N ratios: QY01 was inoculated into fermentation medium containing 530 mg / L ammonia nitrogen and C / N ratios of 0 (no carbon source), 1:1, 2:1, 5:1, 10:1, and 15:1, respectively. After 72 h of shake flask fermentation at 32°C and 160 r / min, the ammonia degradation effects at different C / N ratios were measured. Figure 2 (b) By Figure 2 (b) It can be seen that at an initial ammonia nitrogen concentration of 530 mg / L, B.spizizenii has a higher ammonia nitrogen removal efficiency in the low C / N range, i.e. C / N<5, with a degradation rate of more than 40%. Even without providing a carbon source, B.spizizenii still has a degradation rate of about 10% for ammonia nitrogen, which is different from most strains with the optimal C / N=10 or even C / N=15. Under high C / N, i.e. C / N>5, the ammonia nitrogen degradation rate is significantly reduced, below 30%. Under C / N=1:1, the highest ammonia nitrogen degradation rate of about 60% is reached after 72 hours of fermentation.

[0036] (IV) Ammonia nitrogen degradation capacity at different fermentation temperatures:

[0037] QY01 was inoculated into fermentation medium containing 530 mg / L ammonia nitrogen and C / N of 10, and the ammonia nitrogen degradation effect under different temperature conditions was measured after shaking flask fermentation at 25℃, 30℃, 35℃, and 40℃ and 160r / min for 72h. Figure 2 (c) By Figure 2 (c) It can be seen that at an initial ammonia nitrogen concentration of 530 mg / L, B.spizizenii has a certain degradation rate within the fermentation temperature range of 25°C-40°C. As the temperature increases, the degradation effect increases. When the temperature is greater than 30°C, the ammonia nitrogen degradation rate decreases as the temperature increases. At a fermentation temperature of 30°C, the highest degradation rate of >30% is reached after 72 hours of fermentation.

[0038] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A low carbon-nitrogen ratio ammonia-nitrogen-degrading bacterium, characterized in that: This strain is Bacillus subtilis subsp. Bacillus spizizenii ) QY01, the deposit number is: GDMCC No.63869, deposited in Guangdong Provincial Microbiological Culture Collection Center on October 11, 2023; the carbon-nitrogen ratio is 1:1 or 2:

1.

2. An application of the low carbon-nitrogen ratio ammonia nitrogen degrading bacteria as claimed in claim 1 in reducing ammonia nitrogen pollutants in a low carbon-nitrogen ammonia nitrogen environment, characterized in that: The carbon-nitrogen ratio is 1:1 or 2:1.

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