A Bacillus cereus strain with high environmental adaptability for nitrification and denitrification, microbial agent and application thereof

Abstract

The invention belongs to the technical field of microorganisms and aquaculture, and discloses a Bacillus cereus strain with high environmental adaptability for nitrification and denitrification, a microbial agent and its application. Bacillus cereus HCCN1 was deposited in the Guangdong Provincial Microbial Strain Collection Center on November 13, 2024, with a deposit number of GDMCC No: 65477. The present invention also discloses a microbial agent comprising the strain HCCN1 as an active ingredient and its application. The strain HCCN1 of the present invention has high stress resistance and high environmental adaptability, and has heterotrophic nitrification and aerobic denitrification functions. It can efficiently degrade nitrogen and phosphorus under harsh environmental conditions such as a wide pH range, temperature range, and high salt and alkali concentrations, and purify aquaculture water bodies. It has high biosafety and can meet the industry needs of efficient, healthy and sustainable aquaculture.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and aquaculture, and in particular to a strain of Bacillus cereus with high environmental adaptability for nitrification and denitrification, a microbial agent and application thereof. Background Art

[0002] Aquaculture is one of the fastest growing food production systems in the world. In order to obtain higher yields and benefits, the aquaculture industry has developed in the direction of high density and intensiveness in recent years, which has made problems such as eutrophication of water bodies and frequent diseases increasingly prominent. At present, my country's aquaculture model is mainly high-density and intensive. This model is easy to operate, has high yields and good benefits, but it is also easy to cause the growth of various pollutants in the aquaculture water, seriously affecting the yield and benefits of aquaculture. Healthy aquaculture is a complex issue, among which the purification of aquaculture water and the treatment of aquaculture tailwater are key aspects. At present, the deterioration of aquaculture water quality and wastewater discharge have seriously restricted the sustainable development of the aquaculture industry.

[0003] According to relevant statistics, the discharge of pollutants from aquaculture tailwater in my country accounts for about 6% of the total agricultural discharge. The pollutants are mainly nitrogen and phosphorus, and the main impact is eutrophication of water bodies. The development of aquaculture is bound to lead to an increase in breeding density and feed consumption. Relevant studies have shown that only a small part of the phosphorus and nitrogen in the feed can be assimilated by animals, and the rest of the phosphorus and nitrogen are discharged into the environment in the form of effluent, resulting in an increase in the content of phosphorus and nitrogen in the surrounding environment and destroying the ecological balance. Therefore, it is urgent to solve the problem of phosphorus and nitrogen accumulation in aquaculture wastewater.

[0004] Bacillus cereus in the prior art Bacillus cereus ), also known as Bacillus cactus, is a common Gram-positive bacteria. It belongs to the genus Bacillus and is widely found in soil, water, air, and animal intestines. There are many types of Bacillus cereus, which usually refer to Bacillus cereus microorganisms with similar physiological and biochemical characteristics and genetic characteristics. Among them, only a very small number of Bacillus cereus strains can be used for wastewater treatment. For example, a Bacillus cereus ( Bacillus cereus )LYS bacteria and its application, this invention is to overcome the current high temperature and high salt inhibit denitrifying bacteria, the shortcomings in the field of high temperature and high salt wastewater denitrification, by controlling the process parameters can effectively degrade the nitrogen compounds in the influent of high temperature and high salt sewage treatment plants, without pollution to the environment, such as degrading nitrogen compounds in coking wastewater, printing and dyeing wastewater, pharmaceutical wastewater, petrochemical wastewater, food wastewater, aquaculture wastewater, and landscape water. However, this bacterium can only denitrify nitrogen compounds in water bodies, but cannot nitrify phosphides at the same time, and its better working environment conditions are: pH value 7.5-8.5, water temperature 50℃, total salinity 6%.

[0005] In summary, the Bacillus cereus in the prior art is generally not applicable to sewage treatment, and conventional denitrification or phosphorus removal strains generally come from high-intensity wastewater or activated sludge, and have poor environmental adaptability. They are difficult to adapt to the harsh aquaculture water environment of high acidity, high alkalinity, high salt, and low nutrient ratio in coastal areas and saline-alkali areas for aquaculture, which seriously limits its scope of application and cannot meet the industry needs of efficient, healthy, and sustainable aquaculture. Summary of the invention

[0006] In view of the above problems, the purpose of the present invention is to provide a Bacillus cereus with high environmental adaptability to nitrification and denitrification, a microbial agent and its application, to screen and cultivate a denitrification and phosphorus removal strain with high stress resistance, high environmental adaptability, and the ability to simultaneously have heterotrophic nitrification and aerobic denitrification functions, so as to expand its use in aquaculture water treatment under a variety of harsh environmental conditions, so as to meet the industry needs of efficient, healthy and sustainable aquaculture.

[0007] The purpose of the present invention is achieved by the following technical solutions:

[0008] A Bacillus cereus strain with high environmental adaptability to nitrification and denitrification, which is a Bacillus cereus strain Bacillus cereus HCCN1 was deposited in the Guangdong Provincial Microbiological Strain Collection Center (GDMCC) on November 13, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province. The deposit number is GDMCC No: 65477, and its deposit status is alive.

[0009] The strain HCCN1 is Gram-positive, has dry colonies, and has a 16S rDNA sequence length of 716 bp.

[0010] The strain HCCN1 was isolated and screened from water samples and mud samples from aquaculture pond in the south of Doumen District, Zhuhai City, Guangdong Province.

[0011] A microbial agent, which is based on the Bacillus cereus strain Bacillus cereus HCCN1 as the active ingredient.

[0012] The highly environmentally adaptable nitrifying and denitrifying Bacillus cereus or the microbial agent is used in biological denitrification and phosphorus removal of nitrogen- and phosphorus-containing wastewater.

[0013] The carbon source for biological treatment of nitrogen-phosphorus-containing wastewater is at least one of sodium citrate, sodium succinate, sodium oxalate and glucose.

[0014] The nitrogen-phosphorus-containing wastewater has a carbon-nitrogen ratio of 0-20, a phosphorus-nitrogen ratio of 0-1.2, and a temperature of 20°C-40°C.

[0015] The pH value of the nitrogen and phosphorus-containing wastewater is 5-12.

[0016] The salinity of the nitrogen and phosphorus-containing wastewater is 0% to 10%.

[0017] The strain Bacillus cereus HCCN1, with sodium citrate as carbon source, C / N=15, P / N=0.8, pH=8, T=30℃, NH4 + -N, NO3 - -N, NO2 - -N degradation efficiency reaches the highest level, and the degradation efficiency of PO4 3- The total removal rate of -P was 100%.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] (1) The present invention provides a highly environmentally adaptable nitrifying and denitrifying Bacillus cereus strain HCCN1, which is screened and cultivated from an aquaculture pond. It has high stress resistance and high environmental adaptability. It is a denitrification and phosphorus removal strain with heterotrophic nitrification and aerobic denitrification functions. It can efficiently degrade NH4 in a wide pH range, temperature range, and harsh environmental conditions such as high salt and acid-base concentrations. + -N, NO3 - -N, NO2 - -N and PO4 3- -P, can purify aquaculture water under a variety of harsh environmental conditions, with high biological safety, can better meet the industry needs of efficient, healthy and sustainable aquaculture; in particular, it can meet the needs of biological denitrification and phosphorus removal of aquaculture effluent or other nitrogen-phosphorus-containing high-alkali wastewater in areas where fresh and salt water meet and saline-alkali tidal flats.

[0020] (2) The present invention provides a salt-tolerant and acid-tolerant Bacillus cereus strain HCCN1 having heterotrophic nitrification and aerobic denitrification functions. Under completely aerobic conditions, the strain can utilize NH4 + -N, NO3 - -N and NO2 - -N is used as the only inorganic nitrogen source for aerobic nitrification and denitrification; its degradation efficiency can reach 95.87%, 90.62% and 100.00% respectively; corresponding to PO4 3- The total removal rate is 100%.

[0021] (3) The Bacillus cereus HCCN1 of the present invention has both heterotrophic nitrification and aerobic denitrification functions, can utilize a variety of organic carbon sources, has a strong tolerance to high concentrations of organic carbon, and has a good ability to remove organic carbon from water bodies. This strain is particularly suitable for the treatment of high C / N nitrogen-containing wastewater. The Bacillus cereus HCCN1 provided by the present invention can better utilize organic substrates, has a fast growth rate, and can easily reach a high biomass concentration, achieving efficient removal of organic carbon while maintaining a high denitrification rate.

[0022] (4) The Bacillus cereus HCCN1 of the present invention has a strong ability to adapt to the environment and can tolerate a pH value of 5 to 11. The denitrification and phosphorus removal effect does not change much within this pH value range. The bacterium can not only be used in the pH value range of conventional aquaculture water bodies, but also can be used for denitrification and phosphorus removal treatment of highly alkaline or acidic water bodies, breaking through the application limitations of highly alkaline or acidic water bodies on the treatment of alkaline and acidic wastewater by microorganisms.

[0023] (5) The Bacillus cereus HCCN1 of the present invention can tolerate a salinity of 0-5%, and the denitrification and phosphorus removal effects within this salinity range do not vary much. Therefore, the strain can not only be used in the salinity range of conventional aquaculture water bodies, but also can be used for denitrification and phosphorus removal in high-salinity water bodies, breaking through the application limitations of high-salinity water bodies on the treatment of saline wastewater by microorganisms.

[0024] (6) The Bacillus cereus strain HCCN1 of the present invention can tolerate a phosphorus-nitrogen ratio range of 0-1.0%, and the denitrification and phosphorus removal effect within this phosphorus-nitrogen ratio range does not vary much. The strain can not only be applied to the phosphorus-nitrogen ratio range of conventional aquaculture water bodies, but is also expected to be used for denitrification and phosphorus removal in water bodies with high phosphorus-nitrogen ratios, which to a certain extent breaks through the limitations of high phosphorus-nitrogen ratio water bodies on the treatment of phosphorus-containing wastewater by microorganisms.

[0025] (7) The Bacillus cereus strain HCCN1 provided by the present invention can overcome the incompatibility problem of nitrification and denitrification caused by different oxygen demands, making it possible for nitrification and denitrification to proceed simultaneously in the same aerobic reactor. The application of this strain in the microbial denitrification process of aquaculture water is conducive to reducing the equipment footprint and construction costs, improving the treatment efficiency, and significantly reducing the periodic water changes in the aquaculture process. It has good economic and environmental benefits and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention is further described using the accompanying drawings, but the embodiments in the accompanying drawings do not constitute any limitation to the present invention. A person skilled in the art can obtain other drawings based on the following drawings without creative work.

[0027] Figure 1 This is a colony morphology diagram of Bacillus cereus HCCN1 on a nutrient agar plate according to an embodiment of the present invention;

[0028] Figure 2 is a Gram staining image of Bacillus cereus HCCN1 of an embodiment of the present invention;

[0029] Figure 3 The scanning electron microscope images of Bacillus cereus HCCN1 of the embodiment of the present invention include AC, wherein A is the scanning electron microscope image of HCCN1, and B and C are the length and width ranges of HCCN1;

[0030] Figure 4 is a graph showing the time-survival rate of zebrafish in the bacterial solution of Bacillus cereus HCCN1 of an embodiment of the present invention;

[0031] Figure 5 The growth and denitrification effects of Bacillus cereus HCCN1 under different organic carbon sources and different inorganic nitrogen sources are compared, including AC, where the only nitrogen source of A is NH4 + -N, B's only nitrogen source is NO2 - -N, C The only nitrogen source is NO3 - -N;

[0032] Figure 6 The growth and denitrification effects of Bacillus cereus HCCN1 under different C / N and different inorganic nitrogen sources are compared, including AC, where the only nitrogen source of A is NH4 + -N, B's only nitrogen source is NO2 - -N, C The only nitrogen source is NO3 - -N;

[0033] Figure 7 The growth and denitrification effects of Bacillus cereus HCCN1 under different P / N and different inorganic nitrogen sources are compared, including AC, where the only nitrogen source of A is NH4 + -N, B's only nitrogen source is NO2 - -N, C The only nitrogen source is NO3 - -N;

[0034] Figure 8 The growth and denitrification effects of Bacillus cereus HCCN1 under different pH and different inorganic nitrogen sources are compared in Figure AF, where the only nitrogen source of A and B is NH4 + -N, the only nitrogen source for C and D is NO2 - -N, E, F the only nitrogen source is NO3 - -N;

[0035] Fig. 9The growth and denitrification effects of Bacillus cereus HCCN1 under different salinities and different inorganic nitrogen sources are compared in Figures AF, where the only nitrogen source for A and B is NH4 + -N, the only nitrogen source for C and D is NO2 - -N, E, F the only nitrogen source is NO3 - -N;

[0036] Fig.10 The growth and denitrification effects of Bacillus cereus HCCN1 under different temperatures (°C) and different inorganic nitrogen sources are compared, including AC, where the only nitrogen source of A is NH4 + -N, B's only nitrogen source is NO2 - -N, C The only nitrogen source is NO3 - -N;

[0037] Fig.11 It is a graph showing the antibacterial effect of commonly used clinical antibiotics on Bacillus cereus HCCN1 of the embodiment of the present invention. DETAILED DESCRIPTION

[0038] The following is combined with Figure 1-11 The present invention is further described in detail with reference to the accompanying drawings and embodiments.

[0039] Example 1

[0040] See attached Figures 1 to 3 , the highly environmentally adaptable nitrification and denitrification Bacillus cereus HCCN1 strain was collected, isolated, cultivated and identified.

[0041] The present embodiment provides a highly environmentally adaptable nitrification and denitrification Bacillus cereus strain, which is a Bacillus cereus strain. Bacillus cereus HCCN1 was deposited in the Guangdong Provincial Microbiological Strain Collection Center (GDMCC) on November 13, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province. The deposit number is GDMCC No: 65477, and its deposit status is alive.

[0042] See attached Figure 1-3 The strain HCCN1 is Gram-positive, the colonies are dry and slightly raised, easy to pick up with an inoculation loop, round and light yellow, with a transparent ring around the colonies, a diameter of 4-5 mm, and no special smell; its 16S rDNA sequence length is 716bp.

[0043] The strain HCCN1 was isolated from water samples and mud samples from aquaculture pond in Doumen District, Zhuhai City, Guangdong Province. More specifically, the following steps were used:

[0044] 1. Sample collection

[0045] The bacterial Bacillus cereus strain HCCN1 of the embodiment of the present invention was screened and separated from water samples and mud samples from aquaculture pond in the south of Doumen District, Zhuhai City, Guangdong Province; the sample was collected according to the "Mixed Sample Collection Method" in the "Technical Specifications for Soil Environmental Monitoring" (HJ / T 166-2004), and the plum blossom point sampling method was used for point sampling. The surface, middle and deep water bodies and bottom mud were collected from the aquaculture pond in sterile sampling bags, and stored in a refrigerated manner at 4°C for transportation and storage.

[0046] 2. Preparation of culture medium and solution

[0047] (1) Salt solution (g / L): NaCl 2.5g, MgSO4·7H2O 2.5g, MnSO4·4H2O 0.05g, FeSO4·7H2O0.05g; adjust pH to 6, store in a sealed container at 4℃ and away from light;

[0048] (2) Trace element solution (g / L): MgSO4·7H2O 50.0g, CaCl2·2H2O 5.5g, CuSO4·5H2O1.57g, ZnSO4·7H2O 2.2g, FeSO4·7H2O 5.0g, MnCl2·4H2O, CoCl2·6H2O 1.60g, Na2EDTA50.0g;

[0049] (3) Enrichment medium (g / L): sodium succinate 5.62 g, KH2PO4 0.087 g, (NH4)2 SO4 0.472 g, NaNO3 0.24 g, NaNO2 0.165 g, salt solution 50 mL;

[0050] (4) BTB medium (g / L): 5.66 g of anhydrous sodium citrate, 1.5 g of KH2PO4, 0.2 g of MgSO4·7H2O, 7.9 g of Na2HPO4, 0.842 g of NaNO3, 0.362 g of NaNO2, 0.192 g of NH4Cl, 25 g of agar, 1 mL of 1% BTB ethanol solution, 2 mL of trace element solution, adjust the pH to 7.0-7.5;

[0051] (5) Single nitrogen source fermentation medium (DMⅠ) (g / L): 4.56 g sodium citrate, 0.352 g KH2PO4, 0.472 g (NH4)2 SO4, 2 mL trace element solution, pH 7.0;

[0052] (6) Single nitrogen source fermentation medium (DMⅡ) (g / L): 4.56 g sodium citrate, 0.352 g KH2PO4, 0.607 g NaNO3, 2 mL trace element solution, pH 7.0;

[0053] (7) Single nitrogen source fermentation medium (DMⅢ) (g / L): 4.56 g sodium citrate, 0.352 g KH2PO4, 0.4928 g NaNO2, 2 mL trace element solution, pH 7.0.

[0054] The basal culture medium used in the experiment was sterilized by high-pressure steam at 121°C for 20 minutes before use.

[0055] 3. Enrichment, separation and screening of heterotrophic nitrification-aerobic denitrification strains

[0056] (1) Sample pretreatment: Weigh 10g of sediment sample into a conical flask with 90mL of sterile saline and a small amount of sterilized glass beads, which is a 10-fold diluted suspension, and plug it with cotton (this process is performed in a clean bench). Oscillate at a speed of 160r / min for 1h to evenly disperse the sediment sample and fully suspend the microorganisms in the saline. (The same applies to water samples);

[0057] (2) Enrichment culture: Take 22.2 mL of the above mixture (10 -1 10 mL of water sample was added to 90 mL of enrichment medium and cultured at 30°C and 160 r / min for 24 h. This step mainly increases the number of target strains to facilitate the subsequent separation and purification of strains.

[0058] (3) Sample enrichment solution plate coating: Take 1 mL of the water sample and mud sample after the above enrichment culture, add them to 9 mL of sterile saline and dilute them to 10 -2 , take 55 test tubes each containing 9 mL of sterile water, numbered as "dilution multiple + water sample / mud sample", use a pipette to draw 1 mL of the suspension in the test tube and add it to the tube numbered as "mud sample - 10 -2 " in the test tube, and gently blow and wash it several times in the test tube to make 10 -2 The mud sample dilution was diluted to 10 -10 Repeat the process to dilute the water sample and the bottom mud to 10 -10 . Take 10 respectively -7 ~10 -10Take 100 μL of the dilution solution from the prepared BTB plate (three parallels for each gradient and one blank control) and spread it evenly in one direction with a burned applicator. Then place it in a 30℃ constant temperature biochemical incubator and incubate it upside down for 2 days;

[0059] (4) Separation and purification: Observe the plates at different dilutions, select the plates with colony counts between 30 and 300, and discard the plates that do not meet the requirements. Use an inoculation loop to pick up single colonies of different shapes and textures, and use the plate to draw four zones in a zigzag shape. Use an inoculation loop to pick up colonies in zone one and draw lines several times, and then draw lines in zones two, three, and four in turn. After each zone is drawn, sterilize the inoculation loop once and cool it before drawing the next zone (repeat 3 to 4 times). After drying at room temperature, turn it upside down and culture it in a 30°C constant temperature biochemical incubator for 2 to 3 days. Pick a single colony from the third or fourth zone, stain it with ammonium oxalate crystal violet, and then check it for purity under a microscope. If it is not pure, continue to repeat the zoning and drawing lines, or increase the dilution multiple to enrich the culture again until a purified single colony is obtained. Finally, observe with a 100× oil immersion lens and take photos for record;

[0060] (5) Initial screening by spot inoculation: Use an inoculation needle to pick up the purified strain and spot inoculate it in the BTB denitrification identification medium for 2-3 days. Select strains with high denitrification ability based on the growth of the colony and the size of the blue halo in the BTB medium around the colony. Generally speaking, the larger the blue halo, the higher the denitrification ability. After inoculation on the slant and constant temperature culture at 30℃ for 2-3 days, the test tube is stored at 4℃;

[0061] (6) Strain expansion culture: Prepare nutrient broth medium and sterilize it with high pressure steam at 121°C for 20 min. Inoculate the four strains into the sterilized nutrient broth medium respectively and culture them at 30°C and 160 rpm for 16 h to allow the strains to grow to the logarithmic growth phase.

[0062] (7) Rescreening of nitrification and denitrification performance: Prepare rescreening medium and sterilize it. Add four strains to the rescreening medium respectively, with three replicates in each group, and culture at 30°C and 160rpm. Take appropriate amount of samples at 0h, 12h, 24h, 36h, and 48h, and measure OD 600 After ultra-high speed centrifugation at 8000 rpm and 4 °C for 5 min, the supernatant NH4 + -N, NO3 - -N, NO2 - -N and PO4 3- -P content.

[0063] 4. Identification of heterotrophic nitrification-aerobic denitrification strain HCCN1

[0064] (1) Morphological identification: After the above screening and separation, a heterotrophic nitrification-aerobic denitrification strain HCCN1 was obtained. The Gram reaction of this strain was positive, the colonies were dry, slightly raised, and easy to pick up with an inoculation loop. They were round and light yellow, with a transparent ring around the colonies, with a diameter of 4-5mm, and no special smell. Under a scanning electron microscope, the bacteria were rod-shaped, without flagella, and were about 2.13±0.04μm in length.

[0065] (2) Molecular biological identification: DNA of strain HCCN1 was extracted using Takara Lysis Buffer for Microorganism to Direct PCR lysase. Its 16S rDNA was amplified using this template, and a pair of universal primers were used for amplification: upstream primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3'; downstream primer (1492R): 5'-GGCTACCTTGTTACGACTT-3'. The universal primers were synthesized by Shanghai Biotechnology Co., Ltd. PCR reaction system (25μL): 2×Unique TM Taq Master Mix (With Dye) 12.5μL, upstream primer and downstream primer 1μL each, DNA template 1μL, ddH2O 9.5μL. The PCR program is as follows: ①94℃, 5 min; ②94℃ pre-denaturation, 1min; ③55℃ annealing, 1min; ④72℃ extension 1.5min; ⑤72℃, 10min; ②~④ cycles 30 times. 1% agarose gel electrophoresis analysis results. Sequencing of PCR-amplified 16SrDNA products was completed by Shanghai Bioengineering Co., Ltd.

[0066] After the above screening and separation, a heterotrophic nitrification-aerobic denitrification strain HCCN1 was obtained. The 16SrDNA sequence length of strain HCCN1 was 716 bp, and its physiological and biochemical characteristics are shown in Table 1.

[0067] Table 1

[0068]

[0069] Note: “+” means positive; “-” means negative

[0070] 5. Conclusion

[0071] After the above steps of collection, screening, cultivation, identification, etc., a preferred heterotrophic nitrification-aerobic denitrification strain HCCN1 with strong stress resistance was obtained; based on its 16SrDNA, bacterial morphology, colony morphology, physiological and biochemical identification items, the strain HCCN1 was judged to be Bacillus cereus ( Bacillus cereusThe strain has been deposited in Guangdong Microbiological Strain Collection Center (GDMCC), with the deposit address at 5F, Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, with the deposit number 65477 and the deposit status as alive.

[0072] Example 2

[0073] This example is an environmental safety evaluation of the strain HCCN1 provided in Example 1, as a basis for further application, including the following contents:

[0074] (1) Fish toxicity test: Select healthy zebrafish with a body length of 3 ± 1 cm ( Danio Rerio ), and temporarily cultured in a large water body with continuous aeration for 30 days, during which they were fed normally and water was changed regularly; after the state stabilized, they were randomly assigned to 15L glass tanks, and an experimental group with bacterial solution was set up ( Bacillus cereus HCCN1) and a control group (CRT) with an equal volume of sterile water added, with 30 zebrafish in each experimental group and 3 replicates in each group; take the overnight cultured bacterial solution, centrifuge at 8000r / min for 5min, discard the supernatant, resuspend in sterile PBS buffer, repeat 1-2 times, and then resuspend in sterile water. The OD value was obtained according to the standard curve of the determination. 600 The relationship between the bacterial concentration and the experimental water bacterial concentration was adjusted to about 1×10 6 CFU / mL, and the blank control group was added with the same amount of sterile water; during the experiment, the experimental subjects were fed normally and the experimental water was completely replaced every three days. After the water was changed, the above method was repeated to add bacterial solution and sterile water respectively, and the survival rate of zebrafish in each group was recorded. The experiment lasted for 14 days;

[0075] See attached Figure 4 , under normal conditions for 14 days, the survival rate of zebrafish in the control group was 100%, and the concentration of test bacteria in the water of the experimental group was 10 6 CFU / mL, which is higher than the pathogenic dose of common pathogens (10 4 CFU / mL); the survival rate of zebrafish in the experimental group was 100%, which was not significantly different from that in the control group (p>0.05); it was preliminarily judged that Bacillus cereus HCCN1 had high aquatic biological safety.

[0076] (2) Commonly used antibiotic resistance experiments: The experimental methods, steps and evaluation criteria of antibiotic resistance experiments (antibiotic paper susceptibility tests) are based on the "M100 Antimicrobial Susceptibility Test Implementation Standard (13th Edition)" and "M02-A10 Antimicrobial Susceptibility Test Paper Method Implementation Standard (Vol. 32 No. 1)". The specific steps are as follows:

[0077] a) Prepare MHA (Guangdong Huankai Biotechnology Co., Ltd.) plate and adjust the pH to 7.2~7.4;

[0078] b) Use sterile blank drug sensitivity paper (Hangzhou Microbiological Reagent Co., Ltd.) to prepare paper with corresponding drug content;

[0079] c) Inoculate the strain into nutrient broth medium, culture at 30°C, 180r / min until the logarithmic phase, and dilute to the specified concentration before the experiment;

[0080] d) Use a disposable sterile cotton swab to dip into the diluted bacterial solution, squeeze out the water from the centrifuge tube wall, and evenly spread it on the MHA plate, and dry it at room temperature for 5 minutes;

[0081] e) Use sterile tweezers to stick the paper containing antibiotics in the center of the MHA plate. Set up 3 parallel replicates for each experiment;

[0082] f) Invert the plate within 15 minutes and incubate at 30°C for 18 hours;

[0083] g) Use IP54 metal shell digital display vernier caliper (Yongkang Jingsida Trading Co., Ltd.) to measure the diameter of the inhibition zone;

[0084] Observe the size of the inhibition zone. The results are shown in the attached Fig.11 , In the figure, AP stands for cefalotin, metronidazole, penicillin, oxacillin, sulfonamide, erythromycin, ciprofloxacin, neomycin, streptomycin, tetracycline, cefazolin, norfloxacin, polymyxin B, imipenem, amoxicillin, cefoxitin, and aztreonam, respectively. The results showed that the strain HCCN1 was sensitive to ciprofloxacin, neomycin, streptomycin, tetracycline, cefazolin, norfloxacin, polymyxin B, imipenem, amoxicillin, cefoxitin, and aztreonam, and was resistant to cefalotin, metronidazole, penicillin, oxacillin, sulfonamide, and erythromycin.

[0085] Therefore, strain HCCN1 is sensitive to multiple antibiotics, which provides a reference for the placement of antibiotics in aquaculture ponds in future practical applications, provides a means of prevention and control for the killing of strains, and improves the biosafety of strains. The results of the HCCN1 antibiotic resistance test are shown in Table 2. Therefore, strain HCCN1 improves its resistance to environmental stress and significantly improves its drug resistance, and has good environmental safety.

[0086] Table 2

[0087]

[0088] Example 3

[0089] In this example, the Bacillus cereus strain provided in Example 1 Bacillus cereus HCCN1 is used as an active ingredient in the preparation of a microbial agent.

[0090] Specifically, the HCCN1 strain is directly prepared as a suspension, or is prepared as a fixed bacterial agent using porous materials such as biochar as a carrier, so as to improve the microscopic growth environment of the strain and accelerate the growth rate of the strain.

[0091] It can be prepared by conventional techniques, so it will not be described in detail.

[0092] Example 4

[0093] In this example, the microbial agent (suspension) prepared by the HCCN1 strain provided in Example 3 was applied to biological denitrification and phosphorus removal of nitrogen- and phosphorus-containing wastewater, and the efficiency and effect of purifying wastewater under various harsh conditions were specifically tested.

[0094] The carbon source used in the biological treatment process of nitrogen and phosphorus-containing wastewater is at least one of sodium citrate, sodium succinate, sodium oxalate and glucose, which can be selected according to actual needs.

[0095] In this embodiment, the nitrogen-phosphorus-containing wastewater used has a carbon-nitrogen ratio of 0-20, a phosphorus-nitrogen ratio of 0-1.2, a temperature of 20°C-40°C, a pH value of 5-12, and a salinity of 0%-10%, and is tested using multiple carbon sources.

[0096] Optimal growth and denitrification conditions of Bacillus cereus HCCN1

[0097] (1) Testing the effects of different organic carbon sources on the growth and denitrification performance of Bacillus cereus HCCN1

[0098] Sodium oxalate, sodium succinate, glucose and sodium citrate were selected as four carbon sources, and C / N was fixed at 10, 30℃, 160r / min and pH = 7.0. Based on DM fermentation medium, the addition amount of sodium oxalate, sodium succinate, glucose and sodium citrate per liter of medium was 5.58g, 5.62g, 2.50g and 4.08g respectively; the addition amount of (NH4)2SO4, NaNO3 and NaNO2 as single inorganic nitrogen sources per liter of medium was 0.472g, 0.607g and 0.493g respectively.

[0099] The candidate strains were inoculated into nutrient broth medium, cultured at 30°C, 160r / min for 16h, and then inoculated into the above denitrification medium with different organic carbon sources at a 1% inoculation rate. The culture fluid was taken at 0h, 4h, 8h, 12h, 24h, 36h, and 48h to measure its OD 600 , 8000r / min, 4℃, high-speed centrifugation for 3 min, and then the supernatant was taken to determine NH4 + -N, NO3 - -N, NO2 - -N three nitrogen elements and PO43- The experiment set up three technically repeated experimental groups and a blank control group, and the control group was added with an equal amount of physiological saline.

[0100] Finally, the effects of four different organic carbon sources, sodium oxalate, sodium succinate, glucose and sodium citrate, on the growth and nitrogen and phosphorus removal of HCCN1 were analyzed. The test results are as follows Figure 5 shown.

[0101] (2) Testing the effects of different C / N ratios on the growth and denitrification performance of HCCN1

[0102] Sodium citrate was selected as the carbon source of the denitrification medium, and the conditions were fixed at 30°C, 160r / min, P / N=0.2, pH=7.0, and the C / N gradient was set to 0, 2, 5, 10, 15, 18, and 20. The amount of sodium citrate added to the medium for each gradient was 0g / L, 0.817g / L, 2.042g / L, 4.083g / L, 6.127g / L, 7.353g / L, and 8.169g / L; the amount of (NH4)2SO4, NaNO3, and NaNO2 added per liter of medium as a single inorganic nitrogen source was 0.472g, 0.607g, and 0.493g, respectively. The candidate strains were inoculated into nutrient broth medium, cultured at 30°C, 160r / min for 16h, and then inoculated into the above medium at a 1% inoculation rate. The culture fluid was taken at 0h, 4h, 8h, 12h, 24h, 36h, and 48h to measure its OD 600， 8000r / min, 4℃ high speed centrifugation for 3 min, and then the supernatant was taken to determine NH4 + -N, NO3 - -N, NO2 - -N three nitrogen elements and PO4 3- The experiment set up three technically repeated experimental groups and a blank control group, and the control group was added with an equal amount of physiological saline.

[0103] Finally, the effects of seven different C / N ratios (0, 2, 5, 10, 15, 18, and 20) on the growth and nitrogen and phosphorus removal of HCCN1 were analyzed. The results are as follows: Figure 6 shown.

[0104] (3) Testing the effects of different P / N ratios on the growth and denitrification performance of HCCN1

[0105] Sodium citrate was selected as the carbon source of the denitrification medium, and the conditions were fixed at 30°C, 160r / min, C / N=15, pH=7.0, and the P / N gradient was set to 0, 0.1, 0.2, 0.5, 0.8, 1.0, and 1.2. The amount of KH2PO4 added to the medium for each gradient was 0.0000g / L, 0.0439g / L, 0.0870g / L, 0.1755g / L, 0.3510g / L, 0.4387g / L, and 0.5265g / L; the amount of (NH4)2SO4, NaNO3, and NaNO2 added per liter of medium as a single inorganic nitrogen source was 0.472g, 0.607g, and 0.493g, respectively. The candidate strains were inoculated into nutrient broth medium, cultured at 30°C, 160r / min for 16h, and then inoculated into the above medium at a 1% inoculation rate. The culture fluid was taken at 0h, 4h, 8h, 12h, 24h, 36h, and 48h to measure its OD 600 , 8000r / min, 4℃ high speed centrifugation for 3 min, and then the supernatant was taken to determine NH4 + -N, NO3 - -N, NO2 - -N three nitrogen elements and PO4 3- The experiment set up three technically repeated experimental groups and a blank control group, and the control group was added with an equal amount of physiological saline.

[0106] Finally, the effects of seven different P / N ratios (0, 0.1, 0.2, 0.5, 0.8, 1.0, and 1.2) on the growth and nitrogen and phosphorus removal of HCCN1 were analyzed. The results are as follows: Figure 7 shown.

[0107] (4) Testing the effects of different pH on the growth and denitrification performance of HCCN1

[0108] The conditions of C / N=15, P / N=0.8, 30℃, 160r / min, sodium citrate as the single organic carbon source, etc. were fixed, and the pH gradient was set to 5, 6, 7, 8, 9, 10, 11, and 12. As the single inorganic nitrogen source, (NH4)2SO4, NaNO3, and NaNO2 were added in an amount of 0.472g, 0.607g, and 0.493g per liter of culture medium, respectively. The candidate strains were inoculated into nutrient broth medium, cultured at 30℃ and 160r / min for 16h, and then inoculated into the above culture medium with an inoculum of 1%, and the culture fluid was taken at 0h, 4h, 8h, 12h, 24h, 36h, and 48h to determine its OD 600 , 8000r / min, 4℃ high speed centrifugation for 3 min, and then the supernatant was taken to determine NH4 + -N, NO3 - -N, NO2 --N three nitrogen elements and PO4 3- The experiment set up three technically repeated experimental groups and a blank control group, and the control group was added with an equal amount of physiological saline.

[0109] The effects of eight different pH values ​​(5, 6, 7, 8, 9, 10, 11, and 12) on the growth of HCCN1 and the effects of nitrogen and phosphorus removal were analyzed. Figure 8 shown.

[0110] (5) Testing the effects of different temperatures on the growth and denitrification performance of HCCN1

[0111] The conditions of C / N=15, pH=8.0, 160r / min, sodium citrate as the single organic carbon source were fixed, and the temperature gradient was set to 20℃, 25℃, 30℃, 35℃, and 40℃. As the single inorganic nitrogen source, (NH4)2SO4, NaNO3, and NaNO2 were added in an amount of 0.472g, 0.607g, and 0.493g per liter of culture medium, respectively. The candidate strains were inoculated into nutrient broth medium, cultured at 30℃ and 160r / min for 16h, and then inoculated into the above culture medium with an inoculum of 1%, and the culture fluid was taken at 0h, 4h, 8h, 12h, 24h, 36h, and 48h to determine its OD 600 , 8000r / min, 4℃ high speed centrifugation for 3 min, and then the supernatant was taken to determine NH4 + -N, NO3 - -N, NO2 - -N three nitrogen elements and PO4 3- The experiment set up three technically repeated experimental groups and a blank control group, and the control group was added with an equal amount of physiological saline.

[0112] Finally, the effects of five different temperatures (20℃, 25℃, 30℃, 35℃, and 40℃) on the growth of HCCN1 and the effects of nitrogen and phosphorus removal were analyzed. Fig.10 shown.

[0113] (5) Effects of different salinities on the growth and denitrification performance of HCCN1

[0114] The conditions of C / N=15, P / N=0.8, pH=8.0, 160r / min, sodium citrate as the sole organic carbon source, etc. were fixed, and the salinity gradient was set to 0%, 1.0%, 1.5%, 2.0%, 3.0%, 5.0%, 7.0%, and 10.0%. The salinity was controlled by the additional addition of NaCl, and the corresponding addition amount per liter was 0g, 1.0g, 1.5g, 2.0g, 3.0g, 5.0g, 7.0g, and 10.0g. As the sole inorganic nitrogen source, the addition amount of (NH4)2SO4, NaNO3, and NaNO2 per liter of culture medium was 0.472g, 0.607g, and 0.493g, respectively. The candidate strains were inoculated into nutrient broth medium, cultured at 30°C, 160r / min for 16h, and then inoculated into the above medium at a 1% inoculation rate. The culture fluid was taken at 0h, 4h, 8h, 12h, 24h, 36h, and 48h to measure its OD 600 , 8000r / min, 4℃ high speed centrifugation for 3 min, and then the supernatant was taken to determine NH4 + -N, NO3 - -N, NO2 - -N three nitrogen elements and PO4 3- The experiment set up three technically repeated experimental groups and a blank control group, and the control group was added with an equal amount of physiological saline.

[0115] Finally, the effects of eight different salinities, 0%, 1.0%, 1.5%, 2.0%, 3.0%, 5.0%, 7.0%, and 10.0%, on the growth of HCCN1 and the effects of nitrogen and phosphorus removal were analyzed. The results are as follows Fig. 9 shown.

[0116] Through the above-mentioned various test results of this embodiment, and the attached Figure 5-Figure 10 It can be seen that the strain HCCN1 provided by the present invention can grow with a variety of organic carbon sources such as sodium citrate and sodium succinate. When growing with sodium citrate, the growth and nitrogen and phosphorus removal effects are the best. It can grow under C / N of 0~20, P / N of 0~1, pH of 5~11, salinity of 0%~5%, and temperature of 20℃~35℃. When C / N is 0, HCCN1 grows poorly, indicating that it needs a carbon source as the basis of energy and structure during growth and metabolism. Insufficient carbon source will lead to limited energy metabolism, thereby affecting growth; when P / N is 0, the nitrogen removal effect is significantly worse than when the P / N ratio is 0.1, indicating that P is an essential element for HCCN1 growth metabolism; when the salinity increases from 0 to 5%, the growth of the strain improves accordingly, indicating that the bacteria are suitable for high-salt conditions and grow better under high-salt conditions. When NH4 +When -N is the only nitrogen source, it can grow normally at low temperatures, indicating that the bacteria has a certain preference for ammonia nitrogen under this condition. The optimal denitrification conditions are sodium citrate as the carbon source, C / N=15, P / N=0.8, pH=8, and T=30℃. The denitrification rates of HCCN1 were 95.87%, 90.62%, and 100.00%, respectively, and the corresponding phosphorus removal rates were all 100%. Under the optimal denitrification and phosphorus removal conditions, the pH value increased from 7 to 11, and the corresponding nitrogen and phosphorus removal rates had no statistically significant difference, indicating that it has good tolerance to alkaline environments.

[0117] The above embodiments show that the strains selected by the present invention have improved the stress resistance to survival and reproduction in harsh environments, especially the stress resistance to high salt and extreme acid-base environments. However, environmental safety can still be guaranteed. The strain can not only perform a good heterotrophic nitrification and aerobic denitrification process under high alkaline conditions, but also effectively remove phosphate. The alkali and salt resistance of the strain are helpful to promote aquaculture in saline-alkali land, tidal flats and other areas, increase the land area for aquaculture, prevent water pollution, and improve ecological benefits.

[0118] In summary, the strain HCCN1 of the present invention can play a key role in the field of high-salt and high-alkali nitrogen and phosphorus aquaculture tailwater treatment. The strain has no adverse effects on aquaculture objects, has a high biosafety of aquatic organisms, and is suitable for application in most aquaculture water bodies; it also has heterotrophic nitrification, aerobic denitrification and dephosphorization functions; it can use a variety of organic carbon sources while having strong tolerance to high concentrations of organic carbon, salinity and extreme acid-base environments, and has good water body organic carbon removal capabilities. The strain can also be used in some food processing wastewater treatment or aquaculture tailwater projects. For wastewater containing a large amount of sugars and organic acids, the strain HCCN1 can quickly adapt to and utilize these organic carbon sources and convert them into energy required for its own growth. The function of the strain HCCN1 is comprehensive and unique. It not only has heterotrophic nitrification and aerobic denitrification capabilities, but also can efficiently dephosphorize. In the face of high concentrations of organic carbon, high salinity and extreme acid-base environments, the strain HCCN1 is extremely tolerant. The strain is particularly suitable for the treatment of nitrogen-containing sewage with high C / N; under completely aerobic conditions, the strain can use NH4 + -N, NO3 - -N and NO2 - -N is used as the only inorganic nitrogen source for aerobic nitrification and denitrification; the strain can overcome the incompatibility problem of nitrification and denitrification caused by different oxygen demands, making it possible for nitrification and denitrification to proceed simultaneously in the same aerobic reactor, which can comprehensively improve the quality of aquaculture water environment, reduce eutrophication of water bodies, promote the sustainable utilization of water resources, has good economic and environmental benefits, and has broad application prospects.

[0119] It should be particularly noted that within the scope of the above embodiments of the present invention, the technical solutions obtained by selecting other specific or similar components, specific or similar processes, parameters, etc. can all achieve the technical effects of the present invention, so they are no longer listed one by one.

[0120] Finally, it should be noted that, although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as limiting the present invention. After reading the above content, various modifications and substitutions of the present invention will be obvious to those skilled in the art. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A Bacillus cereus strain with high environmental adaptability to nitrification and denitrification, characterized in that: Bacillus cereus Bacillus cereus HCCN1 was deposited in the Guangdong Microbial Strain Collection Center (GDMCC) on November 13, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the deposit number is GDMCC No: 65477.

2. The Bacillus cereus strain with high environmental adaptability to nitrification and denitrification according to claim 1, characterized in that: The strain HCCN1 was isolated and screened from water samples and mud samples from aquaculture pond in the south of Doumen District, Zhuhai City, Guangdong Province.

3. A microbial agent, characterized in that: It is the Bacillus cereus strain according to claim 1 Bacillus cereus HCCN1 as the active ingredient.

4. Use of the Bacillus cereus strain with high environmental adaptability to nitrification and denitrification according to claim 1, or the microbial agent according to claim 3, in biological denitrification and phosphorus removal treatment of nitrogen- and phosphorus-containing wastewater.

5. The use according to claim 4, characterized in that: The carbon source for biological treatment of nitrogen- and phosphorus-containing wastewater is at least one of sodium citrate, sodium succinate, sodium oxalate and glucose.

6. The use according to claim 4, characterized in that: The nitrogen- and phosphorus-containing wastewater has a carbon-nitrogen ratio of 0-20, a phosphorus-nitrogen ratio of 0-1.2, and a temperature of 20°C-40°C.

7. The use according to claim 4, characterized in that: The pH value of the nitrogen and phosphorus-containing wastewater is 5-12.

8. The use according to claim 4, characterized in that: The salinity of the nitrogen and phosphorus-containing wastewater is 0% to 10%.

9. The use according to any one of claims 4 to 8, characterized in that: The strain Bacillus cereus HCCN1, with sodium citrate as carbon source, C / N=15, P / N=0.8, pH=8, T=30℃, NH4 + -N、NO3 - -N, NO2 - -N degradation efficiency reaches the highest level, and the degradation efficiency of PO4 3- The total removal rate of -P was 100%.

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