Paraburkholderia sp. and application thereof

Abstract

The application relates to the field of microorganisms, and particularly discloses a Paraburkholderia sp. and application thereof. The strain is isolated from an environment sample of a breeding farm, can efficiently degrade ammonia and hydrogen sulfide, and is identified as Paraburkholderia sp. The strain is a non-pathogenic bacterium, has good degrading capacity (the removal rates are all above 80%) on ammonia and hydrogen sulfide, can inhibit harmful microorganisms in a breeding environment to a certain extent, can be developed into a deodorant for efficiently removing ammonia and hydrogen sulfide, and has a wide application prospect.

Description

Technical Field

[0001] This application relates to the field of microbiology, and more specifically, to a species of *Paraburkholderia* SP. and its applications. Background Technology

[0002] With the development of society and the economy, the foul odors from factories, garbage dumps, sewage treatment plants, farms, and pet feces have had a very serious impact on agricultural production and people's normal lives. Common odorous gases include nitrogen compounds, sulfides, aldehydes, and fatty acids. Nitrogen compounds are mostly ammonia and methylamine, which are mainly produced in toilets, animal feces, and farms; sulfides are mainly hydrogen sulfide and thiols, which are mainly produced in household garbage and sewers.

[0003] To effectively control odorous gases in the environment, various deodorization technologies have been developed, which can be categorized into physical, chemical, and biological deodorization technologies based on their deodorization principles. Physical deodorization methods mainly include using adsorption and masking deodorants to reduce odor concentration or smell, and also include ventilation devices for livestock and poultry farming. Chemical deodorization methods primarily employ oxidants and sterilizing agents with oxidizing effects for oxidation-reduction deodorization. Biological deodorization methods include microbial inoculant methods, insect deodorization, and soil modification methods; among these, microbial inoculant methods mainly rely on the decomposition and transformation effects of highly efficient microorganisms, and are characterized by high efficiency and cost-effectiveness.

[0004] Currently, there is a lack of microbial agents that can efficiently degrade ammonia and hydrogen sulfide. Therefore, this application is hereby submitted. Summary of the Invention

[0005] To address the aforementioned technical problems, this application provides a species of *Paraburkholderia* SP. and its applications.

[0006] The technical solution adopted in this application is as follows:

[0007] In the first aspect, this application provides a species of *Paraburkholderia* SP. deposited by the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32506 and deposit date of November 6, 2024.

[0008] Secondly, this application provides a microbial inoculant containing the aforementioned *Paraburkholderia* SP.

[0009] Thirdly, this application provides a deodorant containing the aforementioned microbial agent.

[0010] Furthermore, the viable count of *Paraburkholderia* SP. in the aforementioned deodorant is 1 × 10⁻⁶. 7 CFU / mL ~ 1×10 9 CFU / mL.

[0011] Fourthly, this application provides a method for preparing the above-mentioned deodorant, comprising:

[0012] (1) The above-mentioned Paraburkholderia SP. was inoculated into nutrient broth medium for activation;

[0013] (2) The activated Paraburkholderia SP. was inoculated into beef extract peptone medium and cultured for 1-3 days to obtain fermentation broth;

[0014] (3) Mix the fermentation liquid with other auxiliary materials.

[0015] Furthermore, the viable count of *Paraburkholderia* SP. in the fermentation broth was 1 × 10⁻⁶. 7 CFU / mL ~ 1×10 9 CFU / mL.

[0016] Fifthly, this application provides the use of the aforementioned *Paraburkholderia* SP. in products for removing ammonia and / or hydrogen sulfide.

[0017] Furthermore, the above applications are for removing ammonia and / or hydrogen sulfide from composting plants, landfills, sewage treatment plants, or livestock farms.

[0018] In summary, this application has the following beneficial effects:

[0019] This application, through screening, isolated a strain of bacteria capable of efficiently degrading ammonia from environmental samples of aquaculture farms. The strain was identified as *Paraburkholderia* SP. This non-pathogenic strain not only exhibits excellent degradation capabilities for ammonia and hydrogen sulfide (removal rates exceeding 80% for both), but also shows some inhibitory effect on harmful microorganisms in the aquaculture environment. Taking wastewater deodorization in wastewater treatment plants as an example, verification showed that adding a bacterial agent containing *Paraburkholderia* SP. to wastewater can effectively degrade ammonia and hydrogen sulfide, reducing their concentrations and removing odors. Therefore, this strain can be developed into a highly efficient deodorant for removing ammonia and / or hydrogen sulfide, with broad application prospects. Attached Figure Description

[0020] Figure 1 The results show the ammonia nitrogen removal rates of the 13 strains obtained in the initial screening in Example 1 of this application.

[0021] Figure 2 A morphological diagram of a strain of *Paraburkholderia* SP. provided in Example 1 of this application;

[0022] Figure 3 This is a diagram of the hemolytic experiment of *Paraburkholderia* SP. provided in Example 2 of this application;

[0023] Figure 4 The cumulative emission of NH3 in the composting deodorization experiment provided in Example 5 of this application;

[0024] Figure 5 The cumulative H2S emissions during the composting deodorization experiment provided in Example 5 of this application. Detailed Implementation

[0025] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0026] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0027] Example 1

[0028] This embodiment provides a screening process for ammonia-removing bacteria strains.

[0029] 1. Preparation of culture medium:

[0030] (1) Selective culture medium for ammonia removal bacteria: 5g glucose, 1.0g NaCl, 0.6g (NH4)2SO4, 1.0g K2HPO4, 0.5g MgSO4·7H2O, 0.05g FeSO4·7H2O, 1L distilled water.

[0031] (2) Basic culture medium: 10g peptone, 2g beef extract, 5.0g NaCl, 1L distilled water, pH 6.5-7.5.

[0032] (3) Slant culture medium: basal culture medium and 20g agar.

[0033] 2. Enrichment and purification:

[0034] Samples were collected from chicken manure, cow manure, pig manure, sludge, sewage, and compost. 10 ml of each fresh sample was added to 100 ml of ammonia-removing liquid selective culture medium ((NH4)2SO4 0.6 g·L⁻¹). -1 After culturing at 30℃ and 120 rpm for 5 days, 10 ml of the culture medium was transferred to a new 100 ml selective medium ((NH4)2SO4 1 g·L⁻¹). -1 After culturing for 5 days, transfer 10 ml to a new 100 ml selective medium ((NH4)2SO4 2 g·L⁻¹). -1 At this concentration, the culture medium was transferred to a new culture medium every 5 days, and the culture was continuously acclimatized for one month to obtain an enriched bacterial solution.

[0035] Take 1 ml of the enriched bacterial culture and perform serial dilutions, adding 10... -4 10 -5 10 -6 0.1 ml of bacterial suspension was inoculated onto ammonia-free solid selective medium plates, with each gradient replicated three times, and incubated upside down at 30°C for 5 days. Single colonies were repeatedly streaked for purification until no contaminants were observed under a microscope to obtain pure strains. A total of 253 strains were isolated.

[0036] 3. Initial screening:

[0037] Based on 253 bacterial strains, the preserved strains were activated in LB medium for 24 hours and inoculated into ammonia-removing selective medium at an inoculum rate of 0.5%. The medium was incubated at 180 rpm and 30°C. At 24 h and 48 h of incubation, 5 mL of bacterial solution was taken and mixed with equal volumes of Griess reagent A and B. 1 mL of the mixed reagent was added to the bacterial solution. At the same time, the medium without bacteria was used as a blank control (CK). Based on the color intensity of the reaction, strains with high ammonia removal capacity were quickly screened for further experiments.

[0038] Results: A total of 13 bacterial strains with high ammonia removal efficiency were obtained. The ammonia nitrogen removal rates of these 13 strains were measured at 24h and 48h, and the results are as follows: Figure 1 As shown.

[0039] Depend on Figure 1 As can be seen, after initial screening, 7 strains of the 13 strains had ammonia nitrogen removal rates of more than 60% at both 24h and 48h. Therefore, the 7 strains with the highest degradation rates (R1, R2, R3, R4, W1, W4, W7) were selected for subsequent experiments.

[0040] 4. Secondary screening:

[0041] The seven strains preserved above were activated in LB medium for 24 hours and inoculated into ammonia-removing selective medium at an inoculum rate of 0.5%. The medium was incubated at 30°C and 180 rpm. After 24 hours of incubation, 5 mL of bacterial solution was taken and centrifuged at 12000 rpm for 2 min. The supernatant was then subjected to Nessler's reagent colorimetric method, and the ammonia nitrogen content in the solution was measured at a wavelength of 420 nm using a spectrophotometer. At the same time, a blank control (CK) medium without bacteria was used and placed under the same conditions to measure the ammonia nitrogen content in CK. The ammonia nitrogen removal rate in the medium can be calculated by comparing the sample with CK, thereby comparing the ammonia removal ability of each strain.

[0042] The results are shown in Table 1:

[0043] Table 1. Effects of different bacterial strains on ammonia removal.

[0044]

[0045] As shown in Table 1, the R3 strain was found to have the strongest ability to degrade ammonia nitrogen through secondary screening, with a removal rate of 86.78%.

[0046] 5. Strain identification:

[0047] (1) Bacterial morphological characteristics:

[0048] After incubating strain R3 on NB plates at a constant temperature (30℃) for 24 hours, as follows... Figure 2 As shown, its bacterial cell morphology is characterized by being round, white, opaque, and having smooth edges.

[0049] (2) Molecular identification:

[0050] Genomic DNA was extracted from strain R3 using a 2×T5 Direct PCR Kit (Plant). PCR amplification of the ITS fragment of the DNA was performed using universal primers.

[0051] The PCR amplification product was sequenced to obtain the 16SRNA sequence of strain R3. The sequencing results were compared with the NCBI database using BLAST analysis to confirm that the strain belongs to the genus Paraburkholderia sp.

[0052] Example 2

[0053] This embodiment evaluates the safety of Paraburkholderia SP.

[0054] 1. Evaluation Method:

[0055] Hemolytic test: The selected microbial strain R3 with high efficiency in ammonia deodorization was streaked on a blood agar plate, with Bacillus cereus as a positive control. The plate was incubated in a constant temperature incubator at 30℃ for 24 hours. The presence of a transparent or translucent hemolytic ring around the microbial strain was observed to determine whether the strain was hemolytic.

[0056] 2. Experimental Results:

[0057] like Figure 3 As shown, no hemolytic zone appeared after culturing this strain, indicating that this strain of *Paraburkholderia* SP. is non-hemolytic and has high safety during production and use.

[0058] Example 3

[0059] This embodiment provides a simulation experiment of the ammonia removal effect of the above-mentioned strains on chicken manure.

[0060] (1) Activation of microbial strains:

[0061] The strain Paraburkholderia SP. was inoculated into NB medium and activated overnight.

[0062] (2) Preparation of microbial agents:

[0063] The activated bacterial strain was inoculated into beef extract peptone medium and cultured at 30°C and 120 rpm for 48 hours. The fermentation broth was used as the inoculum treatment solution, and the viable cell count was determined to be 4.1 × 10⁻⁶ using the plate count method. 8 CFU / mL.

[0064] (3) Chicken manure simulation experiment:

[0065] Add 200g of fresh chicken manure sample to a 2L sealed plastic box. Place a 100mL beaker containing 50mL of 0.01mol / L sulfuric acid solution (to absorb the generated NH3) into the plastic box. Cover the beaker with two layers of gauze and secure with a rubber band. After sealing the box, incubate at 50℃ (NH3 production approximately 30ppm). After 3 hours, remove the plastic box and spray with bacterial agent using a sprayer. Spray 1ml of bacterial solution into the plastic box, repeating 3 times. Simultaneously, spray an equal volume of sterile water as a control. The ammonia release was determined using Nessler's reagent spectrophotometry. Calculate the ammonia removal rate using the following formula:

[0066] Ammonia removal rate = Difference in ammonia release between the blank control group and the bacterial agent treatment group / Ammonia release of the blank control group × 100%.

[0067] (4) Experimental results:

[0068] Table 2. Ammonia removal efficiency of treatment group and control group

[0069]

[0070] As shown in Table 2, this strain of *Paraburkholderia* SP. can effectively inhibit the release of ammonia from chicken manure, with an inhibition rate of over 85%. Therefore, this strain can effectively inhibit ammonia released from animal excrement and holds promise for development as a highly efficient deodorizer for removing ammonia.

[0071] Example 4

[0072] This embodiment uses wastewater from different wastewater tanks in a wastewater treatment plant as the substrate for a closed deodorization experiment.

[0073] Wastewater from wastewater tanks 1-3 was divided into 500ml Erlenmeyer flasks, with 50ml of wastewater in each flask. Three replicates were set up for each experiment. The prepared bacterial treatment solution (same as in Example 3) was then diluted with water at a ratio of 1:100 and sprayed onto each flask at a dosage of 5ml. The control group was sprayed with an equal volume of sterile water. The flasks were then sealed with three layers of plastic wrap and secured with rubber bands. The flasks were placed in a 37℃ incubator, and the changes in ammonia and hydrogen sulfide concentrations were measured after 24 hours. The experimental results are shown in Table 3.

[0074] Table 3. Degradation effects of microbial agents on ammonia and hydrogen sulfide in different wastewaters

[0075]

[0076] As shown in Table 3, the bacterial agent provided in this application has a significant inhibitory effect on ammonia and hydrogen sulfide released from sewage in the sewage tank, with a removal rate of over 80%, significantly reducing the concentration of ammonia and hydrogen sulfide in the sewage tank.

[0077] Example 5

[0078] This embodiment provides a composting deodorization experiment.

[0079] A 20L plastic bucket was used. Kitchen waste composted separately served as the control group, while kitchen waste composted with added microbial treatment solution (provided in Example 3) served as the experimental group. Approximately 15kg of compost material was added to each of the control group (K) and experimental group (S) buckets and stirred thoroughly. A hole was punched in the bucket lid to connect a conduit. One end of the conduit was sealed with hot melt adhesive, and the other end was connected to an alkaline zinc ammonium complex solution and a boric acid solution, respectively, for the determination of H2S and NH3.

[0080] The experiment lasted 20 days. Absorbance samples were taken every 2 days to measure the absorbance, and the concentrations of NH3 and H2S were calculated. The experimental results are as follows: Figures 4-5 As shown.

[0081] The cumulative emissions of NH3 are as follows Figure 4 As shown, the cumulative emissions of H2S are as follows: Figure 5 As shown, the peak emissions of NH3 and H2S both occurred during the high-temperature period of composting, followed by a gradual decrease in emission rate and a gradual plateauing of cumulative emissions. Throughout the composting process, the cumulative emissions of NH3 and H2S in the control group were higher than those in the experimental group. At the end of composting, the cumulative release of NH3 in the control group reached 4577.26 mg / m³. 3 The experimental group had a concentration of 1936.77 mg / m³. 3 After adding the bacterial agent, the NH3 removal rate reached 57.69%. Meanwhile, the cumulative release of H2S in the control group reached 400.63 mg / m³. 3 The experimental group had a concentration of 143.01 mg / m³. 3 After adding the microbial agent, the H2S removal rate reached 66.30%. This indicates that the microbial agent provided in this application can significantly remove NH3 and H2S generated during composting, exhibiting unexpected effects.

[0082] The specific embodiments described in this invention are merely explanations of this application and are not intended to limit it. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A species of *Paraburkholderia* SP., characterized in that, It is deposited by the China General Microbiological Culture Collection Center (CGMCC) at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32506 and deposit date of November 6, 2024. The *Burkholderia paraknegriensis* can simultaneously degrade ammonia and hydrogen sulfide, with removal rates exceeding 80%.

2. A microbial inoculant, characterized in that, The microbial agent contains *Paraburkholderia* SP. as described in claim 1.

3. A deodorant, characterized in that, The deodorant contains the microbial agent as described in claim 2.

4. The deodorant according to claim 3, characterized in that, The viable count of *Paraburkholderia* SP. in the deodorant is 1 × 10⁻⁶. 7 CFU / mL ~ 1×10 9 CFU / mL.

5. A method for preparing the deodorant according to claim 3 or 4, characterized in that, It includes: The *Paraburkholderia* SP. strain described in claim 1 was inoculated into a nutrient broth medium for activation. The activated Paraburkholderia SP. was inoculated into beef extract peptone medium and cultured for 1-3 days to obtain the fermentation broth; The fermentation broth is mixed with other auxiliary materials.

6. The method for preparing the deodorant according to claim 5, characterized in that, The viable count of *Paraburkholderia* SP. in the fermentation broth was 1 × 10⁻⁶. 7 CFU / mL ~ 1×10 9 CFU / mL.

7. The use of Paraburkholderia SP. as described in claim 1 in products for removing ammonia and / or hydrogen sulfide.

8. The application according to claim 7, characterized in that, The application is for removing ammonia and / or hydrogen sulfide from composting plants, landfills, sewage treatment plants or farms.

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