Bacillus luchonensis and application thereof in treating aquaculture wastewater

Abstract

The application discloses a bacillus marinus and application thereof in treatment of aquaculture wastewater, and belongs to the technical field of wastewater purification. The bacillus marinus W202 has been preserved in the China General Microbiological Culture Collection Center on August 8, 2016, and the address of the preservation center is No. 1, Xibanchenli, Jia 3, Chaoyang District, Beijing, and the preservation number is CGMCC No. 12818. The bacillus marinus W202 strain is separated from marine waters, and experiments prove that the strain has good heterotrophic nitrification and aerobic denitrification capacity, and it is speculated that the strain can be used as a new strain for purifying aquaculture wastewater. In combination with treatment experiments on pig farm aquaculture wastewater, it is proved that the bacillus marinus W202 can remove total nitrogen, nitrate nitrogen, total phosphorus, active phosphate and other nitrogen and phosphorus pollutants in the aquaculture wastewater, and the purification effect is remarkable, thereby providing a new idea for purification treatment of livestock and poultry aquaculture wastewater.

Description

Technical Field

[0001] This invention relates to the field of wastewater purification technology, and in particular to a Bacillus chrysogenum and its application in treating aquaculture wastewater. Background Technology

[0002] In recent years, my country's livestock industry has developed rapidly, transforming from scattered, small-scale farming to large-scale, intensive, and large-scale operations. The number of livestock and poultry raised has increased year by year. Data shows that from 2005 to 2014, the number of pigs raised increased annually, with pork production showing an overall upward trend, rising from 45.28 million tons in 2005 to 54.04 million tons in 2018, an increase of 19.3%. Beef production also gradually increased, from 5.49 million head in 2005 to 7.26 million head in 2017, an increase of 32.2%. By the end of 2019, the number of pigs slaughtered was 544.192 million, and the number of cattle raised was 91.383 million. However, in the process of large-scale farming, the increased number of livestock leads to the daily generation of large amounts of manure and wastewater from pen flushing, and this wastewater typically contains high concentrations of pollutants such as nitrogen, phosphorus, heavy metals, and organic matter. According to data from the National Environmental Statistics Bulletin, approximately 10.686 million tons and 726,000 tons of ammonia nitrogen emissions annually originate from agricultural production, accounting for about 48% and 32% of the total national emissions, respectively. A staggering 95% of these emissions come from livestock and poultry farming. Livestock and poultry farming pollutants have undoubtedly become the primary culprit for agricultural pollution. Therefore, finding a highly efficient, low-cost, and widely applicable wastewater treatment technology is of great significance for the large-scale development of the livestock industry. Summary of the Invention

[0003] The purpose of this invention is to provide a Bacillus huanghaiensis and its application in treating aquaculture wastewater, in order to solve the problems existing in the prior art. The Bacillus huanghaiensis W202 obtained by this invention can remove nitrogen and phosphorus pollutants such as total nitrogen, nitrate nitrogen, total phosphorus, and reactive phosphate from aquaculture wastewater, effectively purifying livestock and poultry aquaculture wastewater and providing a new approach to the treatment of aquaculture wastewater.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides a Bacillus chrysogenum (Bacillus chrysogenum) Bacillus marisflavi W202 was deposited on August 8, 2016 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 12818.

[0005] The present invention also provides a microbial agent comprising the strain of Bacillus huanghaiensis W202 or its fermentation broth.

[0006] Furthermore, the bacterial count (OD) of Bacillus huanghaiensis W202 in the fermentation broth... 600nm The value is 0.6-1.3.

[0007] The present invention also provides an application of the aforementioned Bacillus huanghaiensis W202 or the aforementioned bacterial agent in the treatment of aquaculture wastewater.

[0008] The present invention also provides a preparation for purifying aquaculture wastewater, comprising the aforementioned Bacillus chrysogenum W202 or the aforementioned bacterial agent.

[0009] Furthermore, the aquaculture wastewater is livestock and poultry aquaculture wastewater.

[0010] Furthermore, the livestock and poultry breeding wastewater includes pig farm breeding wastewater.

[0011] The present invention discloses the following technical effects: This invention isolated a strain of Bacillus huanghaiense W202 from marine waters. Experiments confirmed that this strain possesses good heterotrophic nitrification and aerobic denitrification capabilities, suggesting its potential as a novel strain for purifying aquaculture wastewater. Combined with treatment experiments on pig farm wastewater, it was demonstrated that Bacillus huanghaiense W202 can remove nitrogen and phosphorus pollutants such as total nitrogen, nitrate nitrogen, total phosphorus, and reactive phosphate from aquaculture wastewater, exhibiting significant purification effects and providing a new approach for the purification and treatment of livestock and poultry farming wastewater. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 Gram staining image of strain W202; Figure 2 This is a scanning electron microscope image of strain W202; Figure 3 This is a growth curve of strain W202; Figure 4 The growth of strain W202 at different temperatures; Figure 5 The growth of strain W202 under different pH conditions. Detailed Implementation

[0014] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0015] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0016] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0017] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0018] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0019] Example 1: Screening, identification and preservation of Bacillus huanghaiensis W202 1. Samples, reagents, and instruments 1.1 Sample Collection Sea mud and seawater samples were collected from the waters near Fuzhou, Fujian Province, and left to stand for 7 days for later use.

[0020] 1.2 Culture medium LB liquid medium: Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl. Add distilled water or deionized water to a final volume of 1 L. Stir and heat to boiling until completely dissolved. Dispense into 100 mL Erlenmeyer flasks, 20 mL per flask. Autoclave at 121 °C for 15 min. LB solid medium: Same as LB liquid medium, except add 15g of agar; Nitrification medium (per liter): 4.0 g sodium citrate, 0.5 g (NH4)2SO4, 50 mL Vickers salt solution; Vickers salt solution: 6.5 g K2HPO4 4`3H₂O, 2.5g MgSO₄ 4` 7H₂O, 2.5g NaCl, 0.05g FeSO₄ 4` 7H2O, 0.04g MnSO 4` H2O; the final pH of the culture medium is 7.0.

[0021] Denitrification medium (per liter): 0.721g KNO3, 10.55g Na2HPO4 4` 12H2O, 1.5g KH2PO4, 0.1gMgSO 4` 7H₂O, 4.0g sodium citrate, 2mL trace element solution; Trace element solution: 50.0g EDTA-Na₂, 5.5g CaCl₂, 5.06g MnCl₂ 2` 4H₂O, 5.0g FeSO₄ 4` 7H2O, 2.2 g ZnSO4, 1.61 gCoCl 2` 6H₂O, 1.57g CuSO₄ 4` 5H2O; adjust the pH of the aerobic denitrification medium to 7.0.

[0022] 1.3 Consumables and Instruments 10 mL test tubes and stoppers, 150 mL conical flasks, 1 mL pipettes and tips, petri dishes, shaker, SPX intelligent biochemical incubator (Ningbo Jiangnan Instrument Factory), SE402F electronic scale (Aohao Instruments Shanghai Co., Ltd.), SW-CJ-1F clean bench (Suzhou Antai Air Technology Co., Ltd., Sujing Group), LDZX-5KBS vertical pressure steam sterilizer (Shanghai Shenan Medical Instrument Factory), JSM-6380LV scanning electron microscope (Nippon Electron Corporation).

[0023] 2 Experimental Methods 2.1 Isolation and Identification of Bacillus huanghaiensis Bacteria in marine soil were isolated using a soil dilution method. 1g of marine silt was diluted with 9mL of sterile seawater to prepare a solution of 10... -2 -10 -5 A suspension of marine mud was taken at a rate of 1.0 mL and spread onto an LB agar plate, then incubated at 37°C for 3 days. Single colonies were selected and streaked onto new LB agar plates. The obtained monoclonal strains were then streaked a second time to obtain purebred strain W202. Gram staining and scanning electron microscopy were used to observe the bacterial morphology. The results are as follows: Figure 1 and Figure 2 Strain W202 is a Gram-positive bacterium with rod-shaped cells, 1-2 μm long and 0.3-0.5 μm wide.

[0024] The method of directly amplifying 16S rDNA from bacterial cells was adopted. Universal bacterial primers for 16S rDNA amplification were used, specifically 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID No. 2) and 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No. 3). The 25 μL PCR reaction system contained: 1 μL DNA template, 3 μL dNTPs (10 mM), 1 μL each of primers (10 mM), 7 μL 10x PCR Buffer, 2 μL MgSO4 (50 mM), and 5 U Taq enzyme. -1 0.5 μL of amplified DNA and 9.5 μL of ultrapure water were added. PCR reaction conditions: pre-denaturation at 95℃ for 5 min, followed by thermal cycling: denaturation at 94℃ for 30 s, annealing at 57℃ for 30 s, extension at 72℃ for 90 s, for 30 cycles; final extension at 72℃ for 10 min. After amplification, 5 μL of the amplified sample was electrophoresed on a 1% agarose gel at 60 V. The target DNA fragment was recovered and sent to a sequencing company for sequencing. The sequencing results are as follows (SEQ ID No. 1):

[0025] The assembled sequence was compared with the NCBI database using BLAST, and combined with morphological identification results, strain W202 was identified as Bacillus huanghaiense (Bacillus). Bacillus marisflavi ).

[0026] 2.2 Preservation of strain W202 The strain W202 is classified as Bacillus huanghaiensis ( Bacillus marisflavi It was deposited on August 8, 2016 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 12818.

[0027] 3. Culture and performance verification of the strain 3.1 Growth curve determination of strain W202 The strain was inoculated at a 1% inoculum in LB liquid medium and activated for 24 h to obtain W202 cell stock solution. 100 μL of the cell stock solution was inoculated into 39 test tubes containing 10 mL of LB liquid medium and incubated at 37℃ for static fermentation. Using uninoculated medium as a control, samples were taken at 0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, 84, and 96 h of fermentation to measure the OD (600 nm) value of the cells (3 replicates). Growth curves of the cells were plotted. Results are as follows: Figure 3 The results showed that strain W202 entered the logarithmic growth phase at 2 h, reached its growth peak at 24 h (at which time the bacterial cell OD (600nm) value was 1.24±0.01), entered the stationary phase after 24 h, and gradually entered the death phase.

[0028] 3.2 Determination of the optimal growth temperature for strain W202 100 μL of the bacterial stock solution was inoculated into test tubes containing 10 ml of LB liquid medium. After static fermentation at 10, 20, 30, 40, and 50 °C for 24 h and 48 h, the OD (600 nm) value of the fermentation broth was measured (three replicates). After determining the optimal growth temperature range of the strain to be 30-40 °C, static fermentation was carried out at 31, 33, 35, and 37 °C for 24 h, and the OD (600 nm) value of the fermentation broth was measured (three replicates) to determine the optimal growth temperature. The results are as follows: Figure 4 The results showed that strain W202 did not grow at 10℃ or 50℃, and grew slowly at 20℃. Its suitable growth temperature was 30-40℃, and the optimal growth temperature was 33℃ (the bacterial cell OD (600 nm) value was 1.21±0.01).

[0029] 3.3 Determination of the optimal pH for the growth of strain W202 100 μL of the bacterial stock solution was inoculated into test tubes containing 10 mL of LB liquid medium at pH 3, 4, 5, 6, 7, and 8. After static fermentation for 24 h and 48 h, the OD (600 nm) value of the fermentation broth was measured (three replicates). The results are as follows: Figure 5 The results showed that strain W202 grows slowly at pH 3 or 4, and its suitable growth pH is 5-8, with the optimal growth pH being 7.

[0030] 3.4 Determination of denitrification performance of strain W202 The bacterial culture in the logarithmic growth phase was centrifuged at 7000 rpm for 4 min. The cells were then washed and resuspended with sterile 1×PBS, and inoculated into 30 mL of sterile denitrification medium. The culture was incubated at 150 rpm and 33 °C for 72 h, and the NO3 content in the culture medium was measured. - -N,NH4 + -N, NO2 - -N, total nitrogen (TN), and OD (600nm) values ​​were measured. Each experimental group was set up in triplicate. The results are shown in Table 1.

[0031] Table 1. Denitrification performance of strain W202 As can be seen from Table 1, strain W202 operates with nitrate nitrogen (NO3) as the primary nitrogen source. - The organism remained stable after 72 hours of cultivation in a medium with nitrogen (NO3-N) as the sole nitrogen source (OD(600nm) = 1.04), achieving a nitrate nitrogen removal rate of up to 97.7%. Furthermore, a small amount of ammonia nitrogen was observed during denitrification, resulting in a slightly higher TN value than NO3-. - -N, Overall, this strain can effectively degrade nitrate nitrogen and has denitrification capabilities.

[0032] 3.5 Determination of nitrification performance of strain W202 The difference was that the inoculation was done into 30 mL of sterile nitrification medium, otherwise the same as in 3.4. The results are shown in Table 2.

[0033] Table 2 Nitrification performance of strain W202 As can be seen from Table 2, strain W202 operates with ammonia nitrogen (NH4+). +The strain can still grow stably (OD(600nm) is 0.98) after 72 hours of cultivation in a medium with N as the sole nitrogen source. It has a high ammonia nitrogen removal rate of up to 93%. During the nitrification process, intermediate products nitrite nitrogen and nitrate are produced, but the intermediate products do not accumulate in large quantities. This is because the strain has both nitrification and denitrification capabilities and can directly utilize the intermediate products nitrite nitrogen and nitrate for denitrification. Overall, the strain has good heterotrophic nitrification and aerobic denitrification capabilities and has the potential to purify aquaculture wastewater.

[0034] Example 2: Treatment effect of Bacillus huanghaiensis W202 on livestock and poultry breeding wastewater The Bacillus huanghaiensis W202 fermentation broth prepared under the optimal culture conditions in 3.1-3.3 of Example 1 was concentrated, freeze-dried and pulverized to obtain a solid bacterial agent with a bacterial count up to OD (600nm) value of 1.25. The experimental group was treated with this bacterial agent to treat aquaculture wastewater, the control group was treated with Bacillus subtilis bacterial agent with the same bacterial count, and the blank group was not treated with any bacterial agent.

[0035] The wastewater used in the experiment was taken from the pig farm of Fujian Xinxing Pig Breeding Co., Ltd. Specific nitrogen and phosphorus pollutant concentrations are shown in the blank group of Table 3. According to the "Integrated Wastewater Discharge Standard" (GB 8978-1996), the total nitrogen, nitrate nitrogen, total phosphorus, and reactive phosphate in the tested wastewater exceeded the standards. Direct discharge of this type of wastewater would lead to severe eutrophication of water bodies and pollution of the ecological environment. The selected livestock wastewater was treated with Bacillus thuringiensis and Bacillus subtilis inoculants, respectively. The purification experiment was conducted at 27℃ in 250mL Erlenmeyer flasks. 100mL of wastewater and 10g of the corresponding microbial inoculant were added to each flask. The mixture was shaken at 150r / min for 12h, centrifuged at 4000r / min for 15min, and the supernatant was collected. The concentrations of total nitrogen, nitrate nitrogen, total phosphorus, and reactive phosphate were measured.

[0036] The determination methods for nitrogen and phosphorus pollutants were in accordance with the "Methods for Monitoring and Analysis of Water and Wastewater". Total nitrogen was determined using the alkaline potassium persulfate method, nitrate nitrogen using ultraviolet spectrophotometry, total phosphorus using the ammonium molybdate spectrophotometric method, and reactive phosphate using the phosphomolybdic blue method. The experimental results are shown in Table 3. Table 3. Purification effect of microbial inoculants on aquaculture wastewater As can be seen from Table 3, the Bacillus huanghaiensis inoculant prepared by the present invention can significantly remove nitrogen and phosphorus pollutants such as total nitrogen, nitrate nitrogen, total phosphorus, and reactive phosphate from pig farm wastewater. The purification effect is significant and is superior to that of the conventional Bacillus subtilis inoculant used to treat livestock wastewater. It is evident that the present invention provides a new strain with potential application value for the purification and treatment of livestock and poultry farm wastewater.

[0037] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of Bacillus chrysogenum (Bacillus chrysogenum) Bacillus marisflavi W202, characterized in that, It was deposited on August 8, 2016 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 12818.

2. A microbial agent, characterized in that, The strain comprising Bacillus huanghaiensis W202 as described in claim 1 or its fermentation broth.

3. The microbial agent according to claim 2, characterized in that, The OD of the fermentation broth 600nm The value is 0.6-1.

3.

4. The application of Bacillus huanghaiensis W202 as described in claim 1 or the bacterial agent as described in any one of claims 2-3 in the treatment of aquaculture wastewater.

5. A preparation for purifying aquaculture wastewater, characterized in that, It contains the Bacillus huanghaiensis W202 as described in claim 1 or the bacterial agent as described in any one of claims 2-3.

6. The application according to claim 4, characterized in that, The wastewater in question is from livestock and poultry farming.

7. The application according to claim 6, characterized in that, The livestock and poultry breeding wastewater is pig farm breeding wastewater.

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