Brevibacillus parvus and use thereof

Abstract

The present application relates to the field of microorganism, discloses a strain of Brevibacillus laterosporus and application thereof, the preservation number of the Brevibacillus laterosporus is CGMCC NO:31319.The Brevibacillus laterosporus can effectively degrade alkane and remove COD in wastewater.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to *Brevibacillus laterosporus* and its applications. Background Technology

[0002] As a crucial pillar of my country's economy, the petrochemical industry inevitably generates various types of wastewater during its processing and production. With increasing national emphasis on environmental protection, the Beijing-Tianjin-Hebei region has implemented stricter requirements for suspended solids, COD, and ammonia nitrogen in its wastewater discharge. Wastewater undergoing advanced treatment must meet the Class IV surface water standard, making existing wastewater treatment processes in refining and chemical enterprises insufficient to meet these new environmental demands. To satisfy the needs of water resource protection, reclaimed water utilization, and continuous pollutant reduction, the advanced removal of organic pollutants has become a key focus in the water treatment field.

[0003] Wastewater treatment in my country's refining and chemical enterprises is mainly achieved through source reduction, process control, and end-of-pipe treatment. Early wastewater treatment primarily involved a "physicochemical pretreatment + biological organic matter removal" process before discharge. With increasingly stringent wastewater discharge standards, it is necessary to add technologies such as biological total nitrogen removal and advanced oxidation for organic matter removal to the existing processes to achieve deep removal of organic matter and total nitrogen. Deep organic matter removal often employs advanced oxidation processes, first improving the biodegradability of wastewater through oxidation, followed by further biological treatment. Currently, the most researched advanced oxidation technologies include ozone catalytic oxidation, Fenton oxidation, and electrochemical oxidation.

[0004] While ozone oxidation has a strong ability to decolorize and remove organic pollutants, it suffers from high ozone consumption, low utilization rate, long hydraulic retention time, high operating costs, and the risk of ozone emission polluting the atmosphere. Fenton oxidation technology has low investment and relatively low direct operating costs, but the generated sludge is hazardous waste and produced in large quantities, with disposal costs exceeding 20 yuan / ton of water. Electrochemical oxidation is highly effective at removing organic matter from wastewater, but its disadvantages include very high investment costs, high energy consumption, rapid electrode wear, and high operating costs. Although the aforementioned advanced oxidation methods can effectively remove organic matter from wastewater, their generally high treatment costs and the risk of secondary pollution limit their widespread application.

[0005] Biochemical methods for removing organic matter are low-cost and stable, making them the primary technology used in industrial wastewater treatment. However, wastewater typically exhibits low residual pollutant concentrations after two stages of biochemical treatment, resulting in an imbalanced nutrient ratio and persistent, difficult-to-degrade organic matter, thus increasing the difficulty of advanced wastewater treatment. Therefore, conventional microbial treatment methods are insufficient to meet the requirements for advanced removal of recalcitrant organic pollutants from wastewater. It is necessary to develop efficient technologies for removing recalcitrant organic pollutants, and to achieve advanced removal of these pollutants from wastewater by screening for microorganisms with specific degradation effects. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems existing in the prior art and to provide a strain of Bacillus laterosporus and its application, which can effectively degrade alkanes and remove COD from wastewater.

[0007] To achieve the above objectives, the first aspect of the present invention provides a strain of Bacillus retroflexus, wherein the preservation number of Bacillus retroflexus is CGMCC NO.31319.

[0008] A second aspect of the present invention provides a microbial agent containing *Bacillus retroflexus* as described above; preferably, the microbial agent is a liquid microbial agent.

[0009] A third aspect of the present invention provides the use of the previously described Bacillus retroflexus or the previously described bacterial agent in the degradation of alkane.

[0010] The fourth aspect of the present invention provides the application of Bacillus retroflexus or the bacterial agent described above in wastewater treatment.

[0011] The fifth aspect of the present invention provides a method for treating wastewater, the method comprising: contacting the wastewater with Bacillus retroflexus or the bacterial agent as described above.

[0012] There are currently no reports of Bacillus laterosporus degrading alkanes, but the Bacillus laterosporus of this invention can effectively utilize alkanes as a carbon source, and further, achieves effective removal of COD from petrochemical wastewater.

[0013] Biological Preservation

[0014] The *Brevibacillus laterosporus* strain of this invention, with accession number BHYAG-5, was deposited on July 16, 2024, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) (abbreviation of depositary institution: CGMCC), with accession number CGMCCNo. 31319. Attached Figure Description

[0015] Figure 1 The colony morphology of B. hygroscopic B. Brugia lateralis of the present invention is shown below.

[0016] Figure 2 This is a scanning electron microscope image of BHYAG-5, a lateral spore bacillus of the present invention. Detailed Implementation

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] The first aspect of this invention provides a strain of *Bacillus laterosporus*, whose preservation number is CGMCC NO.31319. In this invention, it is designated as BHYAG-5.

[0019] According to the present invention, the 16S rDNA sequence of the *Bacillus laterosporus* is shown in SEQ ID NO: 1.

[0020] SEQ ID NO: 1:

[0021]

[0022] The optimal growth pH for *Bacillus laterosporus* described in this invention is 6-8, and the optimal growth temperature is 30°C. It exhibits white, opaque, dry colonies on beef extract peptone medium. Figure 1 As shown.

[0023] A second aspect of the present invention provides a microbial agent containing Bacillus retroflexus as described above.

[0024] Preferably, the bacterial agent is a liquid bacterial agent.

[0025] In this invention, the preparation method of the bacterial agent may include: culturing Bacillus retroflexus as described above in a liquid culture medium and resuspending it in a buffer solution.

[0026] Preferably, the viable count of *Bacillus laterosporus* in the bacterial agent is not less than 10. 9 CFU / mL, preferably 10 10 -10 12 CFU / mL.

[0027] A third aspect of the present invention provides the use of the previously described Bacillus retroflexus or the previously described bacterial agent in the degradation of alkane.

[0028] Preferably, the alkane is C9-C. 34 At least one of the alkanes.

[0029] The fourth aspect of the present invention provides the application of Bacillus retroflexus or the bacterial agent described above in wastewater treatment.

[0030] In this invention, the wastewater is petrochemical wastewater.

[0031] Furthermore, the pollutants in the wastewater include at least one of the alkanes.

[0032] Preferably, the wastewater contains C9-C 34 At least one of the alkanes.

[0033] Preferably, the alkane may be provided in the form of diesel oil and / or paraffin.

[0034] Preferably, the alkane content in the wastewater is 50-1000 μg / L, more preferably 100-400 μg / L.

[0035] Preferably, the COD of the wastewater is 30-500 mg / L, and more preferably 50-200 mg / L.

[0036] The fifth aspect of the present invention provides a method for treating wastewater, the method comprising: contacting the wastewater with Bacillus retroflexus or the bacterial agent as described above.

[0037] Preferably, the contact method may be to contact the liquid bacterial agent with the wastewater to be treated.

[0038] Preferably, the contact conditions may include a temperature of 25-35°C and a time of 12-48 hours.

[0039] According to the present invention, the dosage of *Bacillus laterosporus* can be determined based on the degree of pollution in the wastewater. In some specific embodiments of the present invention, the dosage of *Bacillus laterosporus*, based on viable count, is not less than 10 per liter of wastewater. 11 CFU, preferably 10 12 -10 13 CFU. The wastewater can be as described above.

[0040] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0041] Unless otherwise specified, all reagents and materials used in the following examples were purchased from reputable chemical reagent suppliers and were of analytical purity.

[0042] The culture medium used in this invention is as follows:

[0043] Inorganic salt liquid culture medium: ammonium chloride 0.67g, sodium nitrate 0.06g, magnesium sulfate heptahydrate 0.1g, calcium chloride 0.1g, dipotassium hydrogen phosphate trihydrate 1.5g, potassium dihydrogen phosphate 0.5g, ferric chloride hexahydrate 0.1g, deionized water 1L, pH 7-7.2.

[0044] Enrichment medium: Add 1 g / L liquid paraffin to the inorganic salt liquid medium.

[0045] Inorganic salt solid culture medium: Add 20g of purified agar to the inorganic salt liquid culture medium.

[0046] Paraffin screening solid culture medium: 1 g / L liquid paraffin was added to the inorganic salt solid culture medium.

[0047] LB liquid medium: 10g tryptone, 10g NaCl, 5g yeast extract, 1L deionized water, pH 7.2.

[0048] Composition of LB plate medium: 20g of purified agar is added to LB liquid medium.

[0049] The composition of beef extract peptone medium is as follows: 5g peptone, 5g NaCl, 30g beef extract, 15g agar, 1L deionized water, pH 7.0-7.2.

[0050] Alkane removal rate = (Alkane content before degradation - Alkane content after degradation) / Alkane content before degradation × 100%.

[0051] The COD test method refers to the rapid digestion spectrophotometric method for the determination of chemical oxygen demand in water quality (HJ / T 399-2007).

[0052] Example 1

[0053] This embodiment illustrates the enrichment, screening, and purification of B. hygroscopic brevicorbacterium BHYAG-5 as described in this invention.

[0054] (1) Enrichment: Take 15 ml of the mixture of sludge and water from the aerobic pool of the saline wastewater biochemical treatment plant of Tianjin Petrochemical and add it to 85 ml of enrichment medium for culture. Incubate at 35℃ and 180 rpm for 7 days.

[0055] Take 15 ml of the mixed culture medium and transfer it to 85 ml of enrichment medium for further culture. Repeat this process three times to complete the enrichment culture.

[0056] (2) Screening and purification: The enriched culture medium from step (1) was serially diluted, and the dilution factor was 10. 7 10 8 and 10 9 The bacterial suspension was evenly spread on LB agar plates and incubated at 35°C for 24 hours. Colony morphology was observed. The isolated colonies were then isolated again using LB agar until a single *Bacillus laterosporus* strain was obtained. This *Bacillus laterosporus* strain was then deposited at a cultural heritage site with the accession number CGMCC No. 31319 and designated BHYAG-5. A scanning electron microscope image of *Bacillus laterosporus* BHYAG-5 is shown below. Figure 2 As shown, the bacterial cells are rod-shaped and approximately 0.9 μm × 2.2 μm in size.

[0057] Example 2

[0058] This embodiment is used to illustrate the ability of the BHYAG-5 bronchodilator described in this invention to degrade alkanes.

[0059] Using a disposable inoculation loop, the glycerol-preserved B. lateral spores BHYAG-5 bacterial suspension was inoculated onto paraffin solid selection medium and cultured at 30°C for 7 days in a biochemical incubator. It was able to grow on paraffin solid selection medium with paraffin as the sole carbon source, therefore it can be considered that this strain has the ability to degrade alkanes.

[0060] Example 3

[0061] This embodiment is used to illustrate the bacterial agent and its preparation method described in this invention.

[0062] After activating and culturing BHYAG-5 slant culture of *Bacillus laterosporus* for 24 hours, one loop was added to LB liquid medium. 100 mL of LB liquid medium was placed in a 250 mL Erlenmeyer flask, and the flask was incubated at 30°C and 180 rpm for 20 hours to obtain the bacterial suspension. The suspension was centrifuged at 5000 rpm for 5 minutes, the supernatant was removed, and the bacterial cells were retained. The bacterial cells were resuspended in sterile 0.01 mol / L PBS buffer to obtain the bacterial agent described in this invention. The OD600 of the bacterial agent was 3.1, and the viable count was 3.3 × 10⁻⁶. 11 CFU / ml.

[0063] Example 4

[0064] This embodiment illustrates the degradation effect of B. hygroscopic BHYAG-5 on alkanes described in this invention.

[0065] (1) Take 100mL of inorganic salt liquid culture medium and place it in a 250mL Erlenmeyer flask. Seal the flask with a breathable sealing film and sterilize it in an autoclave at 121℃ for 20min. After cooling, add 1g / L of paraffin to the Erlenmeyer flask.

[0066] (2) Add the above-mentioned microbial agent to make the viable bacteria count in the system 2 × 10⁻⁶. 10 CFU / mL, sealed with breathable sealing film, and treated on a shaker at 30℃ and 180rpm for 3d, 5d, and 7d to obtain experimental group samples. In step (1), paraffin was added to the inorganic salt culture medium, but no bacterial agent was added. The blank control group was obtained by treating it in the same way for 3d, 5d, and 7d.

[0067] (3) The experimental group and blank control group samples obtained in the above steps were respectively added to n-hexane at a volume ratio of 1:10, extracted for 20 min, and allowed to stand until complete separation. The upper organic phase was taken, and the C9-C content in the blank control group and experimental group was measured by gas chromatography-mass spectrometry. 34 The content of alkanes was determined, and the alkane removal rate at different reaction times was calculated, as shown in Table 1.

[0068] Table 1

[0069]

[0070] As can be seen from Table 1, the BHYAG-5 bronchodilator provided by this invention can fully decompose alkanes.

[0071] Example 5

[0072] This embodiment illustrates the treatment effect of B. hygroscopic brevicorbacterium lateralis-5 on petrochemical wastewater as described in this invention.

[0073] The biochemical effluent containing saline wastewater from a petrochemical enterprise was treated using Bacillus lateralis BHYAG-5. The wastewater had a conductivity of 2800 μS / cm and a COD of 69 mg / L.

[0074] (1) Place 100 mL of the above-mentioned saline wastewater from a petrochemical enterprise into a 250 mL Erlenmeyer flask, and add the above-mentioned bacterial agent to make the viable bacteria count in the reaction system 1.5 × 10⁻⁶. 9 CFU / mL, sealed with a breathable sealing film, and treated on a shaker at 30℃ and 180rpm for 1 day to obtain the experimental group sample. 110mL of biochemical effluent containing saline from a petrochemical plant was treated without adding any bacterial agent and treated in the same way for 1 day to obtain the blank control group.

[0075] (2) Equal amounts of samples from the control group and the experimental group were taken, and the samples from the experimental group and the control group were extracted with dichloromethane, dried with anhydrous sodium sulfate, purged with nitrogen, and analyzed by GC-MS. The concentration of C in the control group sample was measured. 12 -C 34 The content of mixed alkanes was 120.73 μg / L, and the C content in the experimental group sample was... 12 -C 34 The mixed alkane content is 62.78 μg / L.

[0076] (3) Take equal amounts of samples from the experimental group and the control group, centrifuge at 10000r / min for 3min, take the supernatant and filter it through a 0.22-micron membrane to measure the COD result. The COD of the control group was 69mg / L and the COD of the experimental group was 41mg / L.

[0077] The B. hygroscopic Bacillus lateralis-5 provided by this invention has the ability to degrade mixed alkanes in petrochemical wastewater, with a degradation rate of 48% and a COD removal rate of 40.6% in 24 hours.

[0078] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of *Brevibacillus laterosporus*, characterized in that, The preservation number of the *Bacillus lateralis* is CGMCC NO.31319.

2. The *Bacillus laterosporus* according to claim 1, wherein, The 16S rDNA sequence of the *Bacillus lateralis* is shown in SEQ ID NO:

1.

3. A microbial agent, wherein, The microbial agent contains Bacillus retroflexus as described in claim 1 or 2; Preferably, the bacterial agent is a liquid bacterial agent.

4. The microbial agent according to claim 3, wherein, The viable count of *Bacillus laterosporus* in the bacterial agent is not less than 10. 9 CFU / mL, preferably 10 10 -10 12 CFU / mL.

5. The use of the *Bacillus lateralis* as described in claim 1 or 2, or the bacterial agent as described in claim 3 or 4, in the degradation of alkanes.

6. The application according to claim 5, characterized in that, The alkane is C9-C. 34 At least one of the alkanes.

7. The application of the Bacillus lateralis according to claim 1 or 2 or the bacterial agent according to claim 3 or 4 in wastewater treatment.

8. The application according to claim 7, wherein, The wastewater is petrochemical wastewater.

9. The application according to claim 7, wherein, The pollutants in the wastewater include at least one alkane, preferably C9-C6. 34 At least one of the alkanes; Preferably, the alkane content in the wastewater is 50-1000 μg / L, more preferably 100-400 μg / L; Preferably, the COD of the wastewater is 30-500 mg / L, and more preferably 50-200 mg / L.

10. A method for treating wastewater, characterized in that, The treatment method includes contacting the Bacillus lateralis according to claim 1 or 2 or the bacterial agent according to claim 3 or 4 with the wastewater.

11. The processing method according to claim 10, wherein, The dosage of *Bacillus laterosporus*, based on viable count, is not less than 10 per liter of wastewater. 11 CFU, preferably 10 12 -10 13 CFU.

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