Paenibacillus siamensis and uses thereof

Abstract

The present application relates to the field of microorganisms, and discloses a bacillus siamensis and application thereof.The preservation number of the bacillus siamensis is CCTCC NO: M20231213.The bacillus siamensis can effectively remove COD in sewage, and has excellent degradation effect on 2-methyl-3-heptanone, n-tetradecane and mesitylene in the sewage.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a strain of Bacillus sicca and its applications. Background Technology

[0002] The petrochemical industry is one of the important pillar industries of my country's economy, but the production process inevitably generates various types of wastewater. The original wastewater treatment processes of refining and chemical enterprises are no longer able to meet the environmental protection requirements under the new circumstances. In order to meet the needs of water resource protection, reclaimed water utilization, and continuous reduction of pollutants, the deep removal of organic pollutants has become one of the key issues of concern in the field of water treatment.

[0003] Wastewater treatment in my country's refining and chemical enterprises mainly relies on source reduction, process control, and end-of-pipe treatment. Early wastewater treatment primarily achieved compliance through processes such as "collection + transportation + physicochemical pretreatment + biological removal of organic matter." With stricter wastewater discharge standards, advanced organic matter removal and total nitrogen removal processes have been added to achieve compliance. Advanced organic matter removal often employs advanced oxidation processes to improve the biodegradability of wastewater, facilitating subsequent biological treatment, such as 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. 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 treatment costs are generally high, and they pose a risk of secondary pollution.

[0005] While wastewater biological removal of organic matter is low-cost, the residual pollutant concentration is low after primary and secondary treatment, the nutrient ratio in the wastewater is imbalanced, and the remaining organic matter is difficult to degrade. Meanwhile, wastewater with high salinity and poor biodegradability, such as circulating water discharge, chemically neutralized water, and reverse osmosis concentrate, also adversely affect the biological system after entering the wastewater treatment plant. Circulating water discharge contains certain oxidizing and bactericidal agents; after wastewater is treated and reused via dual membranes, the reverse osmosis concentrate has a high salinity, and the content of recalcitrant organic matter in the concentrate is further increased after membrane concentration; the chemically neutralized wastewater produced by acid-base neutralization has a high salinity and a TDS concentration exceeding 10,000 mg / L, further increasing the difficulty of biochemical degradation. Therefore, conventional microbial treatment is insufficient to meet the requirements for deep removal of recalcitrant organic matter from wastewater, necessitating the development of efficient and low-cost organic pollutant removal technologies. Screening for specific microorganisms that degrade characteristic pollutants can achieve low-cost deep removal of organic pollutants from wastewater. Summary of the Invention

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

[0007] To achieve the above objectives, the first aspect of the present invention provides a strain of Bacillus siamensis, wherein the preservation number of Bacillus siamensis is CCTCC NO: M20231213.

[0008] A second aspect of the present invention provides a microbial agent containing Bacillus sicca as described above.

[0009] A third aspect of the present invention provides the application of Bacillus sicca as described above or the bacterial agent as described above in wastewater treatment.

[0010] A fourth aspect of the present invention provides a method for treating wastewater, the method comprising: contacting the wastewater with Bacillus sicca or the bacterial agent described above.

[0011] The Bacillus licheniformis provided by this invention can effectively remove pollutants from wastewater and effectively reduce COD in wastewater. In particular, it has excellent degradation effects on 2-methyl-3-heptanone, n-tetradecane and mesitylene in wastewater, with removal rates of 12.51%, 27.56% and 18.67% per hour, respectively.

[0012] Biological Preservation

[0013] The Bacillus siamensis of this invention, with accession number WW03, was deposited on July 6, 2023, at the China Center for Type Culture Collection (address: No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, postal code: 430072) (abbreviation of depositary institution: CCTCC), with accession number CCTCC NO: M20231213. Attached Figure Description

[0014] Figure 1 The colony morphology of Bacillus WW03 of the present invention on MRS medium;

[0015] Figure 2 The colony morphology of Bacillus WW03 of the present invention on 2216E medium is shown. Detailed Implementation

[0016] 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.

[0017] The first aspect of this invention provides a strain of Bacillus siamensis, with the preservation number CCTCC NO: M20231213. In this invention, it is designated as WW03.

[0018] According to the present invention, the 16S rDNA sequence of the Bacillus sicca is shown in SEQ ID NO: 1.

[0019] SEQ ID NO: 1:

[0020]

[0021] The optimal growth pH for *Bacillus sicca* described in this invention is 6-8, and the optimal growth temperature is 25-30℃. It exhibits raised, irregularly shaped, viscous colonies on MRS solid medium, such as... Figure 1 As shown; on 2216E solid medium, they appear as white, regularly shaped, round colonies, as... Figure 2 As shown.

[0022] A second aspect of the present invention provides a microbial agent containing Bacillus sicca as described above.

[0023] In this invention, the form of the microbial agent is not particularly limited and can be a conventional form of microbial agent in the art, such as a solid, liquid or semi-solid form.

[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 sicca as described above in a liquid culture medium.

[0026] Preferably, the method for preparing the bacterial agent may further include: resuspending the cultured Bacillus sicca in a liquid culture medium or buffer solution.

[0027] According to the present invention, preferably, the viable count of Bacillus sicca in the bacterial agent is not less than 5 × 10⁻⁶. 9 CFU / mL, preferably 8×10⁻⁶ 9 -2×10 10 CFU / mL.

[0028] A third aspect of the present invention provides the application of Bacillus sicca as described above or the bacterial agent as described above in wastewater treatment.

[0029] According to the present invention, preferably, the wastewater is petrochemical wastewater.

[0030] According to the present invention, preferably, the pollutants in the wastewater include at least one of 2-methyl-3-heptanone, n-tetradecane, and mesitylene.

[0031] In this invention, the Bacillus sicca exhibits significant removal effects on 2-methyl-3-heptanone, n-tetradecane, and mesitylene in wastewater.

[0032] More preferably, the content of 2-methyl-3-heptanone in the wastewater is 1-10 mg / L.

[0033] More preferably, the content of n-tetradecane in the wastewater is 20-100 mg / L.

[0034] More preferably, the content of mesitylene in the wastewater is 1-10 mg / L.

[0035] According to the present invention, preferably, the COD of the wastewater is 30-500 mg / L, more preferably 50-200 mg / L.

[0036] In this invention, the COD testing method can be a conventional testing method in the field. Preferably, it is determined by rapid digestion spectrophotometry according to HJ / T 399-2007, "Determination of Chemical Oxygen Demand in Water".

[0037] A fourth aspect of the present invention provides a method for treating wastewater, the method comprising: contacting the wastewater with Bacillus sicca or the bacterial agent described above.

[0038] In this invention, the contact conditions may include: a temperature of 25-35°C and a time of 12 hours to 7 days.

[0039] According to the present invention, the dosage of the *Bacillus sicca* can be determined based on the degree of pollution in the wastewater. In some specific embodiments of the present invention, the dosage of the *Bacillus sicca*, based on the viable count, is not less than 1 × 10⁻⁶ per liter of wastewater. 8 CFU, preferably 1×10 8 -5×10 8 CFU.

[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 components used in the following examples are shown below:

[0043] Bushnell-Haas liquid medium: 0.2g magnesium sulfate, 0.02g calcium chloride, 1g potassium dihydrogen phosphate, 1g ammonium nitrate, 0.05g ferric chloride, 1L deionized water.

[0044] Bushnell-Haas solid medium: Add 20g of purified agar to Bushnell-Haas liquid medium.

[0045] 2216E liquid culture medium: 50g peptone, 1g yeast extract, 0.1g ferric citrate, 19.45g sodium chloride, 5.98g magnesium chloride, 3.24g sodium sulfate, 1.8g calcium chloride, 0.55g potassium chloride, 0.16g sodium carbonate, 0.08g potassium bromide, 0.034g silver chloride, 0.022g boric acid, 0.004g sodium silicate, 0.0024g sodium fluoride, 0.0016g ammonium nitrate, 0.008g disodium hydrogen phosphate, 1L deionized water.

[0046] 2216E solid medium: Add 15g of purified agar to 2216E liquid medium.

[0047] MRS liquid culture medium: 10g peptone, 8g beef meal, 4g yeast powder, 20g glucose, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1g Tween-80, 1L deionized water.

[0048] MRS solid medium: Add 14g of purified agar to MRS liquid medium.

[0049] The COD testing method is as follows: Take 1.5 mL of the treated sample, centrifuge at 1000 r / min for 3 min, take 1 mL of the supernatant and dilute it three times to obtain a diluted solution. Pipette 2 mL of the diluted solution into a pre-prepared COD reagent tube, digest in a metal bath at 165℃ for 20 min, and determine the COD according to the rapid digestion spectrophotometric method for the determination of chemical oxygen demand in water quality (HJ / T 399-2007). The pre-prepared COD reagent tubes were purchased from Hach Company, USA.

[0050] The contents of 2-methyl-3-heptanone, n-tetradecane and mesitylene were determined by gas chromatography-mass spectrometry.

[0051] Example 1

[0052] This example illustrates the degradation capabilities of Bacillus WW03 of Siamese bacteria on 2-methyl-3-heptanone, n-tetradecane, and mesitylene.

[0053] 1000 mg / L 2-methyl-3-heptanone, 1000 mg / L n-tetradecane, and 1000 mg / L mesitylene were added to Bushnell-Haas solid medium. Glyceryl-preserved Bacillus WW03 culture was inoculated onto the Bushnell-Haas solid medium using a disposable inoculation loop. The culture was incubated at 30°C for 7 days. The strain was able to grow on solid medium with 2-methyl-3-heptanone, n-tetradecane, and mesitylene as carbon sources. Therefore, it can be considered that this strain has the ability to degrade 2-methyl-3-heptanone, n-tetradecane, and mesitylene.

[0054] Example 2

[0055] This embodiment is used to illustrate the bacterial agent and its preparation method described in this invention.

[0056] (1) 200 μL of Bacillus WW03 preserved in glycerol was inoculated into 50 mL of MRS liquid medium and cultured on a shaker at 30 °C and 220 rpm for 24 h to obtain a seed culture with an OD600 of 2.38 (using MRS liquid medium as a blank control). 21 mL of the seed culture was centrifuged at 5000 rpm for 3 min, and the supernatant was removed, retaining the bacterial cells. The bacterial cells were washed with sterile 0.01 mol / L PBS buffer and resuspended in 10 mL of Bushnell-Haas liquid medium to obtain bacterial agent 1. The OD600 of bacterial agent 1 was 5, with a viable count of 8 × 10⁻⁶. 9 CFU / mL.

[0057] (2) 200 μL of *Bacillus sicca* WW03 preserved in glycerol was inoculated into 50 mL of MRS liquid medium and cultured on a shaker at 30 °C and 220 rpm for 12 h to obtain a seed culture with an OD600 of 1.74 (using MRS liquid medium as a blank control). 6 mL of the seed culture was centrifuged at 5000 rpm for 3 min, and the supernatant was removed, retaining the bacterial cells. The bacterial cells were washed with sterile 0.01 mol / L PBS buffer and resuspended in 1 mL of PBS buffer to obtain bacterial agent 2. The OD600 of bacterial agent 1 was 10, with a viable count of 1.6 × 10⁻⁶. 10 CFU / mL.

[0058] Example 3

[0059] This example illustrates the degradation effect of Bacillus sicca WW03 on 2-methyl-3-heptanone, n-tetradecane, and mesitylene.

[0060] (1) Add 2-methyl-3-heptanone, n-tetradecane and mesitylene to 10 mL of sterilized Bushnell-Haas liquid medium so that the concentrations of 2-methyl-3-heptanone, n-tetradecane and mesitylene in the system are all 100 mg / L.

[0061] (2) Add 10 mL of the above bacterial solution 1, and the viable bacteria count in the system will be 4 × 10⁻⁶. 9 The experimental group samples were obtained by treating the samples at CFU / mL on a shaker at 30℃ and 220rpm for 3 hours. In step (1), 2-methyl-3-heptanone, n-tetradecane, and mesitylene were added to the Bushnell-Haas liquid medium, but no bacterial agent was added. The medium was replenished with 10 mL of Bushnell-Haas liquid medium, and the samples were treated in the same way for 3 hours to obtain the blank control group. Equal amounts of the blank control group and experimental group samples were taken, and the COD content was tested.

[0062] (3) Take equal amounts of blank control group and experimental group samples, add n-hexane at a volume ratio of 1:1 for extraction, take the upper organic phase, and measure the contents of 2-methyl-3-heptanone, n-tetradecane and mesitylene in the blank control group and experimental group by gas chromatography-mass spectrometry, as shown in Table 1.

[0063] Table 1

[0064] pollutants Blank control group (mg / L) Experimental group (mg / L) n-Tetradecane 93.77 16.24 Trimethylbenzene 44.29 19.48 2-Methyl-3-heptanone 99.01 61.86 COD 243 129.03

[0065] Example 4

[0066] This example illustrates the treatment effect of Bacillus WW03 on wastewater.

[0067] (1) 49 mL of biochemical effluent from a petrochemical plant's wastewater treatment system was placed in a 250 mL Erlenmeyer flask, and 1 mL of the above-mentioned bacterial agent 2 was added to make the viable bacteria count in the reaction system 3.2 × 10⁻⁶. 8 The experimental group samples were obtained after treatment with CFU / mL at 30℃ and 220rpm for 7 days on a shaker. 50mL of the above biochemical effluent without added bacterial agent 2 was treated in the same manner for 7 days to obtain the blank control group.

[0068] (2) Take equal amounts of blank control group and experimental group samples, add n-hexane at a volume ratio of 1:1 for extraction, take the upper organic phase, and measure the content of 2-methyl-3-heptanone in the blank control group by gas chromatography-mass spectrometry. The content of n-tetradecane is 33 mg / L, the content of mesitylene is 6.7 mg / L, and the content of 2-methyl-3-heptanone in the experimental group is 3.75 mg / L, the content of n-tetradecane is 5.7 mg / L, and the content of mesitylene is 2.95 mg / L.

[0069] (3) Take equal amounts of samples from the experimental group and the blank control group, and test the COD content. The COD of the blank control group was 74 mg / L, and the COD of the experimental group was 38 mg / L.

[0070] 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 Bacillus siamensis, characterized in that, The preservation number of the Bacillus sicca is CCTCC NO: M20231213.

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

1.

3. A microbial agent, characterized in that, The bacterial agent contains Bacillus sicca as described in claim 1 or 2.

4. The microbial agent according to claim 3, wherein, The viable count of Bacillus sicca in the bacterial agent is not less than 5 × 10⁻⁶. 9 CFU / mL, preferably 8×10⁻⁶ 9 -2×10 10 CFU / mL.

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

6. The application according to claim 5, wherein, The wastewater is petrochemical wastewater; And / or, the pollutants in the wastewater include at least one of 2-methyl-3-heptanone, n-tetradecane, and mesitylene.

7. The application according to claim 5, wherein, The content of 2-methyl-3-heptanone in the wastewater is 1-10 mg / L; And / or, the content of n-tetradecane in the wastewater is 20-100 mg / L; And / or, the content of mesitylene in the wastewater is 1-10 mg / L.

8. The application according to claim 5, wherein, The COD of the wastewater is 30-500 mg / L, preferably 50-200 mg / L.

9. A method for treating wastewater, characterized in that, The treatment method includes contacting the Bacillus sicca of claim 1 or 2 or the bacterial agent of claim 3 or 4 with the wastewater.

10. The processing method according to claim 9, wherein, The dosage of the *Bacillus sicca*, based on viable count, is not less than 1 × 10⁻⁶ per liter of wastewater. 8 CFU, preferably 1×10 8 -5×10 8 CFU.

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