Bacillus cereus and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus cereus and application thereof. The bacillus cereus DLYBEJ-3 disclosed by the invention is preserved in the China Center for Type Culture Collection, and the preservation number of the bacillus cereus DLYBEJ-3 is CCTCC NO: M 20231931. The strain can grow by taking MDEA as the sole carbon source and nitrogen source, realizes efficient degradation of MDEA, has the capabilities of removing COD, removing nitrogen, degrading MDEA and reducing biotoxicity at the same time, can be used for treating wastewater containing MDEA, is simple to operate, efficient in treatment and low in cost, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a Bacillus cereus strain and application thereof. Background Art

[0002] In the petrochemical industry, N-methyldiethanolamine (MDEA), an alkaline solvent from the alkanolamine family, is often used as a desulfurizer for circulating hydrogen, dry gas, and liquid hydrocarbons due to its excellent selectivity for acidic gases and low corrosion to equipment. In recent years, with the frequent use of MDEA in refineries, MDEA loss into wastewater systems has become a common occurrence, leading to increased COD and ammonia nitrogen levels in refinery wastewater, limiting compliance with wastewater discharge standards. Furthermore, MDEA is highly biotoxic, and because the nitrogen element of tertiary amines lacks active hydrogen, it also exhibits certain antioxidant properties and is difficult to biodegrade, increasing the difficulty of wastewater treatment.

[0003] Currently, MDEA wastewater treatment mainly involves adsorption, electrolysis, chemical oxidation, biological methods, and coupled technology methods. Adsorption suffers from limited capacity, high regeneration costs, and the tendency to generate secondary pollution; electrolysis suffers from low current efficiency, power consumption, and high costs; chemical oxidation, primarily based on advanced oxidation processes, is significantly affected by experimental conditions and is prone to the production of byproducts; biological methods are affected by the toxicity and recalcitrance of MDEA, resulting in low removal efficiency; and methods that couple multiple technologies result in lengthy processes, large footprints, and high costs.

[0004] Patent CN103833166A discloses a method for treating methyldiethanolamine (MDEA) industrial wastewater using a combination of iron-carbon micro-electrolysis and Fenton-like oxidation technology. This method requires hydrogen peroxide as a catalyst and pH adjustment, resulting in a complex and costly process and the production of byproducts. Furthermore, the iron-carbon micro-electrolysis filler has a limited lifespan and produces scrap iron, limiting its application.

[0005] Patent CN111217419A discloses an N-methyldiethanolamine wastewater treatment device and method. The wastewater treatment device includes a filtration unit and a vacuum ultraviolet photocatalytic oxidation unit. This method requires the installation of a separate treatment unit, resulting in a lengthy process, large footprint, and high costs.

[0006] In view of the above-mentioned problems, there is an urgent need to develop a treatment method that can simplify the treatment process and reduce costs while ensuring a high MDEA removal rate and effectively reducing biological toxicity. Summary of the Invention

[0007] The invention provides a Bacillus cereus strain and application thereof.

[0008] Currently, there are no reports of Bacillus that can efficiently remove MDEA and reduce the content of chemical oxygen demand (COD) and Kjeldahl nitrogen (TKN). In response to the problems existing in the prior art, the present invention adapts, domesticates, and enriches activated sludge samples obtained from refinery biological aeration tanks, and finally isolates and screens a strain of Bacillus cereus, which is named DLYBEJ-3. Bacillus cereus DLYBEJ-3 can rapidly degrade MDEA, organic nitrogen, and COD in petrochemical wastewater, adapts to a wide pH range, and is particularly tolerant to alkaline pH. It can effectively reduce the content of MDEA, TKN, and COD in wastewater, achieve functions such as biological toxicity reduction, and does not require adjustment of wastewater pH. It has the characteristics of high efficiency, short process, and low cost.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] The present invention provides Bacillus cereus DLYBEJ-3, which was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, postal code 430072) on October 18, 2023, with a deposit number of CCTCC NO: M 20231931 and a classification name of Bacillus cereus.

[0011] The main morphological characteristics of Bacillus cereus DLYBEJ-3 are: after 18 hours of incubation at 37°C on TSA medium, the colonies are light yellow, round, with a moist, opaque surface and neat edges. Under a microscope, Bacillus cereus DLYBEJ-3 bacteria appear as rods, measuring 0.5-0.8 μm x 2.5-5.0 μm, either singly or in pairs, and are Gram-positive.

[0012] The nucleotide sequence of the 16S rDNA of the above-mentioned Bacillus cereus DLYBEJ-3 is shown in SEQ ID NO.1.

[0013] The above-mentioned Bacillus cereus DLYBEJ-3 can grow with N-methyldiethanolamine as the sole carbon source and / or the sole nitrogen source.

[0014] The present invention provides a microbial preparation, which contains the above-mentioned Bacillus cereus DLYBEJ-3.

[0015] Preferably, in the microbial preparation, Bacillus cereus DLYBEJ-3 exists in the form of live bacteria.

[0016] The microbial preparations described above may be solid preparations (eg bacterial powder) or liquid preparations (eg emulsion preparations).

[0017] The present invention also provides a method for preparing the above-mentioned microbial preparation, which comprises the step of culturing the Bacillus cereus DLYBEJ-3.

[0018] Preferably, the culture temperature is 20-50°C, more preferably 28-32°C.

[0019] In some embodiments of the present invention, the culturing method comprises: using LB medium supplemented with N-methyldiethanolamine (preferably at a concentration of 200-300 mg / L), and culturing to a stationary phase under aerobic conditions; the culture conditions are: pH 5-10, temperature 20-50°C, dissolved oxygen concentration 0.25-3 mg / L, and culture time 18-48 hours.

[0020] The Bacillus cereus DLYBEJ-3 bacterial liquid obtained by the above-mentioned culture method can be stored in a refrigerator at 4° C. for 2-6 months without being inactivated, and has strong vitality and good stability.

[0021] The bacterial liquid obtained by the above culture can be prepared into a liquid preparation directly or by concentration and / or addition of other excipients, or by drying to prepare a solid preparation, or the bacterial cells in the bacterial liquid can be separated and then prepared into a solid preparation.

[0022] Based on the functions of Bacillus cereus DLYBEJ-3, the present invention provides the following applications of the strain:

[0023] The present invention provides use of the above-mentioned Bacillus cereus DLYBEJ-3 or the microbial preparation in degrading N-methyldiethanolamine.

[0024] The present invention provides use of the above-mentioned Bacillus cereus DLYBEJ-3 or the microbial preparation in treating waste containing N-methyldiethanolamine.

[0025] The present invention provides the use of the above-mentioned Bacillus cereus DLYBEJ-3 or the microbial preparation in reducing the content of N-methyldiethanolamine, COD and / or TKN in N-methyldiethanolamine-containing waste.

[0026] The present invention provides the use of the above-mentioned Bacillus cereus DLYBEJ-3 or the microbial preparation in reducing the biological toxicity of waste containing N-methyldiethanolamine.

[0027] Preferably, the waste is wastewater.

[0028] Further preferably, the wastewater is petrochemical wastewater.

[0029] In the wastewater described above, the content of N-methyldiethanolamine is preferably 1-1000 mg / L, more preferably 10-1000 mg / L.

[0030] The present invention provides use of the above-mentioned Bacillus cereus DLYBEJ-3 or the microbial preparation in preparing a wastewater treatment agent.

[0031] The present invention provides a wastewater treatment agent, which comprises the above-mentioned Bacillus cereus DLYBEJ-3 or the microbial preparation.

[0032] Optionally, the active ingredients of the wastewater treatment agent may include, in addition to the Bacillus cereus DLYBEJ-3 or the microbial preparation described above, other microorganisms or chemicals having wastewater treatment functions (e.g., having the function of degrading N-methyldiethanolamine, COD and / or TKN).

[0033] Optionally, the wastewater treatment agent may further contain auxiliary materials required for the preparation.

[0034] The present invention provides a wastewater treatment method, which comprises: inoculating the above-mentioned Bacillus cereus DLYBEJ-3 or the microbial preparation or the wastewater treatment agent into wastewater to be treated, and treating the wastewater under aerobic conditions.

[0035] Preferably, the treatment temperature is 20-50°C; more preferably 25-35°C.

[0036] Preferably, the pH of the treatment is 5-10; more preferably 6-10.

[0037] Preferably, the dissolved oxygen concentration during the treatment is 3-5 mg / L.

[0038] Preferably, the treatment time is 20-60 hours.

[0039] Bacillus cereus DLYBEJ-3 can use MDEA as the sole carbon and nitrogen source for growth. When treating MDEA-containing wastewater, it can achieve simultaneous and efficient removal of MDEA, COD, and TKN. When the MDEA concentration is not higher than 1000 mg / L, the removal rate is greater than 85%. No toxic byproducts are produced during the reaction process, which can effectively reduce the biological toxicity of the wastewater.

[0040] Compared with the prior art, the present invention has at least the following beneficial effects:

[0041] (1) The Bacillus cereus DLYBEJ-3 provided by the present invention can grow using MDEA as the sole carbon and nitrogen source, thereby achieving efficient degradation of MDEA.

[0042] (2) The Bacillus cereus DLYBEJ-3 provided by the present invention has the ability to simultaneously remove COD, denitrify, degrade MDEA, and reduce biological toxicity. It can be used to treat MDEA-containing wastewater (for example, wastewater with substandard COD, ammonia nitrogen, etc. caused by MDEA loss in refineries), and has broad application prospects.

[0043] (3) The Bacillus cereus DLYBEJ-3 provided by the present invention has a wide pH adaptability range and is particularly tolerant to alkaline pH (e.g., alkaline conditions of about pH 10). When treating MDEA-containing wastewater, there is no need to adjust the wastewater pH, saving a large amount of reagents.

[0044] (4) The use of Bacillus cereus DLYBEJ-3 provided by the present invention to treat MDEA-containing wastewater does not require changing the existing sewage treatment plant process. DLYBEJ-3 can be directly added to the wastewater treatment system, or mixed with activated sludge as an inoculum, or formed into a biofilm on various fillers to treat MDEA-containing wastewater. The operation is simple, the treatment is efficient, and the cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0046] Figure 1 The colony morphology of strain DLYBEJ-3 on a plate culture medium in Example 1 of the present invention is shown.

[0047] Figure 2 The bacterial morphology of strain DLYBEJ-3 in Example 1 of the present invention is magnified 1000 times under a microscope.

[0048] Figure 3 This is the NCBI alignment result of the 16S rDNA sequencing sequence of strain DLYBEJ-3 in Example 2 of the present invention.

[0049] Figure 4 This is the 16S rDNA sequence alignment result of strain DLYBEJ-3 in Example 2 of the present invention.

[0050] Figure 5 This is a phylogenetic tree of the 16S rDNA sequences of strain DLYBEJ-3 and related species in Example 2 of the present invention. DETAILED DESCRIPTION

[0051] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0052] In the present invention, Bacillus cereus DLYBEJ-3 is obtained by acclimating, domesticating, and enriching activated sludge obtained from a refinery's biological aeration tank, followed by isolation and screening. The acclimation method for Bacillus cereus DLYBEJ-3 is operated in a sequencing batch reactor. After achieving high and stable COD and organic nitrogen treatment results through three stages of acclimation, domestication, and enrichment, the MDEA-degrading strain is finally domesticated and enriched. The specific steps are as follows:

[0053] S1: Establish a conventional bioelectrochemical device and add sludge containing sewage treatment bacteria at a sludge concentration of 3000-4000 mg / L;

[0054] S2: Prepare 300 mg / L glucose nutrient solution and 200 mg / L MDEA solution as carbon sources. In subsequent steps, the two solutions are added to the bioelectrochemical reactor according to the volume ratio and the MDEA content in the treated liquid is gradually increased to avoid complete inhibition of the biooxidative activity of the dominant functional bacteria.

[0055] S3: In the initial stage, the electrode solution is maintained at 100% glucose nutrient solution;

[0056] S4: When the removal rates of COD and TKN (TKN refers to organic nitrogen because the system does not contain ammonia nitrogen) reach more than 80%, the electrode liquid composition is changed to 20% MDEA solution and 80% glucose nutrient solution;

[0057] S5: When the removal rate of COD and TKN (TKN here refers to organic nitrogen because the system does not contain ammonia nitrogen) reaches more than 80%, the electrode liquid composition is changed to 50% MDEA solution and 50% glucose nutrient solution;

[0058] S6: When the removal rate of COD and TKN (TKN here refers to organic nitrogen because the system does not contain ammonia nitrogen) reaches more than 80%, the electrode liquid composition is changed to 80% MDEA solution and 20% glucose nutrient solution;

[0059] S7: When the removal rate of COD and TKN (TKN here refers to organic nitrogen because the system does not contain ammonia nitrogen) reaches more than 80%, the electrode liquid composition is changed to 100% MDEA solution.

[0060] In the above method, the cathode and anode of the bioelectrochemical device are plate-shaped or mesh-shaped graphite electrodes, carbon felt electrodes, copper-nickel alloy electrodes, titanium electrodes, iron electrodes, copper electrodes or stainless steel plate electrodes.

[0061] The distance between the anode and cathode of the bioelectrochemical device is 2-10 cm, preferably 4-8 cm.

[0062] The anode and cathode plates of the bioelectrochemical device are respectively connected to a DC regulated power supply to adjust the voltage of the processing system to 0.5-3V, preferably 1-2V.

[0063] An aeration device is provided on the side wall of the bioelectrochemical device. The side wall aeration can not only provide dissolved oxygen but also generate vortex to play a stirring role.

[0064] In the above method, during the acclimation process, the activated sludge wastewater needs to be treated with oxygen aeration for 16-20 hours to adjust the dissolved oxygen concentration to 3.0-5.0 mg / L.

[0065] In the present invention, chemical oxygen demand (COD) was determined using the dichromate method (GB / T11914-1989); Kjeldahl nitrogen (TKN) was determined using gas phase molecular absorption spectroscopy (HJ / T196-2005). Biological toxicity was determined using the water quality - determination of the inhibitory effect of water samples on light emission of Vibrio species (luminescent bacteria test) (ISO 11348-3:2007). MDEA was determined using an HPLC-MS method.

[0066] Example 1 Isolation and Screening to Obtain Bacillus cereus DLYBEJ-3

[0067] Bacillus cereus DLYBEJ-3 was isolated and screened from a bioelectrochemical device using the dilution plate separation method. The original activated sludge sample was obtained from a biological aeration tank at a refinery in November 2022. The original activated sludge sample was acclimated and enriched using the aforementioned acclimation method. The strain was then isolated and screened from the bioelectrochemical device using the dilution plate separation method. The isolation and screening methods are as follows:

[0068] (1) Take 10g of the acclimated sample and add it to a 250mL Erlenmeyer flask. Then add 100mL of culture medium A. After culturing at 30℃ and 180rpm for 5 days, take 1% of the enriched solution and transfer it to fresh culture medium A. Continue culturing under the same conditions until the OD value of the bacterial solution reaches 0. 660 When the cell count is >0.8, transfer the cells to fresh medium A. Repeat the above transfer operation three times.

[0069] (2) Transfer to solid culture medium plate (liquid culture medium A + 2% agar) under sterile conditions, and use plate dilution coating method, the dilution concentration is 10 -6 -10 -16 , culture until a single colony appears on the plate, pick a single colony and streak it on the solid culture medium plate, repeat three times.

[0070] (3) The purified single colony was transferred to medium B with MDEA as the sole carbon and nitrogen source, and cultured at 30°C and 180 rpm until the OD of the colony reached 660 >0.8, a purified strain was obtained. Microscopic observation confirmed its purity. If not, the above steps were repeated until it was confirmed to be pure. After repeated cultivation, the dominant pure strain DLYBEJ-3 was obtained.

[0071] The formula of the culture medium A used above is: KH2PO4 8 mg / L, NaCl 5 g / L, yeast extract 5 g / L, tryptone 10 g / L, MDEA 300 mg / L, MgSO4·7H2O 55 mg / L, and CaCl2 25 mg / L.

[0072] The formula of culture medium B is: KH2PO4 8 mg / L, NaCl 5 g / L, MDEA 300 mg / L, MgSO4·7H2O 55 mg / L, and CaCl2 25 mg / L.

[0073] The colony morphology of strain DLYBEJ-3 on the plate culture medium is as follows Figure 1 As shown in the figure, the bacterial morphology under the microscope is magnified 1000 times as shown in the figure. Figure 2 shown.

[0074] Example 2 Identification of strain DLYBEJ-3

[0075] The obtained pure strain DLYBEJ-3 was sent to Hangzhou Yanqu Information Technology Co., Ltd. for strain identification. The results of 16SrDNA gene sequencing analysis showed that the 16S rDNA gene sequence of the strain was as shown in SEQ ID NO.1. The 16S rDNA sequence was compared with the professional database based on the NCBI database (Update 2021.04.21) ( Figure 3 ), and the results showed that it was most similar to Bacillus cereus. Using MEGA software, the neighbor joining method showed the phylogenetic tree of 16S rDNA sequences of "DLYBEJ-3" and related species ( Figure 4 and Figure 5 ), and similarity was calculated 1000 times. Only nodes with bootstrap values ​​greater than 50% are shown in the developmental tree. The superscript "T" indicates the model strain. Strain DLYBEJ-3 was identified as Bacillus cereus.

[0076] Bacillus cereus DLYBEJ-3 was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, Postal Code 430072) on October 18, 2023, with the deposit number CCTCCNO: M 20231931 and the classification name Bacillus cereus.

[0077] Example 3 Application of Bacillus cereus DLYBEJ-3

[0078] In this example, Bacillus cereus DLYBEJ-3 was used to treat MDEA-containing wastewater. The specific method and results are as follows:

[0079] The indicators of the wastewater to be treated are as follows: MDEA: 300 mg / L, COD: 555 mg / L, TKN: 31.3 mg / L, conductivity: 3000 us / cm, acute biological toxicity of luminous bacteria in wastewater is 39%, and pH is 8.13.

[0080] A DLYBEJ-3 bacterial solution was added to the wastewater at a 1% inoculum volume and treated for 36 hours at 30°C, pH 6-10, and aerobic conditions (dissolved oxygen concentration of 3 mg / L). Testing revealed effluent MDEA concentrations of 30 mg / L, COD concentrations of 57 mg / L, and TKN concentrations of 4.67 mg / L. The acute biotoxicity of the luminescent bacteria was 7%.

[0081] Example 4 Application of Bacillus cereus DLYBEJ-3

[0082] In this example, Bacillus cereus DLYBEJ-3 was used to treat MDEA-containing wastewater. The specific method and results are as follows:

[0083] The indicators of the wastewater to be treated are as follows: MDEA: 500 mg / L, COD: 1065 mg / L, TKN: 50.9 mg / L, conductivity: 2900 us / cm, acute biological toxicity of luminous bacteria in wastewater is 51%, and pH is 9.66.

[0084] A DLYBEJ-3 bacterial solution was added to the wastewater at a 5% inoculum volume and treated for 48 hours at 30°C, pH 6-10, and aerobic conditions (dissolved oxygen concentration of 4 mg / L). Testing revealed effluent MDEA concentrations of 47 mg / L, COD concentrations of 100 mg / L, and TKN concentrations of 7.15 mg / L. The acute biotoxicity of the luminescent bacteria was 11%.

[0085] Comparative Example 1

[0086] This comparative example uses a conventional activated sludge process to treat MDEA-containing wastewater. The specific method and results are as follows:

[0087] The wastewater to be treated and the treatment method were the same as in Example 3, except that the DLYBEJ-3 bacterial solution was not added, and conventional activated sludge aerobic treatment (dissolved oxygen concentration of 3 mg / L) was used for 36 hours. After the reaction treatment, the effluent had an MDEA concentration of 200 mg / L, a COD concentration of 315 mg / L, and a TKN concentration of 18.91 mg / L. The acute biotoxicity of the luminescent bacteria was 35%.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Bacillus cereus DLYBEJ-3, characterized in that It is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M20231931.

2. The Bacillus cereus DLYBEJ-3 according to claim 1, wherein The nucleotide sequence of its 16S rDNA is shown in SEQ ID NO.1; And / or, the Bacillus cereus DLYBEJ-3 can grow using N-methyldiethanolamine as the sole carbon source and / or the sole nitrogen source.

3. A microbial preparation, characterized in that The microbial preparation comprises the Bacillus cereus DLYBEJ-3 according to claim 1 or 2.

4. Use of the Bacillus cereus DLYBEJ-3 according to claim 1 or 2 or the microbial preparation according to claim 3 in degrading N-methyldiethanolamine.

5. Use of the Bacillus cereus DLYBEJ-3 according to claim 1 or 2 or the microbial preparation according to claim 3 in the treatment of waste containing N-methyldiethanolamine.

6. Use of the Bacillus cereus DLYBEJ-3 according to claim 1 or 2 or the microbial preparation according to claim 3 in reducing the content of N-methyldiethanolamine, COD and / or TKN in N-methyldiethanolamine-containing waste, or in reducing the biological toxicity of N-methyldiethanolamine-containing waste.

7. The use according to claim 5 or 6, characterized in that The waste is wastewater.

8. Use of the Bacillus cereus DLYBEJ-3 according to claim 1 or 2 or the microbial preparation according to claim 3 in the preparation of a wastewater treatment agent.

9. A wastewater treatment agent, characterized in that The wastewater treatment agent comprises Bacillus cereus DLYBEJ-3 according to claim 1 or 2 or the microbial preparation according to claim 3.

10. A wastewater treatment method, characterized in that: The method comprises: inoculating the Bacillus cereus DLYBEJ-3 according to claim 1 or 2, the microbial preparation according to claim 3, or the wastewater treatment agent according to claim 9 into the wastewater to be treated, and treating the wastewater under aerobic conditions; Preferably, the treatment temperature is 20-50° C., and / or the treatment pH is 5-10.

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