Brevibacterium paragglutinum 2-1 and application thereof in industrial wastewater treatment

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a brevibacterium paragglutinatus 2-1 and application thereof in industrial wastewater treatment. The brevibacterium paracareactor 2-1 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on May 29, 2025, the preservation number is CGMCC No.34719, the brevibacterium paracareactor 2-1 is classified and named as brevibacterium paracareactor, the preservation number is CGMCC No.34719, the preservation number is CGMCC No.34719, the preservation number is CGMCC No.34719, the preservation number is CGMCC No.34719, the preservation number is CGMCC No.34719, the preservation number is CGMCC No.34719, the preservation number is CGMCC No.34719, and the preservation number is CGMCC No.34719. The strain 2-1 can tolerate a high-toxicity environment of residual ammonia water of a coking plant, has a good effect in the aspect of microbiological treatment of phenol-containing industrial sewage, can grow by taking phenol as a unique carbon source, realizes removal of phenol in wastewater under an aerobic condition, and has huge application potential in biological treatment of industrial wastewater.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more particularly to a strain of Brevibacterium paracoagulans 2-1 and its application in industrial wastewater treatment. Background Art

[0002] Compared to domestic and agricultural wastewater, industrial wastewater is characterized by high COD, high ammonia nitrogen content, complex composition, low biodegradability, strong bioinhibition, high salinity, and difficulty in degradation. If discharged without treatment, it poses a serious threat to human health and the ecological environment. With the development of society, people's awareness of environmental protection is growing, and investment in environmental pollution control is increasing, resulting in significant progress in environmental protection. However, the efficient treatment of difficult-to-degrade industrial wastewater remains a challenge that hinders industrial development and environmental protection.

[0003] Phenols are a typical and major pollutant in industrial wastewater, widely distributed in wastewater from industries such as petroleum, chemical, building materials, ceramics, papermaking, coal gas, textiles, plastics, synthetic fibers, and coking. Phenol is the predominant phenolic compound among these phenols. Phenol is extremely harmful to organisms and the environment. It is highly toxic to all organisms, can accumulate along the food chain, and is highly mutagenic and carcinogenic. For industrial wastewaters with high phenol content, such as coking wastewater, phenol removal plays a crucial role in the biological treatment process and effluent quality. Phenol can account for 50% or more of the COD in both the raw water and the influent of biological systems, making its removal significantly lower in the effluent. Furthermore, when phenol concentrations exceed 200 mg / L, it significantly inhibits nitrification, impacting the removal efficiency of pollutants such as ammonia nitrogen in the biological treatment phase. Therefore, phenol removal is crucial for the stable operation of the biological treatment phase.

[0004] As awareness of environmental protection grows, the treatment of industrial wastewater is gaining increasing public attention. Biological methods are currently the ideal and leading method for treating industrial wastewater, offering advantages such as cost-effectiveness, wide application, and no secondary pollution. However, they also suffer from shortcomings such as low tolerance to toxicants, weak shock load resistance, high sludge production, and difficulty treating high-concentration wastewater. Screening and applying bacterial strains with high treatment efficiency for pollutants such as phenol in industrial wastewater is a prerequisite and foundation for bioaugmentation of industrial wastewater biological treatment processes. Currently, there are numerous patents and research reports on bacterial strains with degradation effects on pollutants such as phenol. However, the tolerance and adaptability of these strains in actual industrial wastewater environments are unknown, and they may not achieve the expected treatment effects in actual treatment. Therefore, screening for microorganisms that can tolerate high-concentration industrial wastewater and degrade phenol and applying them to bioaugmented industrial wastewater treatment will improve the system's removal efficiency for pollutants such as phenol in industrial wastewater, which is of great significance for practical industrial wastewater treatment. Based on the above, the present invention provides a strain of Brevibacterium paracoagulans 2-1 and its application in industrial wastewater treatment. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a strain of Brachybacterium paraconglomeratum 2-1 and its application in industrial wastewater treatment.

[0006] In a first aspect, the present invention provides a strain of Brevibacterium paracoagulans 2-1, which adopts the following technical solution:

[0007] A strain of Brachybacterium paraconglomeratum 2-1, wherein the Brachybacterium paraconglomeratum 2-1 was deposited on May 29, 2025 at the General Microbiology Center of the China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 34719 and the classification name Brachybacterium paraconglomeratum.

[0008] In a second aspect, the present invention provides a microbial agent, which adopts the following technical solution:

[0009] A microbial agent, the active ingredient of which is the above-mentioned Brevibacterium paracoagulans 2-1.

[0010] In a third aspect, the present invention provides the use of the aforementioned Brevibacterium paracoagulans 2-1 and microbial agent in industrial wastewater treatment.

[0011] Preferably, the industrial wastewater is phenol-containing industrial wastewater.

[0012] Preferably, the concentration of phenol in the industrial wastewater is 10-1000 mg / L, and the concentration of ammonia nitrogen is 10-2000 mg / L.

[0013] Preferably, the concentration of phenol in the industrial wastewater is 500 mg / L, and the concentration of ammonia nitrogen is 212 mg / L.

[0014] In summary, the present invention has the following beneficial effects:

[0015] 1. The Brachybacterium paraconglomeratum 2-1 provided by the present invention can tolerate the highly toxic environment of residual ammonia water in coking plants and has great application potential in the biological treatment of high-concentration, highly toxic industrial wastewater.

[0016] 2. The Brachybacterium paraconglomeratum 2-1 provided by the present invention has a good effect in the microbial treatment of phenol-containing industrial wastewater. It can grow using phenol as the sole carbon source and has great application potential in the biological treatment of industrial wastewater.

[0017] 3. The Brachybacterium paraconglomeratum 2-1 provided by the present invention can achieve a phenol removal rate of 100% after 96 hours of aerobic treatment in the treatment process of simulated industrial wastewater with an initial phenol concentration of 500 mg / L and an initial ammonia nitrogen concentration of 212 mg / L, thereby achieving the treatment of phenol-containing wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a colony image of Brachybacterium paraconglomeratum 2-1 on LB medium in Example 1 of the present invention;

[0019] Figure 2 The growth of Brachybacterium paraconglomeratum 2-1 in Example 3 of the present invention on a plate of coking residual ammonia culture medium;

[0020] Figure 3 This is the growth of Brachybacterium paraconglomeratum 2-1 on a coking residual ammonia culture plate under a stereomicroscope in Example 3 of the present invention;

[0021] Figure 4The growth of Brachybacterium paraconglomeratum 2-1 in simulated industrial wastewater in Example 4 of the present invention;

[0022] Figure 5 This is a diagram showing the phenol treatment effect of Brachybacterium paraconglomeratum 2-1 in simulated industrial wastewater in Example 4 of the present invention. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0024] The culture medium used in the embodiments of the present invention is as follows:

[0025] LB liquid medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and 1 L distilled water.

[0026] LB solid medium: Add 15g agar to LB liquid medium.

[0027] Residual ammonia culture medium: 250 mL of deionized water, 6 g of agar, sterilize and cool to 50°C, add 250 mL of residual ammonia and mix well.

[0028] Basal medium: 2.25g dipotassium hydrogen phosphate, 2.75g potassium dihydrogen phosphate, 1.00g ammonium sulfate, 0.20g magnesium chloride hexahydrate, 0.10g sodium chloride, 0.02g ferric chloride hexahydrate, 0.01g calcium chloride, 1L distilled water, pH 6.8-7.0. After sterilization, add varying volumes of phenol stock solution depending on the experimental purpose.

[0029] Example 1

[0030] Isolation and screening of strains

[0031] Simulated wastewater: After sterilizing the basal culture medium, a phenol stock solution was added to adjust the phenol concentration to 1000 mg / L. This basal culture medium used 1000 mg / L phenol as the sole carbon source to simulate wastewater. After one week of storage, the synthetic culture medium became turbid, indicating that foreign microorganisms had entered the synthetic culture medium and were able to grow in the presence of 1000 mg / L phenol.

[0032] Enrichment culture: The sample was collected from the above synthetic medium and streaked several times on LB solid medium to obtain pure strains, such as Figure 1 shown.

[0033] Example 2

[0034] Identification of strains

[0035] After the above separation and purification, the obtained strain was subjected to molecular biological identification, and the bacterial 16S rRNA gene sequence (SEQ ID NO.1) was amplified by PCR and then sequenced and compared.

[0036] The 16S rRNA sequence of Brevibacterium paracoagulans 2-1 is shown in SEQ ID NO: 1:

[0037]

[0038] Amplification primers are:

[0039] 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID NO. 2),

[0040] 1492R: TACGGYTACCTTGTTACGACTT (SEQ ID NO. 3);

[0041] The reaction system was as follows: 10× Buffer 2µL, 2.5mM dNTP 1.5µL, Primer1 1µL, Primer2 1µL, template 1µL, enzyme 0.3µL, water 13.2µL, total volume 20µL;

[0042] The reaction conditions were as follows: pre-denaturation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1.5 min, extension at 72°C for 10 min, and insulation at 4°C.

[0043] The PCR products were detected by agarose gel electrophoresis and then sequenced.

[0044] The 16S rRNA gene sequence (SEQ ID NO. 1) of this strain was obtained after forward and reverse sequencing and assembly. Blast alignment of this sequence with the NCBI database revealed that this strain shared the highest homology, at 99.55%, with Brachybacterium paraconglomeratum LMG 19861. Therefore, the strain was named Brachybacterium paraconglomeratum 2-1 and will be referred to herein as strain 2-1.

[0045] The Brachybacterium paraconglomeratum 2-1 of the present invention was deposited on May 29, 2025 at the General Microbiology Center of the China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 34719 and a classification name of Brachybacterium paraconglomeratum.

[0046] Example 3

[0047] Test on the adaptability of strain 2-1 to actual coking wastewater

[0048] Add 6g of agar to 250mL of deionized water, sterilize and cool to 50℃, add 250mL of residual ammonia water and mix well to obtain residual ammonia water culture medium with a pH of 9.4, a conductivity of 10680μs / cm, a phenol concentration of 800mg / L, an ammonia nitrogen concentration of 2260mg / L, and a COD of 4400mg / L. Pour the residual ammonia water culture medium into the culture dish, wait for the plate to cool and solidify, pick up a few colonies of strain 2-1 with the tip of a gun, divide and mark the plate, place the plate at 30℃ and invert for 3 days, observe the growth of the colonies on the plate, and further observe the colonies under a stereo microscope. Figure 2 、 Figure 3 As shown, after culturing for 3 days, strain 2-1 formed translucent film-like colonies on the remaining ammonia culture medium, indicating that strain 2-1 could tolerate the complex high-concentration pollutant environment of actual coking wastewater and grow and reproduce in this environment.

[0049] Example 4

[0050] Phenol degradation effect test of strain 2-1

[0051] Adjust the initial phenol concentration of the basal medium to 500 mg / L and the ammonia nitrogen concentration to 212 mg / L. According to the inoculum volume of 10% (v / v), the bacterial liquid was aspirated and centrifuged at 12000 rpm for 5 minutes to collect the bacteria. After washing with sterile water, the bacteria were inoculated into the basal medium. At this time, the OD 600 The mixture was cultured at 30°C and 180 rpm in a shaker for 96 h, and the residual phenol content was detected.

[0052] The results are as follows Figure 4 、 Figure 5 As shown, strain 2-1 was able to rapidly proliferate after a three-day lag in a culture medium containing phenol as the sole carbon source and ammonia as the sole nitrogen source. While rapidly proliferating, it also completely removed 500 mg / L of phenol within 24 hours. Overall, the degradation rate of 500 mg / L of phenol was 100% within 96 hours.

[0053] This specific embodiment is only an explanation of the present invention, and it is not a limitation of the present invention. After reading this specification, those skilled in the art can make non-creative modifications to this embodiment as needed, but as long as it is within the scope of the claims of the present invention, it is protected by patent law.

Claims

1. A strain of Brevibacterium paracoagulans 2-1, characterized in that: The Brachybacterium paraconglomeratum 2-1 was deposited on May 29, 2025 at the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 34719 and the classification name Brachybacterium paraconglomeratum.

2. A microbial agent, characterized in that: The active ingredient of the microbial agent is the Brevibacterium paracoagulans 2-1 described in claim 1.

3. Use of the Brevibacterium paracoagulans 2-1 according to claim 1 or the microbial agent according to claim 2 in industrial wastewater treatment.

4. The use according to claim 3, characterized in that The industrial wastewater is phenol-containing industrial wastewater.

5. The use according to claim 4, characterized in that The concentration of phenol in the industrial wastewater is 10-1000 mg / L, and the concentration of ammonia nitrogen is 10-2000 mg / L.

6. The use according to claim 4, characterized in that The concentration of phenol in the industrial wastewater is 500 mg / L, and the concentration of ammonia nitrogen is 212 mg / L.

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