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

Abstract

The present application relates to the field of microbial technology, and specifically discloses a strain of Brachybacterium paraconglomeratum 2-1 and its application in industrial wastewater treatment; the Brachybacterium paraconglomeratum 2-1 is preserved in the China General Microbiological Culture Collection Center on May 29, 2025, with a preservation number of CGMCC No.34719 and a classification name of Brachybacterium paraconglomeratum; the strain 2-1 can tolerate the high toxicity environment of residual ammonia water in a coking plant, has a good effect in the microbial treatment of industrial wastewater containing phenol, can grow by taking phenol as the only carbon source, realizes the removal of phenol in wastewater under aerobic conditions, and has great application potential in the biological treatment of industrial wastewater.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a strain of Paraaggregate Microbe 2-1 and its application in industrial wastewater treatment. Background Technology

[0002] Compared to domestic and agricultural wastewater, industrial wastewater is characterized by high COD, high ammonia nitrogen, complex composition, low biodegradability, strong biological inhibition, high salinity, and recalcitrant degradation. If discharged indiscriminately without treatment, it will pose a serious threat to human health and the ecological environment. With social development and increasing environmental awareness, greater investment has been made in environmental pollution control, resulting in significant achievements in environmental protection. However, the efficient treatment of recalcitrant industrial wastewater remains a major challenge restricting industrial development and environmental protection.

[0003] Phenolic compounds 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 form of these pollutants. Phenol poses a significant threat to organisms and the environment, exhibiting strong toxic effects on all living organisms and accumulating along the food chain. It also possesses strong mutagenic and carcinogenic properties. For industrial wastewater with high phenol content, such as coking wastewater, phenol removal plays a crucial role in the biological treatment process and the quality of the effluent. On the one hand, phenol can account for 50% or even higher of the COD in the raw water and influent to the biological system; removing phenol can significantly reduce the COD of the effluent. On the other hand, when the phenol concentration exceeds 200 mg / L, it significantly inhibits the nitrification process, affecting the removal efficiency of pollutants such as ammonia nitrogen in the biological treatment stage. Therefore, phenol removal is essential for the stable operation of the biological treatment stage.

[0004] With increasing public awareness of environmental protection, the treatment of industrial wastewater has gradually gained social attention. Biological methods are currently the ideal and dominant method for industrial wastewater treatment, offering advantages such as economy, high efficiency, wide application range, and no secondary pollution. However, they also have drawbacks, including low tolerance to toxic substances, weak resistance to shock loads, large sludge production, and difficulty in treating high-concentration wastewater. Screening and applying strains with high treatment efficiency for pollutants such as phenol in industrial wastewater is the prerequisite and foundation for bio-enhancing the industrial wastewater biological treatment process. Currently, there are many patents and research reports on 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 their expected treatment effects in practical applications. Therefore, screening microorganisms that can tolerate high-concentration industrial wastewater and degrade phenol, and applying them to the bio-enhanced treatment of industrial wastewater, will improve the system's removal efficiency of pollutants such as phenol in industrial wastewater, which is of greater significance for practical industrial wastewater treatment. Based on the above, this invention provides a strain of *Pseudomonas paraaggregatus* 2-1 and its application in industrial wastewater treatment. Summary of the Invention

[0005] To address the aforementioned technical problems, this 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 *Paracitating Microbe* 2-1, which adopts the following technical solution:

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

[0008] Secondly, the present invention provides a microbial inoculant, which adopts the following technical solution:

[0009] A microbial agent, wherein the active ingredient of the microbial agent is the aforementioned *Paracocylella esculenta* 2-1.

[0010] Thirdly, the present invention provides the application of the aforementioned Paraaggregating Short Bacillus 2-1 and microbial agents 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 this 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 this invention has good effects on the microbial treatment of industrial wastewater containing phenol. It can grow with 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 this invention can achieve a 100% phenol removal rate after 96 hours of aerobic treatment in the treatment of simulated industrial wastewater with an initial phenol concentration of 500 mg / L and an initial ammonia nitrogen concentration of 212 mg / L, thus realizing the treatment of phenol-containing wastewater. Attached Figure Description

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

[0019] Figure 2 This is an example of the growth of Brachybacterium paraconglomeratum 2-1 on a medium plate containing residual ammonia from coking in Example 3 of the present invention.

[0020] Figure 3 This is an example of the growth of Brachybacterium paraconglomeratum 2-1 under a stereomicroscope on a medium plate containing residual ammonia from coking, as shown in Example 3 of the present invention.

[0021] Figure 4This illustrates the growth of Brachybacterium paraconglomeratum 2-1 in simulated industrial wastewater in Example 4 of this invention.

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

[0023] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

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

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

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

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

[0028] Basic culture 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 different volumes of phenol stock solution according to the experimental purpose.

[0029] Example 1

[0030] Isolation and screening of strains

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

[0032] Enrichment culture: Samples were collected from the above-mentioned synthetic medium and streaked several times on LB solid medium to obtain pure strains, such as... Figure 1 As shown.

[0033] Example 2

[0034] Identification of strains

[0035] After the above separation and purification, the obtained strain was identified by molecular biology. The bacterial 16S rRNA gene sequence (SEQ ID NO.1) was amplified by PCR and then sequenced for comparison.

[0036] The 16S rRNA sequence of *Paracitating Microbes 2-1* is shown in SEQ ID NO:1:

[0037]

[0038] The amplification primers are:

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

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

[0041] The reaction system consisted of: 10×Buffer 2µL, 2.5mM dNTP 1.5µL, Primer 1 1µL, Primer 2 1µL, template 1µL, enzyme 0.3µL, water 13.2µL, and a total volume of 20µL.

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

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

[0044] The 16S rRNA gene sequence of this bacterium (SEQ ID NO.1) was obtained after forward and reverse sequencing and splicing. The sequence was then compared with the NCBI database using BLAST. The comparison results showed that this strain had the highest homology (99.55%) with *Brachybacterium paraconglomeratum* LMG 19861. Therefore, the strain was named *Brachybacterium paraconglomeratum* 2-1, and will be referred to as strain 2-1 in this application.

[0045] The *Brachybacterium paraconglomeratum* 2-1 of this invention was deposited on May 29, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34719 and classification name *Brachybacterium paraconglomeratum*.

[0046] Example 3

[0047] Test of 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 a 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 petri dishes. After the plates cool and solidify, use a pipette tip to pick up a small number of colonies of strain 2-1 and streak them on the plate. Incubate the plates upside down at 30℃ for 3 days, observe the colony growth on the plates, and further observe the colonies under a stereomicroscope. Figure 2 , Figure 3 As shown, after culturing for 3 days, strain 2-1 formed a semi-transparent film-like colony on the remaining ammonia water 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] Test of phenol degradation effect of strain 2-1

[0051] The initial concentration of phenol in the basal medium was adjusted to 500 mg / L and the ammonia nitrogen concentration to 212 mg / L. Bacterial cells were collected by centrifuging at 12000 rpm for 5 min at an inoculation rate of 10% (v / v), washed with sterile water, and then inoculated into the basal medium. At this point, the OD... 600 The concentration was 0.4, and the mixture was incubated at 30℃ and 180rpm for 96 hours. The remaining phenol content was then determined.

[0052] The results are as follows Figure 4 , Figure 5 As shown, strain 2-1 was able to rapidly proliferate in a medium with phenol as the sole carbon source and ammonia nitrogen as the sole nitrogen source after a 3-day lag period, and simultaneously completely removed 500 mg / L phenol within 24 hours during rapid proliferation. Overall, the degradation rate of 500 mg / L phenol was 100% within 96 hours.

[0053] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of the present invention.

Claims

1. A strain of *Paracitating Microbe* 2-1, characterized in that, The aforementioned *Brachybacterium paraconglomeratum* 2-1 was deposited on May 29, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 34719 and classification name *Brachybacterium paraconglomeratum*. The aforementioned *Pseudomonas paraaggregatus* 2-1 can grow and reproduce in the complex high-concentration pollutant environment of actual coking wastewater with a pH of 9.4, an ammonia nitrogen concentration of 2260 mg / L, and a COD of 4400 mg / L.

2. A microbial inoculant, characterized in that, The active ingredient of the microbial agent is the paraaggregating short bacillus 2-1 as described in claim 1.

3. The application of the para-aggregating short bacillus 2-1 as described in claim 1 or the microbial agent as described in claim 2 in industrial wastewater treatment.

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

5. The application 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 application 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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