Composite bacterial flora for treating benzotriazole wastewater

Abstract

A composite bacterial flora for treating benzotriazole wastewater. A bacterial strain composition is provided, the composition comprising the following bacterial strains: (a) Bacillus altitudinis (Bacillus altitudinis) with a preservation number of CGMCC 37186 Bacillus altitudinis ) SD1, (b) Saprospira sp. (Saprospira sp.) with a preservation number of CGMCC 37187 Aquamicrobium defluvii ) SD4, and (c) Pseudomonas aeruginosa (Pseudomonas aeruginosa) with a preservation number of CGMCC 37189 Pseudomonas aeruginosa ) SD32.

Description

Technical Field

[0001] This application relates to the field of wastewater treatment, specifically to the field of wastewater treatment containing benzotriazole. Background Technology

[0002] Benzotriazole (BTA) is an important fine chemical product, appearing as white or light brown needle-like crystals. It has wide applications in industry, primarily as a rust inhibitor and corrosion inhibitor for metals such as silver, copper, and nickel. It can also be used as a paint additive, detergent preservative, dye intermediate, and polymer stabilizer. The mainstream synthesis process of benzotriazole involves the reaction of o-phenylenediamine with sodium nitrite. Therefore, the wastewater generated during the synthesis and cleaning stages of benzotriazole contains not only residual BTA but also unreacted o-phenylenediamine, excess sodium nitrite, acidic catalysts (such as hydrochloric acid), and reaction byproducts, resulting in a complex wastewater composition. Typically, this wastewater is characterized by high COD, strong biotoxicity, poor biodegradability, and high salt content. Currently, the main treatment methods are classified into four categories: physical methods, chemical methods, biological methods, and combined processes.

[0003] Biological methods utilize the metabolic processes of microorganisms to degrade BTA, which have advantages such as low cost and environmental friendliness, but the biotoxicity and recalcitrant nature of BTA need to be overcome.

[0004] Therefore, there is still a need in this field for new microorganisms to effectively treat wastewater containing benzotriazole. Summary of the Invention

[0005] The purpose of this application is to provide microorganisms that can effectively treat wastewater containing benzotriazole, thereby achieving efficient pretreatment of wastewater and reducing the treatment burden on enterprise activated sludge systems.

[0006] This application provides a bacterial composition comprising the following strains:

[0007] (a) Bacillus hygroscopicus with accession number CGMCC 37186 ​​( Altitude Bacillus SD1,

[0008] (b) Wastewater microorganisms with accession number CGMCC 37187 ( Water microbe downstream SD4, and

[0009] (c) Pseudomonas aeruginosa with accession number CGMCC 37189 Pseudomonas aeruginosa SD32.

[0010] This application also provides a microbial preparation comprising the strain composition described in this application.

[0011] Another aspect of this application provides a method for treating wastewater containing triazole, the method comprising treating the wastewater containing triazole using the bacterial composition described in this application. Attached Figure Description

[0012] Figure 1 The wastewater treatment effect of one example of this application is described. Detailed Implementation

[0013] Unless otherwise specified in this specification, the components or their preferred components may be combined to form new technical solutions.

[0014] Unless otherwise specified in this specification, all the embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0015] Unless otherwise specified in this specification, all technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0016] The "range" disclosed herein takes the form of a lower limit and an upper limit. It can be one or more lower limits and one or more upper limits, respectively. A given range is defined by selecting a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this way are inclusive and composable, meaning that any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a particular parameter, it is also expected that ranges of 60-110 and 80-120 are also expected. Furthermore, if minimum range values ​​1 and 2 are listed, and if maximum range values ​​3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0017] In this document, unless otherwise specified, the values ​​mentioned, whether or not they are preceded by the word "about", cover a range of ±10%, or may be ±5%, ±3%, ±2%, ±1%, or ±0.5%.

[0018] In this application, unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, if the method includes steps (a) and (b), it means that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, if the method may also include step (c), it means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0019] In this application, unless otherwise specified, the terms "comprising" and "including" as used herein are open-ended or closed-ended. For example, "comprising" and "including" may mean that other components not listed may also be included, or that only the listed components may be included.

[0020] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0021] In this description, unless otherwise stated, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0022] Unless otherwise specified, all procedures in this paper are performed at room temperature and pressure.

[0023] Unless otherwise specified, all solutions in this document refer to aqueous solutions.

[0024] This application provides a bacterial composition comprising the following strains:

[0025] (a) Bacillus hygroscopicus with accession number CGMCC 37186 ​​( Altitude Bacillus SD1,

[0026] (b) Wastewater microorganisms with accession number CGMCC 37187 ( Water microbe downstream SD4, and

[0027] (c) Pseudomonas aeruginosa with accession number CGMCC 37189 Pseudomonas aeruginosa SD32.

[0028] The Bacillus hygroscopicus described in this application ( Altitude Bacillus The strain was named SD1 and was deposited on December 25, 2025, at the China General Microbiological Culture Collection Center in Beijing, China, with accession number CGMCC 37186.

[0029] The wastewater microorganisms described in this application ( Water microbe downstream The strain was named SD4 and was deposited on December 25, 2025, at the China General Microbiological Culture Collection Center in Beijing, China, with accession number CGMCC 37187.

[0030] The Pseudomonas aeruginosa described in this application ( Pseudomonas aeruginosa The strain was named SD32 and was deposited on December 25, 2025, at the China General Microbiological Culture Collection Center in Beijing, China, with accession number CGMCC 37189.

[0031] In one example of this application, the strain may be a live strain or an inactivated strain.

[0032] In one example of this application, Bacillus hygroscopicus (Bt) Altitude Bacillus SD1, wastewater microorganisms ( Water microbe downstream SD4 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa SD32 can be included in the composition in any proportion. In a preferred embodiment of this application, the Bacillus glaberens (SD32) is present in a proportion based on the weight of the strain. Altitude Bacillus SD1, wastewater microorganisms ( Water microbe downstream SD4 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa The weight ratio of SD32 can be 1:(0.1-10):(0.1-10), for example 1:(0.1-5):(0.1-5), 1:(0.1-1):(0.5-1.5).

[0033] This application also provides a microbial preparation comprising the strain composition described in this application.

[0034] In one example of this application, based on 100 parts by weight of the total weight of the microbial preparation, the content of the strain composition described in this application is 0.1-99.9 parts by weight, preferably 10-80 parts by weight, more preferably 20-70 parts by weight, and most preferably 40-60 parts by weight.

[0035] In one example of this application, the microbial preparation may further include a protectant. The protectant may be any protectant commonly used in the art. In one example of this application, the protectant includes glycerol, mannitol, or a combination thereof. In one example of this application, based on 100 parts by weight of the total weight of the microbial preparation, the content of the protectant is 0.1-99.9 parts by weight, preferably 20-90 parts by weight, more preferably 30-80 parts by weight, and most preferably 40-60 parts by weight.

[0036] In this application, the microbial preparation may be in solid form (e.g., lyophilized powder) or liquid form.

[0037] Another aspect of this application provides a method for treating wastewater containing triazole, the method comprising treating the wastewater containing triazole using the bacterial composition described in this application.

[0038] In this application, the triazole-containing wastewater can be any triazole-containing wastewater (e.g., benzotriazole) as described in the art, such as wastewater generated in the benzotriazole production process and wastewater generated during the use of benzotriazole. In one example of this application, the triazole-containing wastewater typically contains benzotriazole, methylbenzotriazole, o-phenylenediamine, methanol, toluene, sodium nitrate, sodium nitrite, and sodium chloride.

[0039] Example

[0040] strain source

[0041] Hangzhou Xiuchuan Technology Co., Ltd. provided the strain for this study. The strain was preserved using a vacuum freeze-drying method.

[0042] Test methods

[0043] COD values ​​were measured according to HJ 828-2017.

[0044] TN values ​​were measured according to HJ 636-2012.

[0045] The NH3-N value was measured according to HJ 535-2009.

[0046] TDS values ​​are measured according to HJ / T 51-1999.

[0047] culture medium

[0048] LB medium: yeast extract (purchased from Bestway Laboratory Supplies) 5 g / L, peptone (purchased from Bestway Laboratory Supplies) 10 g / L, NaCl 10 g / L, pH=7.

[0049] strain screening

[0050] Strains with a growth cycle of less than 3 days, culture temperature of 25-35℃, and simple culture medium were selected from the chemical-derived strain library of Hangzhou Xiuchuan Technology Co., Ltd. as candidate strains. These strains were activated in sterile 96-well plates using LB medium (0.5 g / L yeast extract, 1 g / L peptone, 1 g / L NaCl, pH=7, aerobic culture for 24 h). Wastewater (from a pharmaceutical chemical plant, containing benzotriazole, methylbenzotriazole, o-phenylenediamine, methanol, toluene, sodium nitrate, sodium nitrite, and sodium chloride, etc.; COD 10000 mg / L, NH3-N 100 mg / L, TN 1000 mg / L, TDS 30000 mg / L, pH=7) was filtered through a 0.22 μm membrane for sterilization before being dispensed into sterile 96-well plates. The activated candidate strains were inoculated into the wastewater at 1% and cultured at 30℃ for 3 days. After cultivation, the OD of the strain in the 96-well plate was measured using a microplate reader (Infinite® 200 PRO NanoQuant, purchased from Teco). 600 Value, select OD 600 >0.5 was used as the target strain. The target strain underwent multiple subculturings (i.e., 10% of the bacterial culture from wastewater was subcultured and placed in wastewater with COD of 10000 mg / L, NH3-N of 100 mg / L, TN of 1000 mg / L, TDS of 30000 mg / L, and pH of 7), and those that still maintained stable growth (i.e., OD500) were selected. 600 The bacterial species with a ratio of 1) were used as the test strains.

[0051] Single strain degradation performance evaluation

[0052] The test strains were activated using LB medium (medium: yeast extract 0.5 g / L, peptone 1 g / L, NaCl 1 g / L, pH=7, aerobic culture with shaking for 24 h). Fresh bacterial culture in the logarithmic phase was centrifuged at 6000 r / min for 5 min to obtain bacterial cells, washed twice with 0.9% (w / w) physiological saline, and then resuspended in sterile distilled water to obtain OD. 600 =1.0 seed culture. Take 100 mL of nutrient-adjusted wastewater (containing benzotriazole, methylbenzotriazole, o-phenylenediamine, methanol, toluene, sodium nitrate, sodium nitrite, and sodium chloride, etc. COD 10000 mg / L, NH3-N 100 mg / L, TN 1000 mg / L, TDS 30000 mg / L, pH=7) and place it in a 250 mL Erlenmeyer flask. Inoculate the seed culture into the wastewater at a 10% inoculum rate. Three replicates were set up for each strain, and a control group without the strain was also included. All systems were incubated at 150 r / min and 30℃. COD values ​​were measured periodically (every 2 days) to evaluate the treatment effect of the strains. A total of 4 strains with high degradation efficiency were isolated.

[0053] Single strain identification

[0054] 16S rRNA gene sequencing was performed on bacterial strains with a COD removal rate of over 50% within 5 days. PCR amplification was performed using universal primers 27F (5'-GAGAGTTTGATCMTGGCTCAG-3') (SEQ ID NO:5) and 1492R (5'-TACGGYTACCTTGTTACGAC-3') (SEQ ID NO:6), and the resulting sequences are shown in SEQ ID NO:1 (SD1), SEQ ID NO:2 (SD4), SEQ ID NO:3 (SD5), and SEQ ID NO:4 (SD32), respectively. The sequencing results were submitted to the EzBioCloud database (http: / / www.ezbiocloud.net / eztaxon / identify) for sequence alignment. The 16S rRNA gene alignment information is shown in Table 1.

[0055] All four strains have been deposited, and the deposit information is as follows:

[0056] Highland Bacillus ( Altitude Bacillus SD1 was deposited on December 25, 2025, at the China General Microbiological Culture Collection Center in Beijing, China, with accession number CGMCC 37186.

[0057] Wastewater microorganisms ( Water microbe downstream SD4 was deposited on December 25, 2025, at the China General Microbiological Culture Collection Center in Beijing, China, with accession number CGMCC 37187.

[0058] Pseudomonas aeruginosa ( Pseudomonas aeruginosa SD32 was deposited on December 25, 2025, at the China General Microbiological Culture Collection Center in Beijing, China, with accession number CGMCC 37189.

[0059] Microbes keratolyticus ( Microbacterium keratanolyticum SD5 was deposited on December 25, 2025, at the China General Microbiological Culture Collection Center in Beijing, China, with accession number CGMCC37188.

[0060] Table 1. Sequencing alignment information of 16S rRNA genes in single strains

[0061]

[0062] Construction of complex microbial communities

[0063] The seed cultures of the four strains were mixed in pairs at a 1:1 ratio. Different mixtures were then inoculated at 10% of the total inoculum into nutrient-adjusted wastewater (containing benzotriazole, methylbenzotriazole, o-phenylenediamine, methanol, toluene, sodium nitrate, sodium nitrite, and sodium chloride; COD 10000 mg / L, NH3-N 100 mg / L, TN 1000 mg / L, TDS 30000 mg / L, pH 7). All systems were incubated at 150 r / min and 30℃, with COD values ​​measured periodically (every 2 days) to evaluate the treatment effect of different combinations on the wastewater. The ratio of the COD degradation rate of the combination to the maximum COD degradation rate of a single strain in the combination was denoted as R. If R > 1, the two strains in the combination exhibited synergistic effects; if R ≤ 1, the two strains in the combination exhibited antagonistic effects. The R values ​​among the strains are shown in Table 2. The results indicate... Microbacterium keratanolyticum SD5 exhibits antagonistic effects with the other three strains, thus the complex microbial community becomes... Altitude Bacillus SD1, Aquamicrobe I flowed down SD4 and Pseudomonas aeruginosa SD32.

[0064] Table 2 R values ​​among strains

[0065]

[0066] Seed cultures of strains SD1, SD4, and SD32 were combined in ratios of 1:1:1, 1:2:3, 1:3:2, 2:1:3, 2:3:1, 3:1:2, and 3:2:1, respectively, and the resulting complex bacterial groups were named BAP0, BAP1, BAP2, BAP3, BAP4, BAP5, BAP6, and BAP7. Different compound bacterial groups were inoculated at 10% of the total inoculum into nutrient-adjusted wastewater (including benzotriazole, methylbenzotriazole, o-phenylenediamine, methanol, toluene, sodium nitrate, sodium nitrite, and sodium chloride; COD 10000 mg / L, NH3-N 100 mg / L, TN 1000 mg / L, TDS 30000 mg / L, pH=7). All systems were incubated at 150 r / min and 30℃, with COD values ​​measured periodically (every 2 days) to evaluate the treatment effect of different compound bacterial groups on the wastewater. The results are shown in Table 3. The compound bacterial group showed the best effect when the ratio of SD1, SD4, and SD32 was 3:1:2; therefore, compound bacterial group BAP5 was selected as the test group.

[0067] Table 3. Effects of various microbial communities on COD treatment

[0068]

[0069] Small-scale test of treatment of benzotriazole wastewater with compound microbial community

[0070] A continuous influent / outfluent device was used to simulate an on-site treatment system as closely as possible to test the effect of the compound microbial community on the treatment of COD and TN in wastewater.

[0071] (1) Continuous water inlet and outlet device

[0072] The continuous inlet and outlet water system includes an equalization tank, a biological pretreatment tank 1 (PBR-1), a biological pretreatment tank 2 (PBR-2), and an outlet water tank, which are connected in series. Each container has an effective volume of 3L, and the water inlet method is bottom inlet and top outlet.

[0073] The equalization tank is used to add nutrients to regulate water quality, and a stirring system is installed at the bottom; wastewater in the equalization tank is pumped to the biological pretreatment tank by a peristaltic pump.

[0074] The biochemical pretreatment tank is a composite microbial biochemical treatment tank with an aeration system installed at the bottom.

[0075] The effluent tank is used to collect treated wastewater.

[0076] Initially, water is continuously fed in at a low load. Once the bacterial concentration in the aerated biological treatment tank stabilizes, water is continuously fed in at a high load, and the operation and control phase begins.

[0077] (2) Preparation of strains

[0078] To minimize the introduction of nitrogenous substances from the culture medium into the wastewater, and given the lack of centrifuge equipment on-site, the strain was fermented using 1 / 4 concentration LB medium (i.e., formulation: peptone 2.5 g / L, yeast extract 1.25 g / L, NaCl 2.5 g / L, pH 7.0). Fermentation continued until the OD reached... 600 When the value reaches about 1.0, SD1, SD4, and SD32 are mixed in an equal volume ratio of 3:1:2 to obtain the seed solution.

[0079] (3) Wastewater nutrient regulation

[0080] Benzotriazole wastewater (wastewater from a pharmaceutical chemical plant, composed of benzotriazole, methylbenzotriazole, o-phenylenediamine, methanol, toluene, sodium nitrate, sodium nitrite, and sodium chloride, etc., was pretreated with XDA-1G macroporous adsorption resin purchased from Xi'an Lanxiao Technology to achieve a COD of 6234 mg / L, NH3-N of 143 mg / L, and TN of 518 mg / L) was supplemented with 0.5 g / L of KH2PO4 and the pH of the wastewater was adjusted to 6.0-7.0 using H2SO4.

[0081] (4) Biological pretreatment

[0082] An aerobic process was adopted, with the HRT (hydraulic retention time, the average time wastewater stays in the reactor) of both PBR-1 and PBR-2 stages controlled at approximately 3 days, DO > 2 mg / L, and water temperature at 30-35℃. Seed culture was added at a 30% inoculum level. During the inoculum treatment period, the pH of the inoculum treatment tank would rise to 8.0, requiring the addition of H2SO4 to maintain the pH at 6.0-7.0.

[0083] Basic water quality data from the system are monitored daily, and the average value is calculated using data from the last week of operation to assess COD treatment effectiveness. The results are as follows: Figure 1 As shown in Table 4.

[0084] Table 4 Summary of the treatment effects of the compound microbial community BAP5 on COD in benzotriazole wastewater

[0085]

[0086] The BAP5 microbial community exhibits excellent growth in the adsorbed effluent from benzotriazole wastewater. After a 3-day treatment time (HRT), it reduced the wastewater COD from 6200 mg / L to approximately 2200 mg / L, achieving a COD removal rate of 62.6%. After a 6-day HRT, the COD was further reduced to approximately 800 mg / L, achieving a COD removal rate of 86.7%. Treatment with the BAP5 microbial community effectively removes most of the COD, significantly reducing the biochemical load on the enterprise's activated sludge system.

Claims

1. A bacterial composition comprising the following strains: (a) Bacillus hygroscopicus with accession number CGMCC No. 37186 Bacillus altitudinis SD1, (b) Wastewater microorganisms with accession number CGMCC No. 37187 ( Aquamicrobium defluvii SD4, and (c) Pseudomonas aeruginosa with accession number CGMCC No. 37189 Pseudomonas aeruginosa SD32, Based on the weight of the strain, the Highland Bacillus ( Bacillus altitudinis SD1, wastewater microorganisms ( Aquamicrobium defluvii SD4 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa The weight ratio of SD32 is 1:(0.1-1):(0.5-1.5).

2. A microbial preparation comprising the strain composition of claim 1.

3. The microbial preparation according to claim 2, characterized in that, Based on 100 parts by weight of the total weight of the microbial preparation, the content of the strain composition is 0.1-99.9 parts by weight.

4. The microbial preparation according to claim 2, characterized in that, The microbial preparation also includes a protectant.

5. A method for treating wastewater containing triazole, the method comprising treating the wastewater containing triazole using the strain composition of claim 1 or the microbial preparation of any one of claims 2-4.

6. The method as described in claim 5, characterized in that, The wastewater containing triazole is wastewater containing benzotriazole.

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