Bacterial strain for efficiently treating high-salt pharmaceutical and chemical wastewater and application of bacterial strain

Abstract

The invention is a divisional application, and the application number of the original application is 202410843780X. The invention relates to the technical field of microorganisms, in particular to a salt-resistant and high-temperature-resistant strain PWBX2401 with high COD (Chemical Oxygen Demand) concentration reducing capacity and application thereof. Wherein the classification name of the bacterial strain PWBX2401 is Stutzerimonas balearica, and the preservation number of the bacterial strain PWBX2401 is GDMCC No: 64263. The research shows that the bacterial strain PWBX2401 can efficiently remove COD (Chemical Oxygen Demand), TN (Total Nitrogen) and NH3-N in the high-salinity wastewater.

Description

[0001] This application is a divisional application. The application number of the original application is 202410843780X, the application date is June 27, 2024, and the invention title is a composite bacterium agent for efficiently treating high-salt pharmaceutical and chemical wastewater, its preparation method and application. Technical Field

[0002] The present invention belongs to the field of microbial technology, and specifically relates to a strain for efficiently treating high-salt pharmaceutical and chemical wastewater and its application. Background Art

[0003] With the rapid development of the modern pharmaceutical and chemical industry, the discharge of pharmaceutical and chemical wastewater has been continuously increasing. During the production process of pharmaceutical and chemical products, a large amount of wastewater is generated, with complex components and containing a large number of toxic substances. If directly discharged into natural water bodies without proper treatment, it will cause serious negative impacts on the environment. Therefore, strict treatment of pharmaceutical and chemical wastewater must be carried out to ensure that it meets the specified discharge standards. Biological treatment methods have been widely used in various industries. However, for general biological treatment processes, high-salt and high-temperature environments will inhibit the metabolic functions of many microorganisms, thereby reducing the removal efficiency of pollutants. In addition, during the treatment of high-salt wastewater, a single strain often faces certain limitations and cannot efficiently treat all pollutants. Therefore, in order to improve the treatment efficiency of wastewater, the research on composite bacterium agents is of great significance. Summary of the Invention

[0004] Aiming at the deficiencies in the treatment of high-salt organic wastewater by the microbial method in the prior art, the present invention provides two salt-tolerant and high-temperature-resistant strains, and adopts a method of multi-strain compounding to prepare a composite bacterium agent for efficiently treating high-salt pharmaceutical and chemical wastewater that is salt-tolerant and high-temperature-resistant. By mixing strains with different degradation characteristics, the advantages of each strain can be fully utilized to make up for each other's deficiencies, thereby achieving a better treatment effect. Using the strains and composite bacterium agent of the present invention can significantly improve the degradation efficiency of high-salt organic wastewater.

[0005] The first object of the present invention is to provide a salt-tolerant and high-temperature-resistant strain PWBX2401 with strong ability to reduce the COD concentration, and a salt-tolerant and high-temperature-resistant strain BWBX2402 with strong nitrogen removal ability. Among them, the preservation number of strain PWBX2401 is: GDMCC No: 64263, and it was preserved in the Guangdong Provincial Microbial Culture Collection Center on January 10, 2024, and the classification name is Stutzerimonas balearica. The preservation number of strain BWBX2402 is: GDMCC No: 64264, and it was preserved in the Guangdong Provincial Microbial Culture Collection Center on January 10, 2024, and the classification name is Bacillus paralicheniformis.

[0006] The colony morphology of the strain PWBX2401 provided by the present invention is as follows: cultured on an LB plate medium at 37°C for 24 h, the colony color is light yellow, the colony is round, with a diameter of about 1 mm, raised in the middle, the edge is smooth and regular, the surface is smooth and shiny. After being identified by 16S rDNA gene sequence detection, the sequence is as shown in SEQ ID No.1.

[0007] The colony morphology of the strain BWBX2402 provided by the present invention is as follows: cultured on an LB plate medium at 45°C for 24 h, the colony color is white, the diameter is about 3 mm, umbilicate in the middle, the edge is irregular, the surface is dry, not sticky, and has no luster. After being identified by 16S rDNA gene sequence detection, the sequence is as shown in SEQ ID No.2.

[0008] The medium used for screening and culturing the above strains is as follows:

[0009] LB solid medium: 1 L of ultrapure water, 10 g·L of peptone -1 , 5 g·L of yeast extract -1 , 10 g·L of sodium chloride -1 , 20 g·L of agar powder -1 .

[0010] The second object of the present invention is to provide the application of the salt-tolerant and high-temperature-resistant strains PWBX2401 and BWBX2402 in the treatment of high-salt wastewater, including the application of the strain PWBX2401 in removing organic matter from high-salt pharmaceutical and chemical wastewater, and the application of the strain BWBX2402 in nitrogen removal treatment of high-salt pharmaceutical and chemical wastewater.

[0011] The third object of the present invention is to provide a composite bactericide, and the composite bactericide contains an active ingredient composed of the salt-tolerant and high-temperature-resistant strains PWBX2401 and BWBX2402. Among them, in the composite bactericide, the bacterial liquids of the strain PWBX2401 and the strain BWBX2402 are mixed at a volume ratio of 1:1 to 1:3. Preferably, in the composite bactericide, the volume ratio of the bacterial liquids of the strain PWBX2401 and the strain BWBX2402 is 1:2.

[0012] The specific preparation method of the above composite bactericide is as follows:

[0013] The strains PWBX2401 and BWBX2402 are respectively inoculated into an LB liquid medium, and cultured on a shaker at 37°C and 200 r / min for 24 h to obtain seed bacterial liquids; then the seed bacterial liquids of the two strains are respectively poured into the fermenters of two fermentation media for scale-up culture at an inoculation amount of 3% by volume, and the two bacterial liquids cultured to OD 600 = 1 are mixed and configured into a composite bactericide according to a volume ratio of 1:2, sealed and refrigerated to obtain the composite bactericide.

[0014] The culture medium used for culturing the above-mentioned strains is as follows:

[0015] LB liquid medium: 1 L of ultrapure water, 10 g·L of peptone -1 , 5 g·L of yeast extract -1 , 10 g·L of sodium chloride -1 .

[0016] The fourth object of the present invention is to provide the application of the above-mentioned composite bactericide containing the active ingredient composed of strain PWBX2401 and strain BWBX2402 in treating high-salt wastewater, which can effectively reduce the concentrations of organic matter (COD), total nitrogen (TN), and ammonia nitrogen (NH3-N) in high-salt pharmaceutical and chemical wastewater.

[0017] The present invention has the following beneficial effects:

[0018] (1) The strains provided by the present invention have strong viability in a high-salt environment, fast growth rate, short adaptation period, and long stationary phase, and can better adapt to the high-salt environment in sewage.

[0019] (2) The strain PWBX2401 provided by the present invention can effectively reduce the concentration of COD in high-salt pharmaceutical and chemical wastewater at high temperature, while the strain BWBX2402 has strong nitrogen removal ability.

[0020] (3) The composite bactericide prepared by mixing the bacterial solutions of the strains PWBX2401 and BWBX2402 provided by the present invention in a volume ratio of 1:1 to 1:3 can treat high-salt pharmaceutical and chemical wastewater more efficiently, and achieve a more efficient effect of removing pollutants during the process of treating high-salt wastewater. The prepared composite bactericide not only combines the functions of individual strains, but also the two strains can produce a synergistic effect to efficiently remove COD, TN, and NH3-N in high-salt wastewater. Description of the Drawings

[0021] Figure 1 It is the colony morphology diagram of the strain PWBX2401 of the present invention.

[0022] Figure 2 It is the colony morphology diagram of the strain BWBX2402 of the present invention.

[0023] Figure 3 It is the phylogenetic evolution tree of the strain PWBX2401 of the present invention.

[0024] Figure 4 It is the phylogenetic evolution tree of the strain BWBX2402 of the present invention.

[0025] Figure 5 It is the statistical chart of the treatment effects of different bacteria on COD, TN, and NH3-N in treating high-salt pharmaceutical and chemical wastewater during the screening process of the embodiment of the present invention.

[0026] Figure 6 Statistical chart of the treatment effects of COD, TN, and NH₃-N in high-salt pharmaceutical and chemical wastewater by strains PWBX2401 and BWBX2402 and their mixing ratios.

[0027] Figure 7 Statistical chart of the treatment effects of COD, TN, and NH₃-N after adding a composite bacterial agent when treating high-salt pharmaceutical and chemical wastewater. Detailed implementation manners

[0028] In order to more clearly understand the above objects, features, and advantages of the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners. Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Therefore, the present invention is not limited by the limitations of the specific embodiments disclosed below.

[0029] Example 1: Isolation, screening of strains PWBX2401 and BWBX2402, and compounding of a composite bacterial agent

[0030] 1. Enrichment culture:

[0031] Take an activated sludge sample from the aerobic tank of the pharmaceutical and chemical sewage treatment system of Jiangxi Wobangxing Technology Environmental Protection Co., Ltd., and dilute the sample to 10 -3 using the gradient dilution method. Then, respectively pipette 100 μL of the 10 -1 , 10 -2 , and 10 -3 dilutions into LB liquid medium. After evenly coating each gradient, incubate it at 28 °C, 37 °C, and 45 °C for about 36 h until single colonies grow.

[0032] 2. Isolation and purification:

[0033] At each temperature, select single colonies with different morphological characteristics on the coated plate and transfer them to LB liquid medium for culturing for 24 h. Repeat streaking three times or more. If the bacteria with a single morphology are observed under the microscope, it indicates that the strain has been isolated and purified.

[0034] 3. Screening:

[0035] Inoculate the purified strains at different temperatures with the same inoculation amount (inoculation amount 3%) into the actual high-salt pharmaceutical and chemical wastewater, and detect the COD, TN, and NH₃-N of the wastewater after culturing according to the specified cycle (48 h). The results are as Figure 5As shown in the figure. Select a strain with the highest COD degradation efficiency at high temperature and a strain with the best nitrogen removal effect at high temperature, named PWBX2401 and BWBX2402 respectively, and transfer them to an LB slant and store them in a 4°C refrigerator for standby.

[0036] 4. Compound of microbial agents:

[0037] Cultivate the screened strains PWBX2401 and BWBX2402 in LB liquid medium at 37°C and 200 r / min for 24 h to obtain seed solutions. Dilute the seed solutions of strains PWBX2401 and BWBX2402 with sterile water to OD 600 = 1, mix them respectively according to the inoculation ratios of 1:0, 0:1, 1:1, 1:2, 1:3, 2:1, 3:1, and inoculate them into high-salt pharmaceutical and chemical wastewater at an inoculation volume ratio of 3%. After culturing in a shaker at 45°C and 200 r / min for 48 h, sample to measure COD, TN, and NH3-N to test the treatment effect. The results are as Figure 6 shown. The results show that when strain PWBX2401 is used to treat high-salt pharmaceutical and chemical wastewater alone, the removal rates of COD, TN, and NH3-N are 90.85%, 51.97%, and 69.55% respectively. When strain BWBX2402 is used to treat high-salt pharmaceutical and chemical wastewater alone, the removal rates of COD, TN, and NH3-N are 71.38%, 80.77%, and 86.30% respectively. When the mixing ratio of strain PWBX2401 and strain BWBX2402 is 1:1, the removal rates of COD, TN, and NH3-N are 90.08%, 65.24%, and 72.20% respectively; when the mixing ratio is 1:2, the removal rates of COD, TN, and NH3-N are 89.13%, 80.56%, and 85.47% respectively; when the mixing ratio is 1:3, they are 76.51%, 81.47%, and 86.84% respectively; when the mixing ratio is 2:1, the removal rates of COD, TN, and NH3-N are 89.51%, 48.97%, and 64.20% respectively; when the mixing ratio is 3:1, the removal rates of COD, TN, and NH3-N are 89.63%, 36.02%, and 44.18% respectively. When the mixing ratio is 1:2, it can efficiently treat COD, TN, and NH3-N in high-salt pharmaceutical and chemical wastewater simultaneously, and the removal effect is the best. Finally, it is determined that the most preferred composite microbial agent is prepared by mixing two kinds of bacterial solutions according to a volume ratio of 1:2.

[0038] Example 2: Identification of strains PWBX2401 and BWBX2402

[0039] 1. Colony morphology:

[0040] The colony morphology of strain PWBX2401 is as follows: cultured on an LB plate medium at 37°C for 24 h, the colony color is light yellow, the colony is round, about 1 mm in diameter, bulges in the middle, with a smooth and regular edge, and the surface is smooth and shiny, as Figure 1 shown.

[0041] The colony morphology of strain BWBX2402 is as follows: cultured on an LB plate medium at 45°C for 24 h, the colony color is white, about 3 mm in diameter, umbilicate in the middle, with an irregular edge, and the surface is dry, not sticky, and has no luster, as Figure 2 shown.

[0042] 2. Molecular biological identification:

[0043] (1) Use an inoculation loop to pick an appropriate amount of bacteria from the slants of strain PWBX2401 and strain BWBX2402 and inoculate them into an LB liquid medium. Culture at 37°C and 200 r / min for 24 h. Take 2 - 3 mL of the fermentation broth and centrifuge at 10,000 rpm for 1 min to collect the bacteria.

[0044] (2) Use a bacterial genomic DNA extraction kit from Solarbio to extract the genomic DNA of the strain. The specific steps refer to the instructions in the kit.

[0045] (3) Perform PCR amplification on the total DNA mentioned above. Select the universal bacterial primers 27F and 1492R for amplification. The PCR reaction system is 50 μL: Premix rTaq 25 μL; 27F 1 μL; 1492R 1 μL; template 1 μL; ddH2O2 22 μL. The thermal cycling parameters are pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, 30 cycles, and extension at 72°C for 5 min. Analyze the PCR product results by 1% agarose gel electrophoresis, and then send the product to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0046] (4) The sequences obtained by amplification were sequenced. The length of the 16S rDNA sequence of strain PWBX2401 was 1435 bp, and the sequence was as shown in SEQ ID No.1. The length of the 16S rDNA sequence of strain BWBX2402 was 1332 bp, and the sequence was as shown in SEQ ID No.2. The gene sequences obtained by sequencing were submitted to NCBI, and homology comparison was performed using Blast. Multiple sequence alignment analysis was performed using MEGA 6.0 software, and a phylogenetic tree was constructed using the Neighbor-Joining method. The results showed that the homology of strain PWBX2401 with Stutzerimonas balearica (NR_025972.1) was more than 99%, and the homology of strain BWBX2402 with Bacillus paralicheniformis (NR_137421.1) was 100%. Based on the morphological characteristics, physiological and biochemical characteristics, homology, and phylogenetic analysis of strain PWBX2401 and strain BWBX2402, strain PWBX2401 was identified as Stutzerimonas balearica, and its phylogenetic tree was as shown in Figure 3 shown. Strain BWBX2402 was identified as Bacillus paralicheniformis, and its phylogenetic tree was as shown in Figure 4 shown.

[0047] Strain PWBX2401 was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on January 10, 2024. The address is the Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Guangdong Province, with a postal code of 510070 and a deposit number of: GDMCC No: 64263.

[0048] Strain BWBX2402 was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on January 10, 2024. The address is the Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Guangdong Province, with a postal code of 510070 and a deposit number of: GDMCC No: 64264.

[0049] The 16S rDNA sequence (SEQ ID NO.1) of strain PWBX2401 is:

[0050]

[0051] The 16S rDNA sequence of strain BWBX2402 (SEQ ID NO.2) is as follows:

[0052]

[0053] Example 3: Preparation of Compound Bacterial Agent

[0054] The strains PWBX2401 and BWBX2402 were respectively inoculated into LB liquid medium and cultured on a shaker at 37 °C and 200 r / min for 24 h to obtain seed bacterial solutions. Then, the seed bacterial solutions of the two strains were poured into the fermenters of two fermentation media respectively for scale-up culture according to the inoculation amount of 3% by volume. The two bacterial solutions cultured to OD 600 = 1 were mixed and configured into a compound bacterial agent according to the volume ratio of 1:2, sealed and refrigerated to obtain the compound bacterial agent. The fermentation medium was configured according to the following ratio: 1 L of ultrapure water, 10 g·L -1 of peptone, 5 g·L -1 of yeast powder, 10 g·L -1 of sodium chloride, the pH was controlled at 6.5 - 7.5. Each component was mixed evenly and sterilized at 121 °C for 20 min.

[0055] Example 4: Performance Detection of Compound Bacterial Agent

[0056] Jiangxi Huabang Pharmaceutical Co., Ltd. mainly produces a series of antibiotic raw materials and pharmaceutical intermediates. Currently, its main products include meropenem MAP, meropenem side chain, crude meropenem, sulbactam acid, tazobactam acid, tazobactam acid, etc. A large amount of production wastewater is generated during the production process. The wastewater contains a large amount of organic pollutants and toxic and harmful substances. Some parameters such as the initial COD value of this pharmaceutical chemical wastewater are shown in Table 1.

[0057] Table 1 Initial Parameters of Huabang Wastewater

[0058] Name pH Salinity (mg / L) COD (mg / L) TN (mg / L) <![CDATA[NH3-N (mg / L)]]> Huabang Wastewater 6.5 18060 18000 780 260

[0059] The compound bacterial agent was inoculated into a 50 ml conical flask containing 20 ml of high-salt wastewater at an inoculation amount of 3%. In this example, the diluted 10 -3 times Huabang high-salt pharmaceutical chemical wastewater was selected. After culturing in a shaker at 45 °C and 200 r / min for 48 h, samples were taken to measure COD, TN, and NH3-N to test the treatment effect. The results are as Figure 7 shown.

[0060] From Figure 7It can be seen that when the strain PWBX2401 is used to treat high-salt pharmaceutical and chemical wastewater alone, the removal rates of COD, TN, and NH3-N are 91.37%, 55.14%, and 68.21% respectively. When the strain BWBX2402 is used to treat high-salt pharmaceutical and chemical wastewater alone, the removal rates of COD, TN, and NH3-N are 68.83%, 83.28%, and 88.42% respectively. When the composite bacterium agent is used to treat Huabang high-salt pharmaceutical and chemical wastewater with a salinity of 6020 mg / L and the mixing ratio is 1:2, the removal rates of COD, TN, and NH3-N are 89.25%, 80.52%, and 84.17% respectively. When the mixing ratio is 1:3, the removal rates of COD, TN, and NH3-N are 79.27%, 81.47%, and 85.37% respectively. The results show that the single strains PWBX2401 and BWBX2402 have preferences for certain pollutants (COD or nitrogen) in the wastewater, while the composite bacterium agent can more efficiently take into account the treatment of COD, TN, and NH3-N in high-salt wastewater. The strains provided by the present invention can survive in a high-salt environment compared with fungi, have a fast growth rate, a short adaptation period, and a long stationary phase, and can better adapt to the high-salt environment in sewage. The composite bacterium agent can tolerate a high-salt environment, and when treating high-concentration organic pollutants and toxic and harmful substances, the removal effects of COD, TN, and NH3-N of the composite bacterium agent are all relatively ideal, and it can be used for the treatment of high-concentration pharmaceutical and chemical wastewater.

[0061] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A salt-tolerant and high-temperature-resistant strain PWBX2401 with the ability to reduce COD concentration, characterized in that, The taxonomic name of strain PWBX2401 is Stutzerimonas balearica , and the deposit number is: GDMCC No: 64263.

2. Use of strain PWBX2401 according to claim 1 in treating COD, total nitrogen, and NH3-N in wastewater.

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