A composite bacterial agent for efficiently treating high-salt pharmaceutical and chemical wastewater, and its preparation method and application

Abstract

The present invention relates to the field of microbial technology, and specifically relates to a salt-tolerant and high-temperature-resistant strain PWBX2401 with strong ability to reduce COD concentration, another salt-tolerant and high-temperature-resistant strain BWBX2402 with strong nitrogen removal ability, a composite bacterium agent containing the two strains, a preparation method and an application of the composite bacterium agent. Among them, the preservation number of strain PWBX2401 is: GDMCC No: 64263, and the preservation number of strain BWBX2402 is: GDMCC No: 64264. Research of the present invention shows that the composite bacterium agent prepared by mixing the bacterial solutions of strain PWBX2401 and strain BWBX2402 in a volume ratio of 1:2 can more efficiently treat high-salt pharmaceutical and chemical wastewater. During the process of treating high-salt wastewater, these two kinds of bacteria can produce a synergistic effect and efficiently remove COD, TN, and NH3-N in the high-salt wastewater.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to two salt-resistant and high-temperature-resistant bacterial strains for efficiently treating high-salt pharmaceutical and chemical wastewater, and a composite bacterial agent thereof, a preparation method and application thereof. Background Art

[0002] With the rapid development of the modern pharmaceutical and chemical industry, the discharge of pharmaceutical and chemical wastewater continues to rise. In the production process of pharmaceutical and chemical products, a large amount of wastewater will be generated, which has complex composition and contains a large amount of toxic substances. If it is directly discharged into natural water bodies without proper treatment, it will have a serious negative impact on the environment. Therefore, pharmaceutical and chemical wastewater must be strictly treated to ensure that it meets the prescribed emission standards. Biological treatment has 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, in the process of treating high-salt wastewater, a single strain of bacteria often faces certain limitations and cannot efficiently treat all pollutants. Therefore, in order to improve the treatment efficiency of wastewater, the research on composite bacterial agents is of great significance. Summary of the invention

[0003] In view of the shortcomings of the prior art microbial method for treating high-salinity organic wastewater, the present invention provides two salt-tolerant and high-temperature-tolerant bacteria, and adopts a multi-bacteria compounding method to prepare a salt-tolerant and high-temperature-tolerant composite bacterial agent for efficiently treating high-salinity pharmaceutical and chemical wastewater. By mixing strains with different degradation characteristics, the advantages of each strain can be fully utilized to make up for each other's shortcomings, thereby achieving a better treatment effect. The strain and composite bacterial agent of the present invention can significantly improve the degradation efficiency of high-salinity organic wastewater.

[0004] The first object of the present invention is to provide a salt-tolerant and heat-resistant strain PWBX2401 with a strong ability to reduce COD concentration, and a salt-tolerant and heat-resistant strain BWBX2402 with a strong nitrogen removal ability. Among them, the deposit number of strain PWBX2401 is: GDMCC No: 64263, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 10, 2024, and the classification name is Stutzerimonas balearica. The deposit number of strain BWBX2402 is: GDMCC No: 64264, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 10, 2024, and the classification name is Bacillus paralicheniformis.

[0005] The colony morphology of the strain PWBX2401 provided by the present invention is as follows: after being cultured on an LB plate medium at 37° C. for 24 hours, the colonies are light yellow in color, round in shape, about 1 mm in diameter, raised in the middle, with smooth and neat edges, and a smooth and shiny surface. After 16S rDNA gene sequence detection and identification, the sequence is shown in SEQ ID No. 1.

[0006] The colony morphology of the strain BWBX2402 provided by the present invention is as follows: after being cultured on an LB plate medium at 45° C. for 24 hours, the colonies are white in color, about 3 mm in diameter, umbilical in the middle, irregular in edges, dry, non-sticky and matte on the surface, and identified by 16S rDNA gene sequence detection, the sequence is shown in SEQ ID No. 2.

[0007] The culture medium used for the above strain screening culture is:

[0008] LB solid medium: 1 L ultrapure water, 10 g·L peptone -1 , Yeast powder 5g·L -1 , sodium chloride 10g·L -1 , agar powder 20g·L -1 .

[0009] The second object of the present invention is to provide the use of salt-tolerant and high-temperature-tolerant strains PWBX2401 and strain BWBX2402 in high-salt wastewater treatment, including the use of strain PWBX2401 in removing organic matter from high-salt pharmaceutical and chemical wastewater, and the use of strain BWBX2402 in nitrogen removal treatment of high-salt pharmaceutical and chemical wastewater.

[0010] The third object of the present invention is to provide a composite bacterial agent, which contains active ingredients composed of salt-tolerant and high-temperature-tolerant strains PWBX2401 and strains BWBX2402. Wherein, in the composite bacterial agent, the bacterial liquid of strains PWBX2401 and strains BWBX2402 are mixed in a volume ratio of 1:1 to 1:3. Preferably, in the composite bacterial agent, the bacterial liquid volume ratio of strains PWBX2401 and strains BWBX2402 is 1:2.

[0011] The preparation method of the above-mentioned composite bacterial agent is specifically as follows:

[0012] The strains PWBX2401 and BWBX2402 were inoculated into LB liquid culture medium, respectively, and cultured at 37°C, 200 r / min on a shaking table for 24 h to obtain seed culture liquid; the seed culture liquids of the two strains were then poured into two fermentation medium fermentation tanks at an inoculum volume ratio of 3% for expansion culture, and cultured until OD 600 =1 are mixed in a volume ratio of 1:2 to form a composite bacterial agent, which is sealed and refrigerated to obtain a composite bacterial agent.

[0013] The culture medium used for the above strain cultivation is:

[0014] LB liquid culture medium: 1L ultrapure water, 10g·L peptone -1 , Yeast powder 5g·L -1 , sodium chloride 10g·L -1 .

[0015] The fourth object of the present invention is to provide the use of the above-mentioned composite bacterial agent containing active ingredients composed of strain PWBX2401 and strain BWBX2402 in the treatment of high-salt wastewater, which can effectively reduce the concentration of organic matter (COD), total nitrogen (TN), and ammonia nitrogen (NH3-N) in high-salt pharmaceutical and chemical wastewater.

[0016] The present invention has the following beneficial effects:

[0017] (1) The strain provided by the present invention has strong survival ability in a high-salt environment, fast growth rate, short adaptation period, and long stable period, and can better adapt to the high-salt environment in sewage.

[0018] (2) The strain PWBX2401 provided by the present invention can effectively reduce the COD concentration in high-salt pharmaceutical and chemical wastewater at high temperature, while the strain BWBX2402 has a strong nitrogen removal ability.

[0019] (3) The composite bacterial agent prepared by mixing the bacterial liquid of the strains PWBX2401 and BWBX2402 provided by the present invention at a volume ratio of 1:1 to 1:3 can more efficiently treat high-salt pharmaceutical and chemical wastewater, and achieve a more efficient effect of removing pollutants in the process of treating high-salt wastewater. The prepared composite bacterial agent not only combines the functions of a single strain, but also the two strains can produce a synergistic effect to efficiently remove COD, TN, and NH3-N in high-salt wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a colony morphology diagram of the strain PWBX2401 of the present invention.

[0021] Figure 2 This is a colony morphology diagram of the strain BWBX2402 of the present invention.

[0022] Figure 3 The phylogenetic tree of the strain PWBX2401 of the present invention is shown in FIG.

[0023] Figure 4 The phylogenetic tree of the strain BWBX2402 of the present invention.

[0024] Figure 5 This is a statistical chart of the treatment effects of different bacteria in treating COD, TN, and NH3-N of high-salt pharmaceutical and chemical wastewater during the screening process of an embodiment of the present invention.

[0025] Figure 6 This is a statistical chart of the treatment effects of strains PWBX2401, BWBX2402 and their mixed ratio on COD, TN and NH3-N in high-salt pharmaceutical and chemical wastewater.

[0026] Figure 7 This is a statistical chart of the treatment effects of COD, TN, and NH3-N after adding composite bacterial agents when treating high-salt pharmaceutical and chemical wastewater. DETAILED DESCRIPTION

[0027] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and therefore, the present invention is not limited to the limitations of the specific embodiments disclosed below.

[0028] Example 1: Isolation, screening and compounding of bacterial strains PWBX2401 and BWBX2402

[0029] 1. Enrichment culture:

[0030] The activated sludge sample was taken from the aerobic pool of the pharmaceutical and chemical wastewater treatment system of Jiangxi Wobangxing Technology and Environmental Protection Co., Ltd. and diluted to 10 -3 , absorb 10 -1 , 10 -2 , 10 -3 Add 100 μL of each dilution to LB liquid culture medium, spread each gradient evenly, and then invert and culture at 28°C, 37°C, and 45°C. A single colony will grow in about 36 hours.

[0031] 2. Separation and purification:

[0032] At each temperature, single colonies with different morphological characteristics were selected from the coating plate and inoculated into LB liquid culture medium for 24 h. The streaking was repeated three times or more. If single-morphological bacteria were observed under a microscope, it indicated that the strain had been isolated and purified.

[0033] 3. Screening:

[0034] The purified strains at different temperatures were inoculated into actual high-salt pharmaceutical and chemical wastewater with the same inoculation amount (inoculation amount 3%). After cultivation for a specified period (48h), the COD, TN, and NH3-N of the wastewater were tested. The results are as follows: Figure 5A strain with the highest COD degradation efficiency at high temperature and a strain with the best nitrogen removal effect at high temperature were selected and named PWBX2401 and BWBX2402, respectively, and transferred to LB slant and stored in a 4℃ refrigerator for later use.

[0035] 4. Compounding of bacterial agents:

[0036] The strains PWBX2401 and BWBX2402 obtained by repeated screening were cultured in LB liquid medium at 37°C and 200 r / min for 24 h to obtain seed liquid. The seed liquids of strains PWBX2401 and BWBX2402 were diluted with sterile water to OD 600 =1, and mixed them in the ratio of 1:0, 0:1, 1:1, 1:2, 1:3, 2:1, and 3:1, and inoculated them into high-salt pharmaceutical and chemical wastewater at a volume ratio of 3%. After culturing in a shaker at 45°C and 200r / min for 48h, samples were taken to measure COD, TN, and NH3-N to test the treatment effect. The results are as follows Figure 6 The results showed that when strain PWBX2401 treated high-salinity pharmaceutical and chemical wastewater alone, the removal rates of COD, TN, and NH3-N were 90.85%, 51.97%, and 69.55%, respectively; when strain BWBX2402 treated high-salinity pharmaceutical and chemical wastewater alone, the removal rates of COD, TN, and NH3-N were 71.38%, 80.77%, and 86.30%, respectively. When the mixing ratio of strain PWBX2401 and strain BWBX2402 was 1:1, the COD, TN and NH3-N removal rates were 90.08%, 65.24% and 72.20%, respectively; when the mixing ratio was 1:2, the COD, TN and NH3-N removal rates were 89.13%, 80.56% and 85.47%, respectively; when the mixing ratio was 1:3, 76.51%, 81.47% and 86.84%, respectively; when the mixing ratio was 2:1, the COD, TN and NH3-N removal rates were 89.51%, 48.97% and 64.20%, respectively; when the mixing ratio was 3:1, the COD, TN and NH3-N removal rates were 89.63%, 36.02% and 44.18%, respectively. When the mixing ratio is 1:2, COD, TN and NH3-N in high-salt pharmaceutical and chemical wastewater can be efficiently treated at the same time, and the removal effect is the best. It is finally determined that the most preferred composite bacterial agent is a mixture of two bacterial liquids in a volume ratio of 1:2.

[0037] Example 2: Identification of strains PWBX2401 and BWBX2402

[0038] 1. Colony morphology:

[0039] The colony morphology of strain PWBX2401 is as follows: after culturing on LB plate medium at 37°C for 24 hours, the colony color is light yellow, the colony is round, the diameter is about 1 mm, the middle is raised, the edges are smooth and neat, and the surface is smooth and shiny. Figure 1 shown.

[0040] The colony morphology of strain BWBX2402 is as follows: after being cultured on LB plate medium at 45°C for 24 hours, the colonies are white in color, about 3 mm in diameter, with an umbilical protuberance in the middle and irregular edges, and a dry, non-sticky, and dull surface. Figure 2 shown.

[0041] 2. Molecular biology identification:

[0042] (1) Use an inoculation loop to pick up an appropriate amount of cells from the slant of strain PWBX2401 and strain BWBX2402 and inoculate them into LB liquid culture medium. Cultivate 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 cells.

[0043] (2) The genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit from Solarbio. The specific steps were referred to the instructions in the kit.

[0044] (3) The total DNA mentioned above was subjected to PCR amplification, and bacterial universal primers 27F and 1492R were used for amplification. PCR reaction system 50μL: Premix rTaq 25μL; 27F 1μL; 1492R 1μL; template 1μL; ddH2O2 2μL. Thermal cycle parameters: 95℃ pre-denaturation for 3min, 95℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 60s, 30 cycles, 72℃ extension for 5min. PCR product results were analyzed by 1% agarose gel electrophoresis, and the products were sent to Shanghai Bioengineering for sequencing.

[0045] (4) The amplified sequences were sequenced. The length of the 16S rDNA sequence of strain PWBX2401 was 1435 bp, as shown in SEQ ID No. 1, and the length of the 16S rDNA sequence of strain BWBX2402 was 1332 bp, 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 between strain PWBX2401 and Stutzerimonas balearica (NR_025972.1) was more than 99%, and the homology between strain BWBX2402 and Bacillus paralicheniformis (NR_137421.1) was 100%. Based on the morphological, physiological and biochemical characteristics, homology and phylogenetic analysis of strains PWBX2401 and BWBX2402, strain PWBX2401 was identified as Stutzerimonas balearica. Its phylogenetic tree is shown in the figure. Figure 3 As shown, strain BWBX2402 was identified as Bacillus paralicheniformis, and its phylogenetic tree is shown in Figure 4 shown.

[0046] The strain PWBX2401 was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on January 10, 2024, located at the Microbiological Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, and the deposit number is: GDMCC No: 64263.

[0047] The strain BWBX2402 was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on January 10, 2024, located at the Microbiological Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, and the deposit number is: GDMCC No: 64264.

[0048] The 16S rDNA sequence of strain PWBX2401 (SEQ ID NO.1) is:

[0049]

[0050] The 16S rDNA sequence of strain BWBX2402 (SEQ ID NO. 2) is:

[0051]

[0052] Example 3: Preparation of composite bacterial agent

[0053] The strains PWBX2401 and BWBX2402 were inoculated into LB liquid medium, and cultured at 37°C, 200 r / min on a shaking table for 24 h to obtain seed culture. The seed culture of the two strains was then poured into two fermentation tanks containing fermentation medium at a volume ratio of 3% for expansion culture. The culture was continued until OD 600 =1, the two bacterial liquids are mixed in a volume ratio of 1:2 to prepare a composite bacterial agent, which is sealed and refrigerated to obtain a composite bacterial agent. The fermentation medium is prepared in the following proportions: 1 L of ultrapure water, 10 g·L of peptone -1 , Yeast powder 5g·L -1 , sodium chloride 10g·L -1 , pH is controlled at 6.5-7.5, all ingredients are mixed evenly, and sterilized at 121℃ for 20 minutes.

[0054] Example 4: Performance testing of composite bacterial agents

[0055] Jiangxi Huabang Pharmaceutical Co., Ltd. takes antibiotic series APIs and pharmaceutical intermediates as its leading products. Its main products include meropenem MAP, meropenem side chain, meropenem crude, 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 the pharmaceutical chemical wastewater are shown in Table 1.

[0056] Table 1 Initial parameters of Huabang wastewater

[0057] name pH Salinity (mg / L) COD(mg / L) TN(mg / L) <![CDATA[NH3-N(mg / L)]]> Huabang Wastewater 6.5 18060 18000 780 260

[0058] The composite bacterial agent was inoculated into a 50 ml conical flask containing 20 ml of high-salt wastewater at an inoculum volume of 3%. -3 The high-salt pharmaceutical chemical wastewater from Huabang was cultured in a shaker at 45°C and 200r / min for 48 hours, and then samples were taken to measure COD, TN, and NH3-N to test the treatment effect. The results are as follows Figure 7 shown.

[0059] Depend on Figure 7It can be seen that when strain PWBX2401 treats high-salinity pharmaceutical and chemical wastewater alone, the removal rates of COD, TN, and NH3-N are 91.37%, 55.14%, and 68.21%, respectively. When strain BWBX2402 treats high-salinity 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 bacterial agent treats Huabang high-salinity pharmaceutical and chemical wastewater with a salinity of 6020 mg / L, the removal rates of COD, TN, and NH3-N are 89.25%, 80.52%, and 84.17%, respectively, when the mixing ratio is 1:2, and the removal rates of COD, TN, and NH3-N are 79.27%, 81.47%, and 85.37%, respectively, when the mixing ratio is 1:3. The results show that the single strain PWBX2401 and strain BWBX2402 have preferences (COD or nitrogen) for treating certain pollutants in wastewater, while the composite bacterial agent can more efficiently treat COD, TN, and NH3-N in high-salt wastewater. Compared with fungi, the strain provided by the present invention can survive in a high-salt environment, has a fast growth rate, a short adaptation period, and a long stable period, and can better adapt to the high-salt environment in sewage. The composite bacterial agent can tolerate a high-salt environment. When treating high-concentration organic pollutants and toxic and harmful substances, the COD, TN, and NH3-N removal effects of the composite bacterial agent are relatively ideal, and can be used for high-concentration pharmaceutical and chemical wastewater treatment.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A salt-tolerant and heat-tolerant strain BWBX2402 with nitrogen removal ability, characterized in that: The taxonomic name of strain BWBX2402 is Bacillus paralicheniformis , the deposit number is: GDMCC No: 64264.

2. A composite bacterial agent, characterized in that: The invention comprises the salt-tolerant and heat-tolerant strain PWBX2401 and the strain BWBX2402 as active ingredients; the classification name of the strain PWBX2401 is Stutzerimonas balearica The deposit number of strain PWBX2401 is GDMCC No: 64263; the classification name of strain BWBX2402 is Bacillus paralicheniformis The deposit number of strain BWBX2402 is: GDMCC No: 64264.

3. The composite bacterial agent according to claim 2, characterized in that: In the composite bacterial agent, the volume ratio of the bacterial solution of strain PWBX2401 to the bacterial solution of strain BWBX2402 is 1:1-1:

3.

4. The composite bacterial agent according to claim 3, characterized in that: In the composite bacterial agent, the volume ratio of the bacterial solution of strain PWBX2401 to the bacterial solution of strain BWBX2402 is 1:

2.

5. A method for preparing the composite bacterial agent according to claim 4, characterized in that: The following steps are involved: The strains PWBX2401 and BWBX2402 were inoculated into LB liquid culture medium, respectively, and cultured at 37°C, 200 r / min on a shaking table for 24 h to obtain seed culture liquid. The seed culture liquids of the two strains were then poured into two fermentation medium fermentation tanks at an inoculation rate of 3% by volume for expansion culture. The culture was continued until OD 600 =1 are mixed in a volume ratio of 1:2 to form a composite bacterial agent, which is sealed and refrigerated to obtain a composite bacterial agent.

6. Use of the strain BWBX2402 according to claim 1 in treating COD, total nitrogen and NH3-N in wastewater.

7. Use of the composite bacterial agent according to any one of claims 2 to 4 in treating COD, total nitrogen and NH3-N in wastewater.

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