Method for domesticating functional microbiota that degrade phthalate esters
The domestication and application of functional microbiota using activated sludge and inorganic salts efficiently decompose phthalate esters, addressing environmental contamination and health risks by achieving high decomposition rates in both laboratory and field conditions.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- NANJING AGRICULTURAL UNIVERSITY
- Filing Date
- 2024-11-14
- Publication Date
- 2026-07-02
AI Technical Summary
Phthalic acid esters (PAEs) are widely used in plastic products, leading to environmental contamination and human health risks, and existing methods are inadequate for effectively decomposing these harmful substances.
A method involving the domestication of functional microbiota that degrade phthalate esters, using activated sludge and inorganic salts to cultivate bacteria capable of breaking down PAEs, followed by a gradient domestication process and mixing with glycerol for preservation, and applying the bacterial suspension to contaminated soil and crops.
The method achieves nearly complete decomposition of PAEs, with rates exceeding 95% in laboratory settings and 85% in actual soil applications, effectively reducing environmental and health hazards.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbial treatment, specifically to a method for domesticating functional bacteria clusters that decompose phthalic acid esters.
Background Art
[0002] Phthalic acid esters (PAEs) are widely used in the production of plastic products to enhance plasticity, softness and multifunctionality. PAEs are non-chemically bonded to polymers and are likely to be released during the production, storage and disposal of plastic products. Through the process of biogeochemical cycles, PAEs further migrate into environmental media such as soil, sediment, water and atmosphere, and are widely detected as environmental hormone pollutants. Among them, six types of PAEs compounds such as dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP) are classified as priority treatment pollutants and endocrine disruptors. PAEs may enter the human body through food intake, inhalation of particulate matter, and percutaneous contact. Related human exposure risk studies have shown that soil ingestion and vegetable ingestion are the main exposure routes of PAEs. Higher concentrations of PAEs have been detected in greenhouse vegetables using plastic films in more regions. PAEs are recognized as toxic and harmful organic substances and potential carcinogens because they have an adverse effect on the endocrine [[ID=3⑨]] system, red blood cell function, and reproductive system of organisms, and act as estrogen [ hormones in the human body, which may lead to the onset of testicular dysgenesis syndrome in men. It has also been found to be harmful to pulsating heart disease, arteriosclerosis, and blood pressure problems. Therefore, Developing effective methods to control the risk of PAE contamination is a matter of urgency. Yes, they are. [Overview of the project]
[0003] To solve the above problems, the present invention relates to the domestication of functional microbiota that degrade phthalate esters. We provide a method, which includes the following steps: S1, S bacterial flora concentration and domestication: 1-1.5 parts activated sludge and 2-3 parts inorganic salt by weight. Place in a culture medium, add the PAEs mixed solution to the inorganic salt medium, and the quality of PAEs in the inorganic salt medium The concentration was adjusted to 6-14 mg / L, and domestication culture was carried out for 3 weeks under room temperature conditions while continuing to stir. , obtain a primary bacterial suspension, S2, S flora gradient domestication: S2-1, Transfer of the primary bacterial suspension: Take 1 to 1.5 parts of the primary bacterial suspension, and 2 to Place 3 parts of inorganic salt medium into the inorganic salt medium, add the PAEs mixed solution to the inorganic salt medium, and stir under room temperature conditions. Continue stirring and culture for 3 weeks to obtain a secondary bacterial suspension. Repeat the transfer operation in step S2-1 five times, and after each transfer, in the inorganic salt medium. The mass concentrations of PAEs were 40 mg / L, 60 mg / L, 80 mg / L, and 100 mg / L, respectively. Adjust the concentration to g / L or 120 mg / L, and continue domestication culture for 3 weeks after the final transfer is complete. Then, obtain a bacterial suspension containing the S flora six times, take 1 to 1.5 parts of the above bacterial suspension six times, and Mix with glycerol at a fraction of 30-40% in a 1:1 volume ratio, store under ultra-low temperature conditions, and store S bacteria. Prepare freeze-dried glycerol containing the stalks, Preparation of bacterial suspension of S3, S flora: Freeze-dried glycerin containing the S flora obtained in step S2 Scrap off 1 to 1.5 parts of the roll with an inoculation ring and inoculate it into 25 to 30 parts of sterilized LB medium. Then, under room temperature conditions, continue stirring and perform activation culture for 24 hours to obtain a pre-activated bacterial suspension. The pre-activated bacterial suspension was placed in a centrifuge and subjected to centrifugation. The supernatant was discarded to obtain the bacterial suspension. The bacterial suspension was washed with an inorganic salt solution, and the centrifugation and inorganic salt solution washing steps were repeated three times. Next, add 18-20 parts of the bacterial suspension to the inorganic salt medium and adjust the OD600 value of the bacterial suspension to 1.0. Allow to stand for approximately 3 hours to obtain a bacterial suspension of the S flora, and store it under low temperature conditions to prepare. Furthermore, the activated sludge was collected from the secondary sedimentation tank of a sewage treatment plant. Description: The activated sludge in the secondary sedimentation tank of the domestic wastewater treatment plant contains a large amount of microorganisms, including PAEs. By adding this, the bacteria can be domesticated and a decomposing microorganism can be obtained. Furthermore, steps S4-S7 are also included: S4, Acquisition of PAEs-contaminated crops: Take PAEs-contaminated soil and prepare it in step S3. A bacterial suspension of the S flora was added, and the mass ratio of the PAEs-contaminated soil to the bacterial suspension of the S flora was 10. The ratio was 0:1, and the PAEs-contaminated soil and the bacterial suspension of the S microbiota were uniformly mixed and left to stand for 48 hours. Afterward, planting of plants began in the PAEs-contaminated soil after purification, and after one cultivation cycle, the PAEs contamination... Harvest the dyed crops, S5, Y microbial community concentration and domestication: PAEs-contaminated crops are sun-dried and then pulverized to 200 meter size. The PAEs-contaminated crop pulverized powder is obtained by passing it through a sieve, and 1 to 1.5 parts of the PAEs-contaminated crop pulverized powder are used. Add the end to 2-3 parts of inorganic salt medium, add the PAEs mixed solution to the inorganic salt medium, and the inorganic salt Adjust the mass concentration of PAEs in the culture medium to 6-14 mg / L, and continue stirring under room temperature conditions for 3 Domestication culture is performed for a week to obtain a primary strengthened bacterial suspension, and the primary strengthened bacterial suspension is used in step S Transfer according to the transfer method in 2, repeat the transfer 5 times, and perform 6-fold enhancement including the Y bacterial flora Obtain a bacterial suspension, mix it with glycerol at a volume fraction of 30% at a volume ratio of 1:1, and under ultra-low temperature conditions Store to obtain and prepare freeze-dried glycerol containing the Y bacterial flora S6. Preparation of the bacterial suspension of the Y bacterial flora: Scrape 1 to 1.5 parts of the freeze-dried glycerol containing the Y bacterial flora obtained in step S5 with an inoculation loop and inoculate it into 25 to 30 parts of sterilized LB medium , continue stirring under room temperature conditions for 24 h for activation culture to obtain a pre-activated enhanced bacterial suspension , put the pre-activated enhanced bacterial suspension into a centrifuge for centrifugation treatment, discard the supernatant to obtain an enhanced bacterial solution, wash the enhanced bacterial solution with an inorganic salt solution, and repeat the centrifugation and inorganic salt solution washing steps 3 times, then put the enhanced bacterial solution into 18 to 20 parts of inorganic salt medium, adjust the OD60 0 value of the enhanced bacterial solution to 1.0, let it stand for 2 to 3 h to obtain a bacterial suspension of the Y bacterial flora, and store it under low temperature conditions to prepare S7. Mixing: Mix the bacterial suspension of the S bacterial flora obtained in step S3 and the bacterial suspension of the Y bacterial flora obtained in step S6 at a mass ratio of 3:1 to obtain a bacterial suspension of the enhanced functional bacterial flora. Explanation: Use the bacterial suspension of the S bacterial flora obtained by preparation for soil treatment, plant plants in the soil, and then Recover the plants and prepare the bacterial suspension again to obtain a bacterial suspension of the Y bacterial flora that is functionally complementary to the bacterial suspension of the S bacterial flora. Mix the two bacterial suspensions at a certain ratio to further improve the decomposition effect of various PAEs pollutions in the soil. Furthermore, the plant is one or more of water spinach, Shanghai green-stemmed cabbage and lettuce . Explanation: The above plants are all common plants, easy to cultivate and recover, and the cultivation cycle The period is short, and the prepared bacterial suspension is effective for the treatment of PAEs in soil. Furthermore, the components and mass concentrations of the inorganic salt medium are as follows: 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HP O4·3H2O, 0.5 g / L of NaCl, 0.2 g / L of MgSO4·7H2O, 1. 5 g / L of agar powder, the solvent is ultrapure water, the pH of the inorganic salt medium is 7. , and the components and mass concentrations of the inorganic salt solution are as follows: 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HPO4·3H2O, 0.5 g / L of NaCl, 0.2 g / L of MgSO4·7H2O, the solvent is ultrapure water, and the pH of the inorganic salt solution is 7.0. Explanation: The inorganic salt medium with the above components and ratios has the advantages of being simple and low-cost to obtain, and can culture the bacterial suspension. Furthermore, the mass ratio of phthalic acid ester DMP, diethyl phthalate DE P, and dibutyl phthalate DBP in the PAEs mixed solution is 1:1: <0001. Explanation: By combining the above three PAEs and growing and <000Agar powder, ultrapure water as solvent, pH of LB medium is 7.0, and the sterilization temperature is 120-125°C. The sterilization time is 20-30 minutes. Furthermore, the centrifugal rotation speed of the centrifugal separation process is 5000 rpm, and the centrifugal separation process The time is 5 minutes. [Effects of the Invention]
[0004] Compared to prior art, the present invention has the following beneficial effects. This invention reuses activated sludge and includes dimethyl phthalate, diethyl phthalate, and dibutyrate. They are raised and propagated using the lizard as their sole carbon source and energy source, and then domesticated using a gradient method in an inorganic salt medium. Activation in LB medium was carried out sequentially to obtain a bacterial suspension of the S community, and this community was fermented for 36 hours. Within an initial concentration of 10 mg / L of mixed PAEs (10 mg / L each of DMP and DEP) It can almost completely decompose (including DBP), with a decomposition rate exceeding 95% for all of them. The application of this technology to the biodegradation of hazardous organic pollutants holds great potential. This invention involves using a bacterial suspension of the S microbiota for soil treatment of PAEs-contaminated soil, followed by planting plants in the soil. Subsequently, the plants were collected and a bacterial suspension was prepared again, and a bacterial suspension functionally complementary to the S flora was prepared. A bacterial suspension of the Y flora can be obtained, and by mixing the two bacterial suspensions in a certain ratio... This further enhances the decomposition of various PAEs contaminations in soil, thereby improving bacterial suspension To further enhance its practicality, it is no longer limited to the laboratory testing stage, but is used in actual soil treatment and secondary processes. By combining treatments, such as in-situ soil treatment and off-site soil treatment, hazardous organic pollutants can be removed. This can further improve the processing effectiveness. [Brief explanation of the drawing]
[0005] [Figure 1]This is a flowchart of the method for domesticating a functional microbiota that degrades phthalate esters, according to the present invention. [Figure 2] This is a scanning electron microscope image of the bacterial suspension of the S microbiota in Experimental Example 1 of the present invention, magnified 5000 times. [Figure 3] This is a scanning electron microscope image of the bacterial suspension of the S microbiota in Experimental Example 1 of the present invention, magnified 20,000 times. [Figure 4] This is a schematic diagram of the bacterial community structure composition of the commensal bacterial community S in the bacterial suspension of the S bacterial community in experimental example 1 of the present invention. [Figure 5] This diagram shows the degradation effect of bacterial flora S on PAEs in an inorganic salt medium in Experimental Example 1 of the present invention. [Figure 6] This diagram shows the decomposition effects of S microbial community and S microbial community + Y microbial community on three types of PAEs in actual soil in Experimental Example 2 of the present invention. [Modes for carrying out the invention]
[0006] To further explain the embodiments adopted in this invention and the effects achieved, the following experiments and Furthermore, the technical solutions of the present invention will be clearly and completely explained. Example 1: This example describes a method for domesticating functional microbiota that degrades phthalate esters. As shown in Figure 1, the following steps are included: S1, S bacterial flora concentration and domestication: In parts by weight, 1 part activated sludge is placed in 2 parts inorganic salt medium, The activated sludge was collected from the secondary sedimentation tank of a sewage treatment plant and mixed with PAEs in an inorganic salt culture medium. The solution was added to bring the mass concentration of PAEs in the inorganic salt medium to 10 mg / L, under conditions of 30°C. Maintain a domesticated culture for 3 weeks while continuing to stir to obtain a primary bacterial suspension. S2, S flora gradient domestication: S2-1, Transfer of primary bacterial suspension: Take 1 part of the primary bacterial suspension and transfer 2 parts of inorganic salt medium. Place in a container, add the PAEs mixed solution to the inorganic salt medium, and continue stirring under 30°C conditions for 3 weeks. Inter-bacterial culture was performed to obtain a secondary bacterial suspension. Repeat the transfer operation in step S2-1 five times, and after each transfer, in the inorganic salt medium. The mass concentrations of PAEs were 40 mg / L, 60 mg / L, 80 mg / L, and 100 mg / L, respectively. Adjust the concentration to g / L or 120 mg / L, and continue domestication culture for 3 weeks after the final transfer is complete. Then, a bacterial suspension containing the S flora was obtained, and a portion of the bacterial suspension was taken, and a volume fraction of 30% was obtained. Mix with glycerol in a 1:1 volume ratio, store at -80°C, and freeze-dry to preserve the S-microbe colony. Prepare glycerol, Phthalate esters DMP, diethyl phthalate DEP, and diethyl phthalate in PAEs mixed solution The mass ratio of Chill DBP is 1:1:1. Preparation of bacterial suspension of S3, S flora: Freeze-dried glycerin containing the S flora obtained in step S2 A portion of the roll was scraped off with an inoculation ring and inoculated into 28 parts of 121°C sterilized LB medium. The bacterial time was 25 minutes, and activation culture was performed for 24 hours under 30°C conditions with continuous stirring. An activated bacterial suspension is obtained, and the pre-activated bacterial suspension is placed in a centrifuge and subjected to centrifugation. The centrifugal separation process uses a rotation speed of 5000 rpm and a centrifugal separation time of 5 minutes. The supernatant is discarded to obtain the bacterial suspension, which is then washed with an inorganic salt solution, followed by centrifugation and washing with the inorganic salt solution. After repeating the step three times, add 20 parts of the bacterial suspension to an inorganic salt medium and measure the OD600 value of the bacterial suspension. The solution was adjusted to 1.0, allowed to stand for 2 hours to obtain a bacterial suspension of the S flora, and then stored at 4°C to prepare the solution. . Here, the components and mass concentrations of the inorganic salt medium are as follows: 1.5 g / L (NH4)2SO4, 0.5g / L of KH2PO4, 1.91g / L of K2HPO4·3 H2O, 0.5 g / L NaCl, 0.2 g / L MgSO4·7H2O, 1.5 g / L The agar powder, with ultrapure water as the solvent and an inorganic salt medium with a pH of 7.0, The components and mass concentrations of the inorganic salt solution are as follows: 1.5 g / L of (NH4 )2SO4, 0.5g / L KH2PO4, 1.91g / L K2HPO4·3H2O, The solution consists of 0.5 g / L NaCl, 0.2 g / L MgSO4·7H2O, and ultrapure water as the solvent. The pH of the inorganic salt solution is 7.0. The stirring speed is 150 rpm. The components and mass concentrations of LB medium are as follows: 5 g / L yeast extract, 1 0 g / L tryptone, 10 g / L sodium chloride, 1.5 g / L agar powder, solvent is super It is pure water, and the pH of the LB medium is 7.0. Example 2: This example describes a method for domesticating functional microbiota that degrades phthalate esters. The method further includes steps S4-S7, following step S3 of Example 1: S4, Acquisition of PAEs-contaminated crops: Take PAEs-contaminated soil and prepare it in step S3. A bacterial suspension of the S flora was added, and the mass ratio of the PAEs-contaminated soil to the bacterial suspension of the S flora was 10. The ratio was 0:1, and the PAEs-contaminated soil and the bacterial suspension of the S microbiota were uniformly mixed and left to stand for 48 hours. Afterward, planting of plants began in the PAEs-contaminated soil after purification, and after one cultivation cycle, the PAEs contamination... The dyed crop was harvested, and the plant was water spinach. S5, Y microbial community concentration and domestication: PAEs-contaminated crops are sun-dried and then pulverized to 200 meter size. The PAEs-contaminated crop pulverized powder is obtained by passing it through a sieve, and 1 part of the PAEs-contaminated crop pulverized powder is divided into 2 parts The PAEs-contaminated crop pulverized powder is placed in an inorganic salt culture medium and then sun-dried. This was obtained by grinding and passing it through a 200-mesh sieve, and then placing it in an inorganic salt medium with a PAEs mixed solution. Add the following to adjust the mass concentration of PAEs in the inorganic salt medium to 10 mg / L, and stir under 30°C conditions. While continuing the domestication culture for 3 weeks, obtain a primary strengthened bacterial suspension, and the primary strengthened bacterial suspension Transfer the samples according to the transfer method in Step S2, repeat the transfer five times, and then transfer the Y fungus. A bacterial suspension containing 6 reinforcings is obtained and mixed with 30% glycerol by volume in a 1:1 volume ratio. Prepare by storing under -80°C conditions to obtain freeze-dried glycerol containing the Y fungus community, Phthalate esters DMP, diethyl phthalate DEP, and diethyl phthalate in PAEs mixed solution The mass ratio of Chill DBP is 1:1:1. S6. Preparation of bacterial suspension of the Y flora: Freeze-dried glycerin containing the Y flora obtained in step S5. One portion of the roll was scraped off with an inoculation ring and inoculated into 27 portions of sterilized LB medium, and the sterilization temperature was 1 The temperature was 23°C, the sterilization time was 27 minutes, and the mixture was activated for 24 hours with continuous stirring under 30°C conditions. The bacteria were cultured to obtain a pre-activated, enhanced bacterial suspension, and the pre-activated, enhanced bacterial suspension was centrifuged. The material is placed in a machine and subjected to centrifugal separation, and the centrifugal separation rotation speed is 5000 rpm. The heart separation treatment time was 5 minutes, the supernatant was discarded to obtain a strengthened bacterial suspension, and the strengthened bacterial suspension was treated with an inorganic salt solution. After washing, repeating the centrifugation and inorganic salt solution washing steps three times, 20 parts of the enhanced bacterial solution are used. Place the bacteria in a hydrochloric acid medium, adjust the OD600 value of the fortified bacterial suspension to 1.0, and let it stand for 2 hours to observe the bacteria of the Y flora. Prepare by obtaining a suspension and storing it at 4°C. S7, Mixing: Bacterial suspension of the S flora obtained in step S3 and Y bacteria obtained in step S6 The bacterial suspensions of the flora are mixed in a mass ratio of 3:1 to obtain a bacterial suspension of the enhanced functional flora. Here, the components and mass concentrations of the inorganic salt medium are as follows: 1.5 g / L (NH4)2SO4, 0.5g / L of KH2PO4, 1.91g / L of K2HPO4·3 H2O, 0.5 g / L NaCl, 0.2 g / L MgSO4·7H2O, 1.5 g / L The agar powder, the solvent being ultrapure water, and the pH of the inorganic salt medium being 7.0, The components and mass concentrations of the inorganic salt solution are as follows: 1.5 g / L of (NH4 )2SO4, 0.5g / L KH2PO4, 1.91g / L K2HPO4·3H2O, The solution consists of 0.5 g / L NaCl, 0.2 g / L MgSO4·7H2O, and ultrapure water as the solvent. The pH of the inorganic salt solution is 7.0. The stirring speed is 150 rpm. The components and mass concentrations of LB medium are as follows: 5 g / L yeast extract, 1 0 g / L tryptone, 10 g / L sodium chloride, 1.5 g / L agar powder, solvent is super It is pure water, and the pH of the LB medium is 7.0. Example 3: This example differs from Example 1 in the following respects: S1, S bacterial flora concentration and domestication: Take 1.2 parts activated sludge and add 2.4 parts inorganic sludge by weight. The activated sludge was collected from the secondary sedimentation tank of a sewage treatment plant and placed in a salt culture medium. Add the PAEs mixed solution to the inorganic salt medium to bring the mass concentration of PAEs in the medium to 6 mg / L, and 3 Domestication culture was carried out for 3 weeks under 0°C conditions with continuous stirring to obtain a primary bacterial suspension. S2, S flora gradient domestication: S2-1, Transfer of primary bacterial suspension: Take 1.2 parts of primary bacterial suspension and 2.4 parts of a solution Place in an inorganic salt medium, add the PAEs mixed solution to the inorganic salt medium, and continue stirring under conditions of 30°C. After 3 weeks of domestication culture, a secondary bacterial suspension was obtained. Repeat the transfer operation in step S2-1 five times, and after each transfer, in the inorganic salt medium. The mass concentrations of PAEs were 40 mg / L, 60 mg / L, 80 mg / L, and 100 mg / L, respectively. Adjust the concentration to g / L or 120 mg / L, and continue domestication culture for 3 weeks after the final transfer is complete. Then, obtain a bacterial suspension containing the S flora six times, take 1.2 parts of the six-times bacterial suspension, and divide by volume 35. Mix with % glycerol in a 1:1 volume ratio and store at -80°C to freeze the S-complex. Prepare by obtaining dry glycerol, Preparation of bacterial suspension of S3, S flora: Freeze-dried glycerin containing the S flora obtained in step S2 Scrap off 1.2 parts of the roll with an inoculation ring and inoculate 25 parts of LB medium that has been sterilized at 120°C. The sterilization time is 30 min, and the bacterial suspension is placed in 18 parts of inorganic salt medium, and the OD600 of the bacterial suspension is Adjust the value to 1.0, let it stand for 2.5 hours to obtain a bacterial suspension of the S flora, and store it under 4°C conditions. I prepared it. Example 4: This example differs from Example 1 in the following respects: S1, S bacterial flora concentration and domestication: Take 1.5 parts activated sludge and culture 3 parts inorganic salt at the weight. The activated sludge was collected from the secondary sedimentation tank of a sewage treatment plant and was mixed with an inorganic salt culture medium containing P Add the AEs mixed solution to bring the mass concentration of PAEs in the inorganic salt medium to 14 mg / L, and 30 Domestication culture was carried out for 3 weeks under °C conditions with continuous stirring to obtain a primary bacterial suspension. S2, S flora gradient domestication: S2-1, Transfer of primary bacterial suspension: Take 1.5 parts of primary bacterial suspension and 3 parts of inorganic salt Place in culture medium, add PAEs mixed solution to inorganic salt medium, and continue stirring under conditions of 30°C. After 3 weeks of domesticated culture, a secondary bacterial suspension was obtained. Repeat the transfer operation in step S2-1 five times, and after each transfer, in the inorganic salt medium. The mass concentrations of PAEs were 40 mg / L, 60 mg / L, 80 mg / L, and 100 mg / L, respectively. Adjust the concentration to g / L or 120 mg / L, and continue domestication culture for 3 weeks after the final transfer is complete. Then, obtain a bacterial suspension containing the S flora six times, take 1.5 parts of the six-times bacterial suspension, and divide by volume 40. Mix with % glycerol in a 1:1 volume ratio and store at -80°C to freeze the S-complex. Prepare by obtaining dry glycerol, Preparation of bacterial suspension of S3, S flora: Freeze-dried glycerin containing the S flora obtained in step S2 Scrap off 1.5 parts of the roll with an inoculation ring and inoculate into 30 parts of LB medium sterilized at 125°C. The sterilization time was 20 min, and the bacterial suspension was placed in 19 parts of inorganic salt medium, and the OD600 of the bacterial suspension was The value was adjusted to 1.0, and after standing for 3 hours, a bacterial suspension of the S flora was obtained. It was then stored at 4°C to prepare the solution. did. Example 5: This example differs from Example 2 in the following respects: The plant in S4 is Shanghai cabbage. S5, Y microbial community concentration and domestication: PAEs-contaminated crops are sun-dried and then pulverized to 200 meter size. PAEs-contaminated crop pulverized powder is obtained by passing it through a sieve, and 1.2 parts of the PAEs-contaminated crop pulverized powder are used. 2.4 parts inorganic salt medium is added, and the ground PAEs-contaminated crop powder is sun-dried. This was obtained by drying, grinding, and passing it through a 200-mesh sieve, and then placing PAEs in an inorganic salt medium. The mixed solution was added to bring the mass concentration of PAEs in the inorganic salt medium to 6 mg / L, under conditions of 30°C. While continuing to stir, the bacteria were cultured for 3 weeks to obtain a primary strengthened bacterial suspension. Transfer the turbid liquid according to the transfer method in step S2, and repeat the transfer five times. A six-stage fortified bacterial suspension containing the colony was obtained and mixed with 30% glycerol in a 1:1 volume ratio. Then, freeze-dried glycerol containing the Y fungus was obtained by storing it under -80°C conditions, S6. Preparation of bacterial suspension of the Y flora: Freeze-dried glycerin containing the Y flora obtained in step S5. Scrap off 1.2 parts of the roll with the inoculation ring, inoculate 25 parts of sterilized LB medium, and set to sterilization temperature. The temperature was 120°C, the sterilization time was 30 minutes, and the enhanced bacterial solution was placed in 18 parts of an inorganic salt medium. The OD600 value of the enhanced bacterial solution was adjusted to 1.0, and it was allowed to stand for 2.5 hours to obtain a bacterial suspension of the Y flora. It was prepared by storing it at 4°C. Example 6: This example differs from Example 2 in the following respects: The plant in S4 is lettuce. S5, Y microbial community concentration and domestication: PAEs-contaminated crops are sun-dried and then pulverized to 200 meter size. PAEs-contaminated crop pulverized powder is obtained by passing it through a sieve, and 1.5 parts of the PAEs-contaminated crop pulverized powder are used. Take the PAEs-contaminated crop powder and place it in a 3 part inorganic salt medium. This is obtained by drying, grinding, and passing it through a 200-mesh sieve, and then adding PAE to an inorganic salt medium. Add the mixed solution to adjust the mass concentration of PAEs in the inorganic salt medium to 14 mg / L, and set it to 30°C. Under these conditions, the bacteria were cultured for three weeks while stirring to obtain a primary strengthened bacterial suspension, and then the primary strengthened bacteria were cultured. Transfer the bacterial suspension according to the transfer method in step S2, and repeat the transfer five times. A six-stage fortified bacterial suspension containing the Y colony was obtained, and mixed with 30% glycerol by volume in a 1:1 volume ratio. Mix and store at -80°C to obtain freeze-dried glycerol containing the Y fungus colony, S6. Preparation of bacterial suspension of the Y flora: Freeze-dried glycerin containing the Y flora obtained in step S5. Scrap off 1.5 parts of the roll with the inoculation ring, inoculate it into 30 parts of sterilized LB medium, and set to sterilization temperature. The temperature was 125°C, the sterilization time was 20 minutes, and the enhanced bacterial solution was placed in 19 parts of an inorganic salt medium. The OD600 value of the enhanced bacterial solution was adjusted to 1.0, and after standing for 3 hours, a bacterial suspension of the Y flora was obtained. It was prepared by storing it under °C conditions. Description: When high temperature is selected for sterilization, the sterilization time can be appropriately shortened, as in Example 1. By arbitrarily selecting one parameter from Examples 3 to 6, a good result can be obtained. Sterilization conditions can be achieved. Experimental Example 1: This experimental example is described based on the scheme described in Example 1 and prepared in accordance with the present invention. This study aims to elucidate the effectiveness of a functional bacterial flora S in the biodegradation of harmful organic pollutants by a bacterial suspension during laboratory experiments. The purpose is to do so. First, the morphology of the bacterial suspension of the S flora was observed using a scanning electron microscope, and the LB medium was incubated at 121°C for 3 minutes. Sterilize for 0 minutes, cool to room temperature, then add the bacterial suspension of the S flora and sterilize for 24 hours (30°C, 15 minutes). (0 rpm) The bacteria are activated by shaking culture, and after activation, 1 ml of the bacterial solution is placed in a 1.5 ml centrifuge tube. Place in a container and centrifuge (5000 rpm, 5 min), discard the supernatant, and collect the soybean-sized bacterial cells. To obtain this, add a 2.5% glutaraldehyde solution to the centrifuge tube and fill the centrifuge tube ( The bacterial suspension of the S flora was stored at 4°C (so that it was immersed in the fixative) and then fixed for 12 hours before being examined. It is sent for examination. Figure 2 is a biological scanning electron microscope image of the bacterial suspension of the final S community. As shown in Figure 3, and as can be seen from the figure (magnified 5000 times and 20000 times), bacteria Most of the bacteria in the S plaque are long and rod-shaped. Subsequently, the bacterial species composition of the S flora bacterial suspension was examined, and 1 mL of the S flora bacterial suspension cultured for 24 hours was examined. Take the turbidity, place it in a 1.5 mL centrifuge tube, and centrifuge for 2 minutes at 4°C under conditions of 5000 r / min. After separating the heart cells, discard the supernatant, collect the bacterial cells, and use a bacterial DNA extraction kit (Omega, USA). Using this method, bacterial genomic DNA is extracted while referring to the instruction manual, and the extracted DNA is diluted to 1%. Galose gel electrophoresis and Nano Drop 2000 (Thermo Scien Quality inspections are conducted by tific (USA), and the above-mentioned Sun is entrusted to Shanghai Biotechnology Co., Ltd. High-throughput sequencing was performed on the 16S rRNA gene of the pull, and the extracted DNA was then processed. As a template, primer 338F(5'-ACTCCTACGGGAGGCAG CAG-3') and Primer 806R (5'-GGACTACHVGGGTWTCTA) Using AT-3'), PCR amplification of the V3-V4 variable region of the bacterial 16S rRNA gene was performed. The PCR product was recovered using a 2% agarose gel, and then processed with NEXTFLEXRapid D A library of PCR products was constructed using NA-SeqKit, and IlluminaPE30 was used. The base sequence was determined using the 0 / PE250 platform. As a result, the resident microbiota S The bacterial community structure and composition were obtained, and the results are shown in Figure 4. Next, we began setting up the experiment to decompose the bacterial suspension of the indoor S microbiota, using 0.1g of each. Weigh out DMP, 0.1g DEP, and 0.1g DBP and place them in a 100ml volumetric flask. Add methanol to a constant volume and prepare a 1g / LPAEs mixed solution (1g / L DMP, 1g / (containing L DEP and 1 g / L DBP) is obtained, and 100 ml of sterilized conical frustus is used. Add 19 ml of inorganic salt medium to the mixture, add 1 ml of bacterial suspension (5% concentration), and mix until the mixture reaches a concentration of 1 g in 200 μL. Add a PAEs mixed solution of 10 mg / L to set the initial concentrations of DMP, DEP, and DBP to 10 mg / L. At the same time, a PAEs-containing inorganic salt medium solution that had not been inoculated with bacteria was set as a control group, and the pH Adjust to 7.0, repeat 3 times for each group, resulting in 4h, 12h, 36h, and 48h respectively. A sample was taken. Using a syringe, 2 ml of supernatant was aspirated and filtered through a 0.22 μm organic phase filtration membrane. After passing through the sample, it was stored in a 2 mL brown liquid phase vial and detected by high-performance liquid chromatography. . High-performance liquid chromatography detection conditions for PAEs: LC-20AT high-performance liquid chromatography Using the Fee (equipped with SPD-2A UV detector), the detection time is 40 min, and the injection The injection volume of the system is 20 μL, the separation system uses acetonitrile-water as the mobile phase, and the initial flow rate is 1 The flow rate was 0 mL / min, and PAEs were separated by gradient elution, with the chromatography column being Φ This is a 4.6 × 250 mm InertsilODS-P liquid chromatography column. The column temperature was 40°C, and the detection system used an ultraviolet detector for detection, and each was 2 A dual-wavelength detection mode was set to 0.5nm and 225nm. Finally, the degradation effect of the S microbiota: Figure 5 shows the degradation effect of the S microbiota on PAEs in inorganic salt medium. As shown, when placed in an inorganic salt medium, the S bacterial community is compared to the three PAEs: DEP, DMP, and DBP. It has a good degradation effect, and in the initial stages of degradation, the DMP alkyl side chain is short and the molecular weight is low, and it is found in the S bacterial community. The DMP decomposition speed is fast, with decomposition rates of DMP, DEP, and DBP being 39.05% each. The percentages were 6.06% and 10.90%, and after 36 hours of incubation, DMP, DEP, and DBP were almost complete. It was completely decomposed, with decomposition rates of 99.85%, 99.85%, and 95.85%, respectively. Based on the above results, the S microbiota has excellent potential for application in the biodegradation of DMP, DEP, and DBP. It was shown that it possesses this characteristic. Experimental Example 2: This experimental example is described based on the scheme described in Example 2 and is prepared in accordance with the present invention. This study aims to elucidate the practical effectiveness of enhanced functional bacterial suspensions in biodegrading harmful organic contaminants. The purpose is to do so. Follow the flow in step S4 to remediate PAEs-contaminated soil, and the soil from the contaminated area will be processed in the laboratory. After collecting the sample and purifying it (heterococcal soil treatment), the overall removal rate of three types of PAEs in the soil was tested. The results are shown in Figure 6, where ΣPAEs represents the total decomposition rate of the three types of PAEs, and from this we can see that The overall removal rate of three types of PAEs in the soil was 68%, and in actual applications, laboratory tests were conducted. Unlike experimental decomposition effects, this is because the actual contaminant components in soil are complex and there is some degree of interference. It is thought that this is because the microbial community is affected by environmental conditions (e.g., temperature, pH value). There is a possibility that some degree of competition may occur with the soil's resident microbiota. Subsequently, the bacterial suspension of the enhanced functional microbiota was prepared according to the flow in steps S5-S7. This enhanced functional bacterial suspension was prepared and applied again to the soil remediation of the contaminated area. Step S4 involves backfilling the purified soil into its original location and burying it, creating a bacterial suspension of the enhanced functional microbiome. The turbid liquid was directly added to the soil in the contaminated area (in-situ soil treatment), with an application rate of 2.5 L / m². 2 and Three weeks later, soil samples were taken to detect the overall removal rate of PAEs, and the results are shown in Figure 6. As can be seen from this, the overall removal rate of PAEs in the soil sample at this time was 85%. Thus, as described above, the bacterial suspension of the S flora obtained in step S3 and the obtained in step S6 A bacterial suspension of the Y microbiota was prepared by mixing the Y microbiota in a 3:1 mass ratio to create an enhanced functional microbiota. The bacterial suspension has higher biodegradability, and when combined with Figure 6, the difference between the two can be grasped more intuitively. This compensates for potential interferences that may occur when using bacterial suspensions of individual S-type microbiota in actual soil, and DB To improve the completeness of P degradation, and to combine in-situ and ectopic soil treatments, hazardous organic pollution This can further improve the efficiency of processing materials.
Claims
1. The following steps are included: Step S1, S bacterial flora concentration and domestication: Add 1 to 1.5 parts activated sludge by weight to 2 to 3 parts Place in an inorganic salt medium, add PAEs mixed solution to the inorganic salt medium, and PA in the inorganic salt medium The mass concentration of Es is adjusted to 6-14 mg / L, and the mixture is stirred continuously at room temperature for 3 weeks. Intermediate domestication culture is performed to obtain a primary bacterial suspension, and the activated sludge is collected from the secondary sedimentation tank of the sewage treatment plant. These were collected, Step S2, S-microbiota gradient domestication: Step S2-1, Transfer of the primary bacterial suspension: Transfer 1 to 1.5 parts of the primary bacterial suspension Take a portion of the solution, place it in 2-3 parts of an inorganic salt medium, add the PAEs mixed solution to the inorganic salt medium, and leave at room temperature. Under these conditions, the bacteria were cultured for three weeks while continuing to stir, and a secondary bacterial suspension was obtained. Repeat the transfer operations in steps S2-2 and S2-1 five times, and after each transfer... The mass concentrations of PAEs in the inorganic salt medium were 40 mg / L, 60 mg / L, and 80 mg / L, respectively. Adjust to L, 100 mg / L, 120 mg / L, and once the final transfer is complete, leave at home for 3 weeks. Continue the livestock culture to obtain six bacterial suspensions containing the S microbiota, and 1 to 1.5 parts of the six bacterial suspensions. Take the mixture and mix it with glycerol at a volume fraction of 30-40% in a volume ratio of 1:1 under ultra-low temperature conditions. Preserve and prepare freeze-dried glycerol containing the S fungal flora. Step S3, Preparation of bacterial suspension of S flora: Freeze-dried suspension containing the S flora obtained in Step S2 Remove 1 to 1.5 parts of dried glycerol using an inoculum ring, and then add 25 to 30 parts of sterilized LB medium. The bacteria were inoculated and activated under room temperature conditions with continuous stirring for 24 hours to obtain a pre-activated bacterial suspension. To obtain the pre-activated bacterial suspension, place it in a centrifuge and centrifuge it, discarding the supernatant. A bacterial suspension is obtained, the bacterial suspension is washed with an inorganic salt solution, and the centrifugation and inorganic salt solution washing steps are repeated three times. After turning the bacterial suspension, add 18-20 parts of inorganic salt culture medium and adjust the OD600 value of the bacterial suspension to 1.
0. Prepare the suspension by stirring and letting it stand for 2-3 hours to obtain a bacterial suspension of the S flora, then storing it under low temperature conditions. Step S4, Acquisition of PAEs-contaminated crops: Take PAEs-contaminated soil and place it in step S3 Add the bacterial suspension of the S flora prepared by the PAEs contaminated soil and the bacterial suspension of the S flora. The ratio was 100:1, and the PAEs-contaminated soil and the bacterial suspension of the S microbiota were uniformly mixed for 48 hours. After standing, planting of plants began in the purified PAEs-contaminated soil, and after one cultivation cycle, P Harvesting AES-contaminated crops, Step S5, Y-complexity concentration and domestication: PAEs-contaminated crops are sun-dried and then pulverized. A pulverized powder of PAEs-contaminated crops is obtained by passing it through a 00-mesh sieve, and 1 to 1.5 parts of PAEs-contaminated crops are used. Place 2-3 parts of the pulverized powder into an inorganic salt medium, and then add the PAEs mixed solution to the inorganic salt medium. Then, adjust the mass concentration of PAEs in the inorganic salt medium to 6-14 mg / L and continue stirring under room temperature conditions. While domesticating the bacteria, a primary strengthened bacterial suspension is obtained by culturing the bacteria for three weeks, and the primary strengthened bacterial suspension is then used to... Transfer the samples according to the transfer method in Step S2, repeating the transfer five times, until the Y fungal colony is included. Six-stage strengthened bacterial suspensions were obtained and mixed with 30% glycerol by volume in a 1:1 volume ratio, and then ultra-low Prepare by storing under temperature conditions to obtain freeze-dried glycerol containing the Y fungal colony, Step S6, Preparation of bacterial suspension of the Y flora: Freeze-dried suspension containing the Y flora obtained in Step S5 Remove 1 to 1.5 parts of dried glycerol using an inoculum ring, and then add 25 to 30 parts of sterilized LB medium. The bacteria were inoculated and activated under room temperature conditions with continuous stirring for 24 hours, followed by a pre-activation and strengthening of the bacterial suspension. A turbidity is obtained, and the pre-activated enhanced bacterial suspension is placed in a centrifuge and subjected to centrifugal separation. Discard the excess to obtain a strengthened bacterial solution, wash the strengthened bacterial solution with an inorganic salt solution, and then perform the centrifugation and inorganic salt solution washing as described above. After repeating the step three times, add 18-20 parts of the enhanced bacterial solution to the inorganic salt medium, and the enhanced bacterial solution The OD600 value was adjusted to 1.0, and after standing for 2-3 hours, a bacterial suspension of the Y flora was obtained, under low temperature conditions. Save and prepare it, Step S7, Mixing: The bacterial suspension of the S flora obtained in Step S3 and the one obtained in Step S6 A bacterial suspension of the Y microbiota was mixed in a mass ratio of 3:1 to create an enhanced functional microbiota bacterial suspension. Obtained, The room temperature is 30°C, the stirring rotation speed is 150 rpm, and the ultra-low temperature The temperature is -80°C, and the low temperature is 4°C. A method for domesticating a functional microbiome that decomposes phthalate esters, characterized by the following features.
2. The plant is one or more of water spinach, Chinese cabbage, and lettuce. A method for domesticating a functional microbiota that decomposes phthalate esters, as described in claim 1.
3. The components and mass concentrations of the inorganic salt medium are as follows: 1.5 g / L (N H ４ ) ２ SO ４ , 0.5g / L KH ２ PO ４ , 1.91 g / L of K ２ HPO ４ 3H ２ O, 0.5 g / L of NaCl, 0.2 g / L of MgSO ４ ·7H ２ O, 1.5 g / L of cold The powder, the solvent is ultrapure water, and the pH of the inorganic salt medium is 7.
0. The components and quality of the inorganic salt solution are as follows: The concentrations are as follows: 1.5 g / L (NH ４ ) ２ SO ４ , 0.5 g / L KH ２ PO ４ , 1.91 g / L of K ２ HPO ４ 3H ２ O, 0.5 g / L NaCl, 0.2 g / L MgSO ４ 7H ２ O. The solvent is ultrapure water, and the pH of the inorganic salt solution is 7.
0. A method for domesticating a functional microbiota that decomposes phthalate esters, as described in item 1. Law.
4. The phthalate ester DMP, diethyl phthalate DEP, and phthalate in the aforementioned PAEs mixed solution. The phthalate according to claim 1, characterized in that the mass ratio of dibutyl DBP is 1:1:
1. A method for domesticating functional microbiota that decomposes acid esters.
5. The components and mass concentrations of the aforementioned LB medium are as follows: 5 g / L yeast extract 10 g / L tryptone, 10 g / L sodium chloride, 1.5 g / L agar powder, solvent The ultrapure water and LB medium have a pH of 7.0, and the sterilization temperature is 120-125°C. The phthalate ester according to claim 1, characterized in that the sterilization time is 20 to 30 minutes. A method for domesticating functional microbiota that decomposes tel.
6. The centrifugal rotation speed of the aforementioned centrifugal separation process is 5000 rpm, and the centrifugal separation process time is 5 min. The functional microbiota that degrades phthalate esters according to claim 1, wherein n is n Domestication methods.