Method for screening phthalate degrading bacteria in plastic water supply pipeline biological membrane

Abstract

The invention discloses a method for screening phthalate degrading bacteria in a plastic water supply pipeline biological membrane. The method comprises the following steps: separating a pipe wall biological membrane, carrying out multi-round culture by using a pollutant as a single carbon source, carrying out enrichment culture by using an LB culture medium, and carrying out culture by using the pollutant as the single carbon source. The phthalate degrading bacteria are screened from a plastic water supply pipeline biological membrane. Technical support is provided for pollution control of the phthalic acid ester in the drinking water.

Description

Technical Field

[0001] The invention relates to the field of environmental microorganisms, and in particular to a method for screening phthalate-degrading bacteria. Background Art

[0002] Due to the many advantages of plastic pipes, they are widely used in tap water distribution systems. In order to improve the physical and chemical properties of plastic pipes and increase the service life of pipes, some organic or inorganic additives are usually used in the production process of plastic pipes. Among them, phthalates are a widely used plasticizer that can increase the flexibility, ductility and durability of pipes. Unlike metal pipes, organic matter leaches out of plastic pipes. Since phthalates are only combined with plastic polymers through physical action rather than chemical bonds, they are easily released from plastic pipes and enter the water.

[0003] The drinking water distribution system is a complex and dynamically changing microbial ecosystem. Most bacteria exist in the biofilm on the inner wall of the water supply pipe. The volatile components of plastic pipes and other additives used to make plastic pipes are also carbon sources for the growth of microorganisms in the water supply pipes. Studies have isolated phthalate-degrading bacteria from soil, sediments, and activated sludge from sewage treatment plants. However, the drinking water distribution system has fewer nutrients and lower phthalate concentrations. Phthalate-degrading bacteria in the biofilm of plastic water supply pipes may have their own unique properties. At present, there is still a gap in research in this area. Therefore, a method for screening phthalate-degrading bacteria in the biofilm of plastic water supply pipes was invented, which is conducive to understanding the characteristics of microorganisms in the biofilm of the pipe, and at the same time developing the potential to use bioremediation methods to degrade phthalates in water bodies to reduce the concentration of pollutants in terminal water, thereby improving water safety. Summary of the invention

[0004] The present invention aims to provide a method for screening phthalate-degrading bacteria in biofilms of plastic water supply pipes. The method uses pollutants as the sole carbon source in a culture medium to carry out multiple rounds of screening and culturing of bacteria in the biofilms of pipe walls, uses Luria-Bertani (LB) culture medium for bacterial enrichment culture, and then uses pollutants as the sole carbon source in the culture medium for single colony culture, thereby achieving the screening of phthalate-degrading bacteria from the biofilms of plastic water supply pipes.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for screening phthalate-degrading bacteria in biofilm of plastic water supply pipes, the method comprising:

[0007] Separation of biofilm: Obtain biofilm from the inner wall of the water supply pipe and wash it, and obtain biofilm suspension by centrifugation;

[0008] Cultivating bacterial suspension: inoculating the biofilm suspension obtained in step 1 into an inorganic salt liquid culture medium, culturing at 150 rpm and 30° C. for 7 days to obtain a bacterial suspension of the first stage, adding the obtained bacterial suspension of the first stage into a new inorganic salt liquid culture medium, culturing again at 150 rpm and 30° C. for 7 days to obtain a bacterial suspension of the second stage;

[0009] Isolating and purifying the bacterial suspension: inoculating the second stage bacterial suspension cultured in step 2 into an inorganic salt solid culture medium, obtaining bacterial cells from the surface of the inorganic salt solid culture medium, replacing the LB solid culture medium, and repeatedly using the three-zone streak method on the LB solid culture medium to separate and purify the bacterial cells to obtain a single colony;

[0010] Enrichment of degradation bacteria: The single colony separated in step 3 was inoculated into LB liquid culture medium for enrichment culture at 150 rpm and 30°C for 36 h to form a bacterial suspension. The bacterial suspension was taken, centrifuged, and sterilized phosphate buffer was added. The suspension was rinsed and centrifuged, and the supernatant was removed. The process was repeated three times to obtain a bacterial suspension of the third stage.

[0011] Screening of phthalate-degrading bacteria: Screening of phthalate-degrading bacteria from the third-stage bacterial suspension obtained in step 4.

[0012] In some embodiments, the biofilm suspension in step 1 is prepared by adding sterilized phosphate buffer, washing the biofilm, centrifuging, removing the supernatant, and repeating three times.

[0013] In some embodiments, the preparation method of the inorganic salt liquid culture medium is: add (NH4)2SO41 g, KH2PO40.8 g, K2HPO40.2 g, MgSO4·7H2O 0.5 g, FeSO40.01 g, CaCl2·2H2O 0.05 g to 1000 mL of distilled water, adjust the pH value to 7-8 with NaOH, sterilize at 121°C for 30 minutes, add phthalate stock solution after cooling, and configure a phthalate concentration of 1 mg / L with phthalate as the single carbon source culture system.

[0014] In some embodiments, the preparation method of the inorganic salt solid culture medium is: add 1.5% agar to the inorganic salt liquid culture medium, sterilize at 121°C for 30 minutes, add phthalate stock solution, configure the phthalate concentration of 1 mg / L as the single carbon source culture system, and then pour it into a glass culture dish to configure it into a solid culture system.

[0015] In some embodiments, the screening of phthalate-degrading bacteria is achieved by identifying degrading bacterial species, and the specific steps include: extracting bacterial DNA, using 5′-end primer 27F5′-AGAGTTTGATCCTGGCTCAG-3′ and 3′-end primer 1492R5′-TACGGCTACCTTGTTACGACTT-3′ for PCR amplification, using agarose gel electrophoresis for separation, sequencing the target fragments, and performing sequence homology retrieval and alignment with the Basic Local Alignment Search Tool program in the National Center for Biotechnology Information database to obtain the identification results of the degrading bacterial species.

[0016] In some embodiments, the method further includes short-term storage of the degrading bacteria, and the specific steps include: sealing the inorganic salt solid culture medium with a sealing film, covering it with tin foil to avoid light, and storing it in a 4°C refrigerator; the specific steps for long-term storage are: after the LB liquid culture medium is enriched again, adding sterilized glycerol, placing it in a cryopreservation tube and storing it in a -80°C refrigerator.

[0017] Compared with the prior art, the advantages and positive technical effects achieved by the present invention are as follows:

[0018] 1) Phthalate-degrading bacteria are screened from biofilms of plastic water supply pipes by separating pipe wall biofilms, using pollutants as a single carbon source for multiple rounds of cultivation, using LB medium for enrichment cultivation, and then using pollutants (phthalates) as a single carbon source for cultivation, so that the obtained degrading bacteria can grow in the oligotrophic environment of the water supply network;

[0019] 2) Moreover, it can degrade phthalates in the water supply network environment, which, on the one hand, helps to fill the gap of phthalate-degrading bacteria in the water supply system, and on the other hand, can also provide new ideas for the pollution control of phthalates in drinking water. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the overall process;

[0021] Figure 2 This is a schematic diagram of the biofilm on the inner wall of the pipeline;

[0022] Figure 3 Schematic diagram of Luria-Bertani (LB) solid culture medium containing a single colony of phthalate-degrading bacteria isolated from a biofilm in a plastic water supply pipe. DETAILED DESCRIPTION

[0023] The technical solution will be clearly described below in conjunction with the accompanying drawings and embodiments.

[0024] like Figure 1 The method of the present invention for screening phthalate-degrading bacteria in biofilm of plastic water supply pipes specifically comprises the following steps:

[0025] Step 1: Separation of biofilm, including the following treatments:

[0026] Step 1.1: Obtain a biofilm. Specifically, obtain a water supply pipe with a biofilm growing on the inner wall, such as Figure 2 As shown, a medicine spoon and a 50 mL centrifuge tube were sterilized by high temperature and high pressure, and the biofilm on the wall of the water supply pipe was scraped with a weighing spoon on a sterile operating table and placed in a centrifuge tube;

[0027] Step 1.2: Clean the biofilm. Specifically, add 10 mL of sterilized phosphate buffer to the centrifuge tube to wash the biofilm, centrifuge at 5000 rpm and 25°C for 2 min, discard the centrifugation clear liquid on the upper layer of the current liquid surface after centrifugation, add phosphate buffer to resuspend the bottom bacterial precipitate and centrifuge, repeat 3 times, discard the centrifugation clear liquid on the upper layer of the current liquid surface after 3 centrifugation, add phosphate buffer again to suspend the bottom bacterial precipitate, and obtain a biofilm suspension;

[0028] Step 2: Cultivating bacterial suspension: configuring a culture system with phthalates as a single carbon source as the first inorganic salt liquid culture medium, adding the biofilm suspension obtained in step 1 to the configured first inorganic salt liquid culture medium, placing it in a shaker and culturing it at 150 rpm and 30° C. for 7 days to obtain a first-stage bacterial suspension; configuring a culture system with phthalates as a single carbon source as the second inorganic salt liquid culture medium again, adding the obtained first-stage bacterial suspension to the second inorganic salt liquid culture medium, placing it in a shaker and culturing it at 150 rpm and 30° C. for 7 days to obtain a second-stage bacterial suspension;

[0029] Step 2.1: Cultivate the first inorganic salt liquid culture medium, including the following treatments:

[0030] Step 2.1.1: configuring a culture system with phthalates as a single carbon source. Specifically, add an inorganic salt liquid culture medium into a container, for example, add 30 mL of inorganic salt liquid culture medium into a 150 mL conical flask; then use a microinjector to add a phthalate stock solution into the inorganic salt liquid culture medium, so that the concentration of the target phthalate in the system is 1 mg / L, and configure a phthalate concentration of 1 mg / L as a single carbon source culture system;

[0031] Step 2.1.2: Shake the phthalate-only carbon source culture system with a phthalate concentration of 1 mg / L, add 1 mL of the washed biofilm suspension, place in a shaker and culture at 150 rpm and 30° C. for 7 days to obtain a bacterial suspension of the first stage;

[0032] Step 2.2: Cultivate the second inorganic salt liquid culture medium, specifically including the following treatments:

[0033] Step 2.2.1: Re-prepare a culture system with phthalates as the sole carbon source. The method is the same as step 2.1. Take fresh inorganic salt liquid culture medium and re-prepare a culture system with phthalates as the sole carbon source at a phthalate concentration of 1 mg / L.

[0034] Step 2.2.2: After the first 7-day culture, take 1 mL of the first-stage bacterial suspension obtained in step 2.1.2 and add it to the culture system configured in step 2.2.1. Place it in a shaker again and culture it at 150 rpm and 30°C for 7 days to obtain the second-stage bacterial suspension;

[0035] Step 3: Isolation and purification of bacterial suspension:

[0036] Step 3.1: Cultivating inorganic salt solid culture medium, specifically including the following treatments:

[0037] Step 3.1.1: Prepare an inorganic salt solid culture medium with phthalate as the sole carbon source, add 1.5% agar to the phthalate as the sole carbon source culture system with a phthalate concentration of 1 mg / L prepared in step 2.1.1, and obtain an inorganic salt solid culture medium with a phthalate concentration of 1 mg / L as the sole carbon source in a glass culture dish;

[0038] Step 3.2: Cultivate in an inorganic salt solid medium with phthalate as a single carbon source. Specifically, after two 7-day cultures, take 100 μL of the second-stage bacterial suspension and apply it to the inorganic salt solid medium with phthalate as a single carbon source. After coating, place it upright for 20 minutes to allow the inorganic salt solid medium to fully absorb the second-stage bacterial suspension, then invert it and place it in a constant temperature incubator at 30° C. for culture, and regularly observe the growth status of bacteria in the inorganic salt solid medium;

[0039] Step 3.3: Obtain bacteria from the surface of inorganic salt solid culture medium. After the bacteria grow to form a single colony, use an inoculation loop to pick bacteria with different visual morphology from the surface of inorganic salt solid culture medium, and separate them using the three-zone streak method on Luria-Bertani (LB) solid culture medium. After the bacteria are placed upright to absorb the liquid for 2 hours, they are placed upside down in a constant temperature incubator at 30°C for culture. In this step, the three-zone streak method is used on the LB solid culture medium to achieve plate streak inoculation. By streaking on the plate, the bacteria are fully dispersed on the surface of the agar plate, so that a single bacterium can be fixed at one point to reproduce and form a single colony, thereby achieving the purpose of separation and purification. The operation used is, for example: in the first zone, repeatedly streak at the starting position, and then streak in a zigzag shape. In the second zone, the plate is rotated 60 degrees and streaked in a zigzag shape. In the third zone, the plate is rotated 60 degrees again, and three-zone streaking is performed, and the rules are the same as the second zone streak;

[0040] Step 3.4: Isolate and purify the bacteria in LB solid medium. After the bacteria grow to form a single colony, pick a single colony from the surface of the LB solid medium with an inoculation loop, separate it using the three-zone streak method on a new LB solid medium, place it upright to allow the bacterial solution to absorb for 2 hours, then invert it and place it in a constant temperature incubator at 30°C for culture. Repeat the separation 3 times to obtain a single colony;

[0041] Step 4: Enrichment of degradation bacteria, including the following treatments:

[0042] Step 4.1: Enrichment culture in LB liquid medium: Add 30 mL of LB liquid medium to a 150 mL conical flask, transfer a single colony to a conical flask containing LB liquid medium, place it in a shaker, and culture at 150 rpm and 30°C for 36 hours to form a bacterial liquid;

[0043] Step 4.2: The LB liquid culture medium is obviously turbid. Place the bacterial suspension in a sterilized 50 mL centrifuge tube and centrifuge it at 5000 rpm and 25°C for 2 min. Discard the supernatant after centrifugation, add 10 mL of sterilized phosphate buffer to the centrifuge tube and centrifuge. Repeat 3 times to obtain the third stage bacterial suspension.

[0044] The present invention further comprises the following processing:

[0045] Step 5: Verify individual colonies, including the following:

[0046] Step 5.1: Perform confirmation culture using an inorganic salt solid culture medium with phthalates as a single carbon source. Specifically, the bacterial suspension of the third stage is applied to an inorganic salt solid culture medium with phthalates as a single carbon source. The strain that can grow is considered to be a strain that has the ability to degrade phthalates.

[0047] Step 5.2: Identify the degrading bacteria. Specifically, select a single colony of phthalate-degrading bacteria on an inorganic salt solid medium, extract bacterial DNA, and use

[0048] PCR amplification was performed using the 5′ end primer 27F5′-AGAGTTTGATCCTGGCTCAG-3′ and the 3′ end primer 1492R5′-TACGGCTACCTTGTTACGACTT-3′, and the target fragment was sequenced. The sequencing results were searched and aligned with the BasicLocalAlignment Search Tool program in the National Center for Biotechnology Information database to obtain the identification results of the degrading bacteria.

[0049] Step 5.3: Preserve the degrading bacteria. Specifically, seal the culture dish containing the inorganic salt solid culture medium containing phthalate degrading bacteria obtained in step 5.1 with a sealing film, cover with tin foil to avoid light, and store in a 4°C refrigerator. If long-term storage is required, after the LB liquid culture medium is enriched again, add sterilized glycerol, place in a cryopreservation tube and store in a -80°C refrigerator.

[0050] Based on the embodiments of the present invention, all other embodiments and technical replacements and modifications of the embodiments obtained by ordinary technicians in the field without departing from the spirit of the present invention and without making creative work shall fall into the protection scope of the present invention.

[0051] The above is only a preferred embodiment of the present invention. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be interpreted as limiting the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications made without departing from the principle of the present invention should also be considered to fall within the scope of protection of the present invention.

Claims

1. A method for screening phthalate-degrading bacteria in biofilm of plastic water supply pipes, characterized in that: The method described is: Separation of biofilm: Obtain biofilm from the wall of water supply pipe and clean it, and obtain biofilm suspension by centrifugation; Cultivating bacterial suspension: inoculating the biofilm suspension obtained in step 1 into an inorganic salt liquid culture medium, culturing at 150 rpm and 30° C. for 7 days to obtain a bacterial suspension of the first stage, adding the obtained bacterial suspension of the first stage into a new inorganic salt liquid culture medium, culturing again at 150 rpm and 30° C. for 7 days to obtain a bacterial suspension of the second stage; Isolating and purifying the bacterial suspension: inoculating the second stage bacterial suspension cultured in step 2 into an inorganic salt solid culture medium, obtaining bacterial cells from the surface of the inorganic salt solid culture medium, replacing the LB solid culture medium, and repeatedly using the three-zone streak method on the LB solid culture medium to separate and purify the bacterial cells to obtain a single colony; Enrichment of degradation bacteria: The single colony separated in step 3 was inoculated into LB liquid culture medium, cultured at 150rpm and 30°C for 36h to enrich and form bacterial liquid, the bacterial liquid was taken, centrifuged, sterilized phosphate buffer was added, rinsed, centrifuged, and the supernatant was removed, and the process was repeated three times to obtain the bacterial suspension of the third stage; Screening of phthalate-degrading bacteria: Screening of single colonies of phthalate-degrading bacteria from the third-stage bacterial suspension obtained in step 4.

2. A method for screening phthalate-degrading bacteria in biofilm of plastic water supply pipes according to claim 1, characterized in that: The preparation method of the biofilm suspension is as follows: adding sterilized phosphate buffer, washing the biofilm, centrifuging, removing the supernatant, and repeating three times.

3. The method for screening phthalate-degrading bacteria in biofilm of plastic water supply pipes according to claim 1, characterized in that: The preparation method of the inorganic salt liquid culture medium is as follows: add (NH4)2SO41 g, KH2PO40.8 g, K2HPO40.2 g, MgSO4·7H2O 0.5 g, FeSO40.01 g, and CaCl2·2H2O 0.05 g to 1000 mL of distilled water, adjust the pH value to 7-8 with NaOH, sterilize at 121°C for 30 minutes, add phthalate stock solution after cooling, and configure a phthalate concentration of 1 mg / L with phthalate as the single carbon source culture system.

4. The method for screening phthalate-degrading bacteria in biofilm of plastic water supply pipes according to claim 1, characterized in that: The preparation method of the inorganic salt solid culture medium is as follows: 1.5% agar is added to the inorganic salt liquid culture medium, sterilized at 121°C for 30 minutes, phthalate stock solution is added, a phthalate concentration of 1 mg / L phthalate is configured as a single carbon source culture system, and the system is poured into a glass culture dish to form a solid culture system.

5. The method for screening phthalate-degrading bacteria in biofilm of plastic water supply pipes according to claim 1, characterized in that: The screening of phthalate-degrading bacteria is achieved by identifying the degrading bacterial strains, and the specific steps are: extracting bacterial DNA, using 5' end primer 27F5'-AGAGTTTGATCCTGGCTCAG-3' and 3' end primer 1492R5'-TACGGCTACCTTGTTACGACTT-3' for PCR amplification, using agarose gel electrophoresis for separation, sequencing the target fragments, and performing sequence homology retrieval and comparison with the Basic Local Alignment Search Tool program in the National Center for Biotechnology Information database to obtain the identification results of the degrading bacterial strains.

6. The method for screening phthalate-degrading bacteria in biofilm of plastic water supply pipes according to claim 1, characterized in that: The method also includes short-term storage of degrading bacteria, specifically comprising the following steps: sealing the inorganic salt solid culture medium with a sealing film, covering it with tin foil to avoid light, and storing it in a 4°C refrigerator; and long-term storage specifically comprising the following steps: adding sterilized glycerol after the LB liquid culture medium is enriched again, placing it in a cryovial and storing it in a -80°C refrigerator.

Images