Mangrove sediment sampling
In October 2020, 10 sampling sites were randomly selected from Gaoqiao Town Mangrove Nature Reserve, Lianjiang City, Zhanjiang City, Guangdong Province. Sediment samples with depths of 3 cm and 5 cm were collected by five-point sampling method and packed in clean glass containers for the isolation and identification of plastic-degrading bacteria.
Reagent and solution configuration
Na2B4O7, CuSO4, CaCl2, ZnSO4, H2SO4, CaCO3, FeSO4, MgSO4, MnSO4 tryptone yeast extract, NaCl, HCI and Agar are all domestic pure reagents for analysis; Low density PE is purchased from Alfa Aesar Co., Ltd for analysis; TIANamp Bacteria DNA Kit (TIANGEN), Green Taq Mix (Novozan), DL2000 DNA Marker (Takara); Ordinary plastic fresh-keeping bag (PE), purchased from the supermarket of Guangdong Ocean University; Glutaraldehyde, absolute ethanol, C12H25SO4Na, NaCl, KCl, KH2PO4, Na2HPO4 and HCl are all domestic pure reagents for analysis.
PBS buffer: Dissolve 8 g NaCl, 1.44 g Na2HPO4, 0.27 g KH2PO4 and 0.2 g KCl in 800 mL distilled water, adjust pH solution to 7.4 with HCl, finally add distilled water to 1 L, sterilize 20 min at 121 ℃, store in 4-guarantee refrigerator, set aside.
2% sodium dodecyl sulfate (SDS): 20 g C12H25SO4Na was dissolved in 1 L distilled water and sterilized for 20 min at 121 ℃ for 0.1 MPa. It was stored in a 4 ℃ refrigerator.
The medium used for the test
Liquid medium
- MM30 liquid medium (g/L): 1 g (NH4)2SO4,3 g KH2PO4,6 g Na2PO4,0.5 mL Trace metal salt solution, pH is 7.1; Mixture 44 solution (mg/100 mL): 17.7 mg Na2B4O7, 39.2 mg CuSO4,20. 1 mg CaCl2, 1095 mg ZnSO4 250 mg EDTA, 100 μ L and 150 μ L concentrated H2SO4 were added to prevent precipitation; Trace metal salt solution (mg/100 mL): 0.5 mg EDTA, 1 mg CaCO3, 0.5 mg FeSO4, 10 mg MgSO4, 10 mg MnSO4, 10 mL Mixture 44.
- LB liquid medium (g/L): 10 g Tryptone, 5 g Yeast extract, 10 g NaCl, pH is 7.0.
Solid medium
18% Agar was added to the liquid medium, sterilized by high pressure steam (121 ℃, 0.1 MPa) for 20 min, then poured into a petri dish and set aside after cooling and solidification.
Screening of PE-degrading microorganisms
PE preprocessing
The PE powder used in the experiment was pretreated by putting it in a sterile petri dish, irradiating it under ultraviolet light for 12 hours, and turning it every 30 min. For PE film treatment, the plastic fresh-keeping bag was cut to the size of 3×5 cm, and then irradiated under ultraviolet light for 12 hours, flipped every 30 min. In order to verify the asepsis, the powder and film irradiated under ultraviolet lamp were inoculated in solid medium and cultured in a constant temperature incubator at 30 ℃ for 3 days. if there is no colony growth around the powder and film, it is considered aseptic.
Enrichment and purification of PE plastic degrading bacteria
Put 10 g of sediment sample into a flask and add 90 mL aseptic distilled water, shake fully for 15 min, take 1 mL supernatant, add it to a 50 mL flask, add 30 mL MM30 and 0.2g PE powder, shake in a constant temperature concussion incubator at 30 ℃ for 180 r/min, put 1 mL bacterial liquid into new MM30 and PE every 7 days, and passage for 7 times. Finally, 10 μ L bacterial solution was diluted to 10-4 with aseptic distilled water, coated on MM30 solid medium, and evenly sprinkled with PE powder. The colonies with different shapes, sizes and colors were selected and purified repeatedly on MM30 solid medium after incubation in a constant temperature incubator at 30 ℃ for 48 h. Finally, the single colony was inoculated in LB broth medium for 12 ~ 24 hours, then 25% glycerol was added and stored in the refrigerator at -80 ℃.
Strain identification
Gram staining kit was used for staining identification and Hukai microbial biochemical identification tube (070010 bacterial biochemical identification tube) was used for biochemical identification. Gram staining was used following the listed steps: (1) fixing: absorbing 10 μ L bacteria on the slide, adding aseptic distilled water, diluting coating, and then fixing by alcohol flame; (2) primary dyeing: adding crystal purple dye, 1 min, rinsing with distilled water; (3) mordant dyeing: adding Gram iodine solution, 1 min, rinsing with distilled water; (4) decolorization: dripping 95% decolorized alcohol for 30 s, rinsing with distilled water. (5) re-staining: drop of Gaza yellow fuhong dye solution, 1 min, rinse with distilled water; (6) microscopic examination: after the glass slides were dried, the staining was observed under the microscope, Gram-positive bacteria were purple and Gram-negative bacteria were red.
Gene sequencing and phylogenetic tree analysis
In order to further determine the species and genus of the isolated bacteria, PCR amplification and sequencing were used to further identify the isolated bacteria at the molecular level. The isolated strains were cultured in LB broth medium for 36 ~ 48 h. DNA was extracted by bacterial DNA extraction kit and amplified by PCR with 515F and 907R primers (Table 1, Table 2 and Table 3). After the PCR product was obtained, 1% agarose gel electrophoresis was performed, and the rest was sent to Guangzhou Tianyi Biotechnology Co., Ltd for sequencing. The homology of the sequences was compared in the BLAST data of NCBI. Finally, the phylogenetic tree was constructed by using the software MEGA 7.0 for analysis.
PE weight loss rate detection
After activating the degradation bacteria and adjusting OD600 to 1, 4 mL solution and 36 mL MM30 medium were inoculated in a 50 mL wide-mouth conical flask, and then 4 portions of PE membrane were exposed for 120 days. One portion of PE membrane was taken out every 30 days. To remove the bacteria on the membrane surface, the specific steps are carried out as follows: (1) rinse with PBS buffer to remove excess culture medium and bacteria; (2) soak with 2% sodium dodecyl sulfate (SDS) solution for 2 hours; (3) rinse with warm distilled water to remove excess SDS; (4) 2% glutaraldehyde fixed solution for 2 hours; (5) 50% ethanol ultrasound twice, 30 min each time; (6) 75% ethanol overnight. (7) 100% anhydrous ethanol ultrasonic 3 times, 30 min each time. (8) after drying to constant weight, weigh with 3 repeats in each group, and calculate the corresponding weight loss rate, weight loss rate = (original weight-degraded weight) / original weight × 100%.
Detection by PE Fourier transform infrared spectrometer
After activating the degrading bacteria and adjusting OD600 to 1, 4 mL solution and 36 mL MM30 medium were inoculated in a 50 mL wide-mouth conical flask, and then put into 0.2 g PE powder. In addition, the blank control group was treated with 4 mL saline and 36 mL MM30 medium, and then added 0.2 g PE powder. After continuous culture in 180 r/min incubator at 30 ℃ for 120 days, a certain amount of MM30 medium was added every 10 days to maintain the final reaction system at 40 mL, with 3 repeats in each group. After the bacteria were removed according to the previous method and dried, the Fourier transform infrared spectrometer was used to detect the changes of functional groups on the surface of PE in the spectral range of 675 ~ 4000 cm-1.