A method for promoting rapid reproduction and attachment of porphyra yezoensis by using seawater bacterial culture waste liquid

Abstract

The present application belongs to the field of marine biotechnology, and particularly relates to a method for promoting rapid reproduction and attachment of Porphyra yezoensis by using seawater bacteria culture waste liquid. The attached seawater bacteria are isolated from the surface of Porphyra yezoensis thalli, cultured after single colony isolation, and identified by cloning 16s rDNA sequence. After obtaining the required seawater bacteria Sulfitobacter sp. or Tenacibaculum sp., the 2216E seawater bacteria liquid medium is continuously cultured at 28 DEG C for 10 hours. The bacteria liquid is centrifuged to remove the bacteria in it, and the supernatant waste liquid is collected and added to the Porphyra yezoensis PES culture medium in proportion. After 2-4 weeks of culture, the release and attachment of a large number of Porphyra yezoensis monospores can be significantly promoted. The present application reuses the waste liquid of seawater bacteria culture, and provides a new way for promoting the attachment of Porphyra yezoensis and rapid and large-scale expansion of biomass. The method can also be used for artificial construction of Porphyra yezoensis algal reef in a marine ranch, so as to achieve the purpose of rapid expansion of biomass and large-scale attachment of algal thalli, and has a wide range of application scenarios.

Description

Technical fields:

[0001] This invention belongs to the field of marine biology and seaweed farm construction, specifically relating to a method for promoting the rapid reproduction and attachment of Porphyra tenuifolia using waste liquid from marine bacterial culture. Technical background:

[0002] *Porphyra yezoensis* is an important economic red algae in my country, widely cultivated in the coastal waters of Jiangsu and Shandong provinces, with an annual output value exceeding 5 billion yuan, demonstrating significant economic value. The life cycle of *Porphyra yezoensis* involves heteromorphic alternation of generations between filamentous sporophytes and thallus gametophytes. Furthermore, the gametophytes can produce single spores for asexual reproduction. This process is very short, bypassing the filamentous stage to directly generate thallus again, which is crucial for increasing the biomass of *Porphyra yezoensis*. Laboratory cultivation of *Porphyra yezoensis* thallus primarily relies on asexual reproduction to rapidly obtain thallus material. In artificial cultivation, asexual reproduction relying on single spores is also an important pathway for the rapid and substantial increase of *Porphyra yezoensis* biomass. After release, single spores need to attach to a substrate to develop; therefore, promoting the large-scale release and attachment of single spores from *Porphyra yezoensis* thallus is of paramount importance for the rapid and substantial acquisition of its biomass.

[0003] Seaweed beds are an important component of marine ranches, but in recent years they have faced the challenge of gradual degradation and shrinking scale. Artificial seaweed beds are an important means of constructing marine ranches by building artificial reefs. The construction of *Porphyra yezoensis* reefs has not yet been reported, likely due to difficulties in increasing *Porphyra yezoensis* biomass and ensuring proper attachment of the algae to the reef. Therefore, developing methods to promote *Porphyra yezoensis* spore release and enhance single-spore adhesion is an effective means of constructing *Porphyra yezoensis* reefs.

[0004] This invention provides a method for promoting the rapid reproduction and attachment of *Porphyra yezoensis* using waste liquid from marine bacterial culture. This method uses waste liquid from laboratory-grown marine bacteria as a raw material, adding it to the normal culture medium of *Porphyra yezoensis*. This significantly promotes the release of monospores from *Porphyra yezoensis* and the attachment of single spores to the substrate, achieving the goal of rapidly and massively generating biomass. This method can be applied to laboratory cultivation of *Porphyra yezoensis* to rapidly obtain large quantities of experimental materials. It can also be applied to the construction of artificial algal reefs. By adding this waste liquid to the reef-building material, it promotes the rapid and massive propagation of *Porphyra yezoensis* biomass, and has broad application scenarios in the construction of artificial algal reefs and seaweed beds. Summary of the Invention:

[0005] The purpose of this invention is to provide a method for promoting the rapid reproduction and attachment of *Porphyra yezoensis* using waste liquid from marine bacterial culture. Marine bacteria *Sulfitobacters* sp. or *Tenacibaculum* sp. were isolated, cultured in 2216E liquid medium, and the supernatant was collected and added to the *Porphyra yezoensis* thallus culture medium. Results showed that this method can significantly promote the release and attachment of *Porphyra yezoensis* spores, and has broad application prospects in the rapid and large-scale proliferation of *Porphyra yezoensis* biomass and the construction of artificial algal reefs in seaweed farms.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for promoting rapid reproduction and attachment of *Porphyra tenuifolia* using marine bacterial culture waste liquid, the specific steps of which are as follows:

[0008] 1. Collect the leaf-like thallus of *Porphyra yezoensis* and wash it 3-5 times with clean seawater in the laboratory to remove any attached algae. Use tweezers and a brush to remove any impurities that are difficult to wash off, obtaining clean *Porphyra yezoensis* leaf-like thallus. Culture it in sterile seawater with PES added as the culture medium.

[0009] 2. Take clean leaves of *Porphyra yezoensis*, cut an appropriate amount of leaves and place them in a 1.5 mL EP tube. Add 1 mL of sterile PES seawater medium and shake on a shaker for 1-2 min. Take 1 μL of the shaken medium and add it to 1 mL of fresh sterile PES medium, shaking to mix thoroughly. Spread 20 μL of the diluted medium onto 2216E solid medium. Incubate at 28°C for 12 h until suitable single colonies appear.

[0010] 3. Pick a single colony and place it in 2216E liquid medium, and incubate it in a shaker at 28°C for 10 hours until the medium becomes turbid.

[0011] 4. Amplify the 16S rDNA sequence of the selected colonies using primers and perform Sanger sequencing, then compare the results with NCBI. Obtain the target strain Sulfitobacters sp. or Tenacibaculum sp.

[0012] The primer pairs used are:

[0013] 27F:5'-AGAGTTTGATCCTGGCTCAG-3'

[0014] 1492R:5'-GGTTACCTTGTTACGACTT-3'

[0015] 5. Expand the culture of the obtained target strain by inoculating it into 2216E liquid medium and culturing it in a shaker at 28°C for 10 hours until the medium becomes turbid.

[0016] 6. Centrifuge the turbid culture medium from step 5 at 10000g for 10 minutes to obtain the supernatant waste liquid.

[0017] 7. Take 100 μL of the waste liquid from step 6 and add it to 30 mL of sterile PES seawater medium. Culture the leaf-like structures of *Porphyra yezoensis* in a cell culture flask.

[0018] 8. Place the above-mentioned Porphyra stripes in a 15℃ constant temperature incubator and set the light conditions to 1000-2000 Lux.

[0019] Culture continuously for 2-4 weeks. Replace with fresh PES medium weekly, adding the above-mentioned proportion of marine bacterial culture waste liquid. Under a microscope, you can observe the release of numerous single spores attaching to the substrate.

[0020] The monoclonal strains of marine bacteria used were one or both of Sulfitobacters p. or Tenacibaculum sp.

[0021] The steps involve completely submerging the leaf-like structures of *Porphyra yezoensis* in sterile seawater.

[0022] The sterile seawater culture medium in step (2) is PES medium, and the culture conditions are: temperature 15-20℃, light intensity 1000-2000 Lux, and photoperiod 12 / 12-14 / 10.

[0023] Step (3) Dilute with sterile seawater to 8000-10000 times the original volume. The isolated strain needs to be expanded by 2216E liquid medium at a temperature of 28-29℃ for 12-24 hours to obtain single colonies.

[0024] The marine bacteria Sulfitobacters p. or Tenacibaculum sp. used to obtain marine bacterial culture waste liquid are isolated from bacteria attached to the surface of the leaf-like body of Porphyra stripes.

[0025] The supernatant obtained in step (4) needs to be added to the PES medium for the leaf-like structure of Porphyra stripes at a ratio of 1:600-800; the culture time is 1-4 weeks (preferably 2-4 weeks), the required temperature is 15℃, the required light intensity is 1000 Lux, and the photoperiod is 12 / 12.

[0026] The invention is described below:

[0027] The strains *Sulfitobacters* sp. or *Tenacibaculum* sp. were isolated from the thallus of *Porphyra yezoensis*. Furthermore, it is not only these two strains that promote the release and attachment of *Porphyra yezoensis* spores among the isolated bacteria; other isolated strains may also have the same effect.

[0028] The waste liquid of the seawater bacteria culture medium can be sterilized by high temperature and high pressure without affecting its addition effect.

[0029] The supernatant obtained from centrifugation has the effect of promoting the release of a large number of single spores, but simply adding the live bacteria in the precipitate to the Porphyra yezoensis culture medium alone does not have the effect of promoting the release of a large number of single spores.

[0030] The volume of the added supernatant waste liquid is not a fixed value. Adding a volume of 50-200uL or even a wider range to 30mL of Porphyra yezoensis culture medium can promote the release of a large number of single spores. In other words, the concentration range of the marine bacteria culture waste liquid is relatively wide, with a concentration of 0.15%-0.66% all having a promoting effect.

[0031] The cultivation conditions for *Porphyra tenuifolia* were as follows: temperature: 15-18℃, light intensity: 150-200 Lux, culture medium: PES medium. The culture medium was replaced weekly with a new one, and a corresponding proportion of waste marine bacterial culture solution was added.

[0032] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0033] 1. The material used in this invention is marine bacterial culture waste liquid, which has the effect of waste recycling, and the effect is significant.

[0034] 2. The method of this invention can significantly promote the release and attachment of Porphyra yezoensis spores, enabling the rapid and large-scale production of Porphyra yezoensis biomass. It has broad application prospects in laboratory cultivation, artificial cultivation in the wild, and in the construction of artificial algal reefs and seaweed farms. Attached Figure Description

[0035] Figure 1 Waste liquid from the culture medium of *Tenacibaculum* sp. was used as an additive to promote the release and attachment of *Tenacibaculum streaksii* spores. A: Macroscopic image of the control group culture. B: Microscopic image of the control group culture. C: Macroscopic image of the experimental group culture. D: Microscopic image of the experimental group culture.

[0036] Figure 2 Waste liquid from the culture medium of *Tenacibaculum* sp. was used as an additive to promote the release and attachment of *Tenacibaculum streaksii* spores. A: Macroscopic image of the control group culture. B: Microscopic image of the control group culture. C: Macroscopic image of the experimental group culture. D: Microscopic image of the experimental group culture. Detailed implementation method:

[0037] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0038] Preparation Example

[0039] 1. Collect the leaf-like thallus of *Porphyra yezoensis* and wash it three times with clean seawater in the laboratory to remove any adhering algae. Use tweezers and a brush to remove any impurities that are difficult to wash off, obtaining clean *Porphyra yezoensis* leaf-like thallus. Culture it in sterile seawater with PES added as the culture medium.

[0040] 2. Take clean leaves of *Porphyra yezoensis*, cut an appropriate amount of leaves and place them in a 1.5 mL EP tube. Add 1 mL of sterile PES seawater medium and shake on a shaker for 5 min. Take 1 μL of the shaken medium and add it to 1 mL of fresh sterile PES medium, shaking to mix thoroughly. Spread 20 μL of the diluted medium onto 2216E solid medium. Incubate at 28°C for 12 h until suitable single colonies appear.

[0041] 3. Pick a single colony and place it in 2216E liquid medium, and incubate it in a shaker at 28°C for 10 hours until the medium becomes turbid.

[0042] 4. Amplify the 16S rDNA sequence of the selected colonies using primers and perform Sanger sequencing, then compare the results with NCBI. Obtain the target strain Sulfitobacter sp. or Tenacibaculum sp.

[0043] The selected colonies were subjected to 16S rDNA sequence amplification using primer pairs. The primer pairs used for sequence amplification were:

[0044] 27F:5'-AGAGTTTGATCCTGGCTCAG-3'

[0045] 1492R:5'-GGTTACCTTGTTACGACTT-3'

[0046] Example 1: Adding waste liquid from Sulfitobacter p. culture medium promotes the release and attachment of large quantities of *Sclerotium streakedense* spores.

[0047] 1. Obtain Sulfitobactersp according to the method described in the preparation example.

[0048] 2. Incubate Sulfitobacter p. in 1.5 mL of 2216E liquid medium in a shaker at 28 °C for 12 h until turbidity is achieved.

[0049] 3. Centrifuge the turbid bacterial solution from step 2 at 10000g for 10 minutes to obtain the supernatant waste liquid.

[0050] 4. Take one *Porphyra yezoensis* thallus (approximately 1.5 cm long and 0.5 cm wide) and place it in a T75 cell culture flask. Add 30 mL of sterile PES seawater medium and 100 μL of supernatant waste liquid. Set up three parallel flasks. This serves as the experimental group. In control group 1, add an equal proportion of sterile 2216E liquid medium without any cultured bacteria. Set up three parallel flasks.

[0051] 5. Place the cell culture flasks of the treatment group and the control group in an incubator at 15-20℃, set the light conditions to 1000-2000 Lux, and the photoperiod to 12 / 12-14 / 10. Culture continuously for 4 weeks, replacing the PES medium weekly with the same proportion of supernatant waste liquid.

[0052] After 4 weeks of culture, the release and attachment of single spores were observed, and the results are shown in the attached figure. Figure 1 The results showed that, compared with the control groups (A and B), the groups (C and D) with added seawater bacteria supernatant waste liquid significantly promoted the release and attachment of *Vibrio spp.* spores.

[0053] Example 2: Adding Tenacibaculum sp. culture medium waste liquid promotes the large-scale release and attachment of streaked purple spores.

[0054] 1. Tenacibaculum sp. was obtained according to the method described in the preparation example.

[0055] 2. Incubate Tenacibaculum sp. in 1.5 mL of 2216E liquid medium in a shaker at 28 °C for 12 h until turbidity is achieved.

[0056] 3. Centrifuge the turbid bacterial solution from step 2 at 10000g for 10 minutes to obtain the supernatant waste liquid.

[0057] 4. Take one *Porphyra yezoensis* thallus (approximately 1.5 cm long and 0.5 cm wide) and place it in a T75 cell culture flask. Add 30 mL of sterile PES seawater medium and 100 μL of supernatant waste liquid. Set up three parallel flasks. This serves as the experimental group. In control group 1, add an equal proportion of sterile 2216E liquid medium without any cultured bacteria. Set up three parallel flasks.

[0058] 5. Place the cell culture flasks of the treatment group and the control group in an incubator at 15-20℃, set the light conditions to 1000-2000 Lux, and the photoperiod to 12 / 12-14 / 10. Culture continuously for 4 weeks, replacing the PES medium weekly with the same proportion of supernatant waste liquid.

[0059] After 4 weeks of culture, the release and attachment of single spores were observed, and the results are shown in the attached figure. Figure 2 The results showed that, compared with the control groups (A and B), the groups (C and D) with added seawater bacteria supernatant waste liquid significantly promoted the release and attachment of *Vibrio spp.* spores.

Claims

1. A method for promoting rapid reproduction and attachment of *Porphyra tenera* using marine bacterial culture waste liquid, characterized in that: The steps are as follows: (1) Collect the leaf-like bodies of Porphyra stripes, rinse them with seawater 3-5 times, place them in sterile seawater, and add PES as a culture medium; (2) Take the cleaned leaf-like algae of Porphyra stripes obtained above and place them in a container. Add sterile seawater and shake with a shaker. (3) The seawater after shaking was diluted and spread on 2216E solid medium for culture to obtain a single clonal strain of seawater bacteria; (4) Isolate and obtain a single clonal strain of marine bacteria, expand the culture and centrifuge to obtain the supernatant waste liquid, add it to the PES medium of Porphyra yezoensis thallus obtained in step (1) for continuous culture for 1-4 weeks. The monoclonal strain of marine bacteria used is Sulfitobacter sp .or Tenacibaculum sp. One or two of them 。 2. The method for promoting rapid reproduction and attachment of *Porphyra tenera* using marine bacterial culture waste liquid according to claim 1, characterized in that: Step (1) The leaf-like structures of Porphyra stripes are completely submerged in sterile seawater.

3. The method for promoting rapid reproduction and attachment of *Porphyra tenera* using marine bacterial culture waste liquid according to claim 1, characterized in that: The sterile seawater culture medium in step (2) is PES medium, and the culture conditions are: temperature 15-20 ℃, light 1000-2000 Lux, photoperiod 12 / 12-14 / 10.

4. The method for promoting rapid reproduction and attachment of *Porphyra tenera* using marine bacterial culture waste liquid according to claim 1, characterized in that: The oscillation time in step (2) is 1-2 minutes.

5. A method for promoting rapid reproduction and attachment of *Porphyra tenera* using marine bacterial culture waste liquid according to claim 1, characterized in that: Step (3) Dilute with sterile seawater to 8000-10000 times the original volume. The isolated strain needs to be expanded by 2216E liquid medium at a temperature of 28-29℃ for 12-24 hours to obtain single colonies.

6. A method for promoting rapid reproduction and attachment of *Porphyra tenera* using marine bacterial culture waste liquid according to claim 1, characterized in that: Seawater bacteria used to obtain seawater bacteria culture waste liquid Sulfitobacter sp .or Tenacibaculum sp. It was isolated from bacteria attached to the surface of the leaf-like body of *Porphyra yezoensis*.

7. A method for promoting rapid reproduction and attachment of *Porphyra tenera* using marine bacterial culture waste liquid according to claim 1, characterized in that: The supernatant obtained in step (4) needs to be added to the PES medium for Porphyra yezoensis thallus at a ratio of 1:600-800; the culture time is 2-4 weeks, the required temperature is 15 ℃, the required light intensity is 1000 Lux, and the photoperiod is 12 / 12.

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