Method for promoting rapid reproduction and attachment of porphyra yezoensis by using seawater bacterium culture waste liquid

Abstract

The invention belongs to the technical field of marine organisms, and particularly relates to a method for promoting rapid reproduction and attachment of porphyra yezoensis by using seawater bacterium culture waste liquid. Attached seawater bacteria are separated from the surface of porphyra yezoensis frond, a single colony is separated and then cultured, and a 16s rDNA sequence is cloned and identified. And after the required seawater bacteria Sulfibacterium sp. Or Tenacibacterium sp. Are obtained, the seawater bacteria are continuously cultured for 10 hours at the temperature of 28 DEG C by using a 2216E seawater bacteria liquid culture medium. And centrifuging the bacterial liquid, removing thalli in the bacterial liquid, collecting supernatant waste liquid, adding the supernatant waste liquid into a porphyra yezoensis PES culture medium in proportion, and culturing for 2-4 weeks, so that the release and attachment of a large amount of spores of the porphyra yezoensis can be remarkably promoted. According to the method, the waste liquid of seawater bacterium culture is recycled, and a new way is provided for promoting porphyra yezoensis adhesion and rapid and large-scale biomass amplification. The method can also be used for artificial construction of porphyra yezoensis algal reefs in marine ranches, achieves the purposes of rapid biomass amplification and large-scale algae attachment of the porphyra yezoensis algal reefs, and has wide application scenarios.

Description

Technical field:

[0001] The invention belongs to the field of marine organisms and seaweed field construction, and in particular relates to a method for promoting the rapid reproduction and attachment of Porphyra yezoensis by utilizing waste liquid of seawater bacteria culture. Technical background:

[0002] Porphyra yezoensis is an important economic red algae in my country. It is widely cultivated in the coastal waters of Jiangsu and Shandong, with an annual output value of over 5 billion yuan, and has great economic value. The life history of Porphyra yezoensis consists of filamentous sporophytes and thallus gametophytes undergoing heteromorphic generation alternation. In addition, the gametophyte of Porphyra yezoensis can also produce monospores for asexual reproduction. This process cycle is very short, and thallus can be directly generated again without going through the filamentous process, which is of great significance for the biomass expansion of Porphyra yezoensis. The laboratory cultivation of Porphyra yezoensis thallus mainly relies on the asexual reproduction process to quickly obtain thallus materials. In the process of artificial cultivation, asexual reproduction relying on monospores is also an important way to rapidly and massively expand the biomass of Porphyra. After the monospores are released, they need to attach to the substrate to develop. Therefore, promoting the release of monospores from the thallus of Porphyra yezoensis in large quantities and promoting their attachment is of great significance for the rapid and massive acquisition of its biomass.

[0003] Seaweed fields are an important part of marine ranches, but in recent years, seaweed fields have been facing the dilemma of gradual degradation and shrinking in size. The artificial creation of seaweed fields by constructing artificial algae reefs is an important means of constructing marine ranches. There are no reports on the construction of Porphyra algae reefs. The main reason may be that there are difficulties in the expansion of Porphyra biomass and the attachment of algae to the algae reefs. Therefore, developing methods to promote the release of Porphyra monospores and enhance the adhesion of monospores is an effective means to construct Porphyra algae reefs.

[0004] The present invention provides a method for promoting the rapid reproduction and attachment of Porphyra yezoensis by utilizing waste liquid from seawater bacteria culture. This method uses waste liquid from laboratory culture of seawater bacteria as raw material, and adds it to the normal culture medium of Porphyra yezoensis, which can significantly promote the release of Porphyra yezoensis monospores, and promote the attachment of monospores to the substrate, thereby achieving the purpose of rapidly and massively generating biomass. This method can be applied to the cultivation of Porphyra yezoensis in the laboratory, and rapidly and massively obtain experimental materials. It can also be applied to the construction of artificial algae reefs, and by adding the waste liquid to the reef construction materials, the rapid and massive propagation of the biomass of Porphyra yezoensis can be promoted, and it has a wide range of application scenarios in the construction of artificial algae reefs and the creation of seaweed fields. Summary of the invention:

[0005] The object of the present invention is to provide a method for promoting the rapid reproduction and attachment of Porphyra yezoensis by using waste liquid of seawater bacteria culture. The seawater bacteria Sulfitobacter sp. or Tenacibaculum sp. is isolated and obtained, and the supernatant is collected after being cultured with 2216E liquid culture medium, and added to the thallus culture medium of Porphyra yezoensis. The results show that the method can promote the release and attachment of Porphyra yezoensis monospores in large quantities, and has broad application prospects in the rapid and large-scale proliferation of Porphyra yezoensis biomass and the construction of artificial algae reefs in seaweed fields.

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

[0007] A method for promoting the rapid reproduction and attachment of Porphyra yezoensis by utilizing wastewater from seawater bacteria culture, the specific steps of which are as follows:

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

[0009] 2. Take clean Porphyra yezoensis leaves, cut an appropriate amount of leaves and place them in a 1.5mL EP tube, add 1mL of sterile PES seawater culture medium, and shake on an oscillator for 1-2 minutes. Take 1μL of the shaken culture medium, add it to 1mL of new sterile PES culture medium, and shake to mix evenly. Take 20uL of the diluted culture medium and spread it on the 2216E solid culture medium. Place it in a 28-degree constant temperature incubator and culture it for 12 hours until a single colony of appropriate size appears.

[0010] 3. Pick a single colony and place it in 2216E liquid culture medium. Place it in a 28℃ shaker and culture it for 10 hours until the culture medium becomes turbid.

[0011] 4. Use primers to amplify the 16S rDNA sequence of the selected colonies and perform Sanger sequencing, and compare them on NCBI to obtain the target strain Sulfitobacter sp. or Tenacibaculum sp.

[0012] The primer pairs used were:

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

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

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

[0016] 6. Centrifuge the turbid culture medium in step 5 at 10,000 g for 10 min to obtain the supernatant waste liquid.

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

[0018] 8. Place the above-mentioned Porphyra yezoensis in a constant temperature incubator at 15°C and set the lighting conditions to 1000-2000 Lux.

[0019] Continue to culture for 2-4 weeks. Replace the PES medium with new one every week and add the above-mentioned proportion of wastewater from seawater bacteria culture. A large number of single spores can be seen released and attached to the substrate under a microscope.

[0020] The used marine bacteria monoclonal strains are one or both of Sulfitobacter sp. and Tenacibaculum sp.

[0021] Step 1: Completely immerse the Porphyra yezoensis thallus in sterile seawater.

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

[0023] Step (3) is diluted with sterile seawater to 8000-10000 times the original volume. The isolated strain needs to be expanded and cultured in 2216E liquid culture medium at a temperature of 28-29° C. for 12-24 hours to obtain a single colony.

[0024] The seawater bacteria Sulfitobacter sp. or Tenacibaculum sp. used for obtaining the seawater bacteria culture waste liquid is isolated from bacteria attached to the surface of the thallus of Porphyra yezoensis.

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

[0026] The following is a description of the invention:

[0027] The strain Sulfitobacter sp. or Tenacibaculum sp. is isolated from the thallus of Porphyra yezoensis. And the isolated bacteria are not only the above two bacteria that have the effect of promoting the release and attachment of Porphyra yezoensis monospores, but other isolated strains may also have the same effect.

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

[0029] The supernatant waste liquid obtained by the centrifugation has the effect of promoting the massive release of single spores, but adding the live bacteria in the precipitate to the culture medium of Porphyra yezoensis alone has no effect of promoting the massive release of single spores.

[0030] The added supernatant waste liquid volume is not a fixed value, 50-200uL, or a wider range of volumes added to 30mL of Porphyra yezoensis culture medium can promote the massive release of single spores, that is to say, the concentration range of the seawater bacteria culture waste liquid is relatively wide, and the concentration of 0.15%-0.66% has a promoting effect.

[0031] The culture conditions of Porphyra yezoensis are as follows: temperature: 15-18°C, light intensity: 150-200 Lux, culture medium: PES culture medium. The culture medium is replaced with a new one every week, and a corresponding proportion of waste liquid from seawater bacteria culture is added.

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

[0033] 1. The material used in the present invention is waste liquid from seawater bacteria culture, which has the effect of waste recycling and the effect is remarkable.

[0034] 2. The method of the present invention can significantly promote the release and attachment of Porphyra yezoensis spores, and can quickly obtain a large amount of Porphyra yezoensis biomass. It has broad application prospects in laboratory cultivation, field artificial cultivation, artificial algae reef construction, and seaweed field construction. Description of the drawings:

[0035] Figure 1 The waste liquid of the culture medium of marine bacteria Tenacibaculum sp. was used as an additive to promote the release and attachment of purple yezoensis monospores. A: Macroscopic picture of the culture of the control group. B: Microscopic picture of the culture of the control group. C: Macroscopic picture of the culture of the experimental group. D: Microscopic picture of the culture of the experimental group.

[0036] Figure 2 The waste liquid of the culture medium of marine bacteria Tenacibaculum sp. was used as an additive to promote the release and attachment of purple yezoensis monospores. A: Macroscopic picture of the culture of the control group. B: Microscopic picture of the culture of the control group. C: Macroscopic picture of the culture of the experimental group. D: Microscopic picture of the culture of the experimental group. Specific implementation method:

[0037] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the present invention equally.

[0038] Preparation Example

[0039] 1. Collect the thallus of Porphyra yezoensis, wash it three times with clean seawater in the laboratory to remove the algae attached to the surface. Use tweezers and a brush to remove impurities that are difficult to wash off, obtain clean thallus of Porphyra yezoensis, and culture it in sterile seawater with PES added as the culture medium.

[0040] 2. Take clean Porphyra yezoensis leaves, cut an appropriate amount of leaves and put them in a 1.5mL EP tube, add 1mL of sterile PES seawater culture medium, and shake on an oscillator for 5 minutes. Take 1μL of the shaken culture medium, add it to 1mL of new sterile PES culture medium, and shake to mix evenly. Take 20uL of the diluted culture medium and spread it on the 2216E solid culture medium. Put it in a 28 constant temperature incubator and culture it for 12h until a single colony of appropriate size appears.

[0041] 3. Pick a single colony and place it in 2216E liquid culture medium. Place it in a 28℃ shaker and culture it for 10 hours until the culture medium becomes turbid.

[0042] 4. Use primers to amplify the 16S rDNA sequence of the selected colonies and perform Sanger sequencing, and compare them on NCBI to obtain the target strain Sulfitobacter sp. or Tenacibaculum sp.

[0043] The 16S rDNA sequence of the picked colonies was amplified using primers. The primer pairs used for sequence amplification were:

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

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

[0046] Example 1: Adding Sulfitobacter sp. culture medium wastewater to promote the massive release and attachment of Pseudomonas yezoensis

[0047] 1. Obtain Sulfitobacter sp according to the method described in the technical scheme of the preparation example.

[0048] 2. Cultivate Sulfitobacter sp. in 1.5 mL 2216E liquid medium in a shaker at 28°C for 12 h until turbid.

[0049] 3. Centrifuge the turbid bacterial solution in step 2 at 10,000 g for 10 min to obtain the supernatant waste liquid.

[0050] 4. Take a thallus of Porphyra yezoensis (about 1.5 cm long and 0.5 cm wide) and place it in a T75 cell culture bottle, add 30 mL of sterile PES seawater culture medium and 100 μL of supernatant waste liquid, and set up 3 parallel bottles. The above is used as the experimental group. Add an equal proportion of 2216E sterile liquid culture medium without any bacteria cultured to the control group 1, and set up 3 parallel bottles.

[0051] 5. Place the cell culture flasks of the above treatment group and control group in a 15-20℃ incubator, set the light conditions to 1000-2000Lux, and the photoperiod to 12 / 12-14 / 10. Cultivate continuously for 4 weeks, replace new PES medium every week and add the same proportion of supernatant waste liquid.

[0052] After 4 weeks of culture, the release and attachment of single spores were observed. Figure 1 The results showed that compared with the control group (A and B), the addition of seawater bacteria supernatant waste liquid groups (C and D) could significantly promote the release and attachment of P. yezoensis spores.

[0053] Example 2: Adding wastewater from Tenacibaculum sp. culture medium to promote the massive release and attachment of Psoralea yezoensis monospores

[0054] 1. According to the method described in the technical scheme of the preparation example, Tenacibaculum sp.

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

[0056] 3. Centrifuge the turbid bacterial solution in step 2 at 10,000 g for 10 min to obtain the supernatant waste liquid.

[0057] 4. Take a thallus of Porphyra yezoensis (about 1.5 cm long and 0.5 cm wide) and place it in a T75 cell culture bottle, add 30 mL of sterile PES seawater culture medium and 100 μL of supernatant waste liquid, and set up 3 parallel bottles. The above is used as the experimental group. Add an equal proportion of 2216E sterile liquid culture medium without any bacteria cultured to the control group 1, and set up 3 parallel bottles.

[0058] 5. Place the cell culture flasks of the above treatment group and control group in a 15-20℃ incubator, set the light conditions to 1000-2000Lux, and the photoperiod to 12 / 12-14 / 10. Cultivate continuously for 4 weeks, replace new PES medium every week and add the same proportion of supernatant waste liquid.

[0059] After 4 weeks of culture, the release and attachment of single spores were observed. Figure 2 The results showed that compared with the control group (A and B), the addition of seawater bacteria supernatant waste liquid groups (C and D) could significantly promote the release and attachment of P. yezoensis spores.

Claims

1. A method for promoting the rapid reproduction and attachment of Porphyra yezoensis by utilizing wastewater from seawater bacterial culture, characterized in that: Here are the steps: (1) collecting Porphyra yezoensis thallus, rinsing with seawater 3-5 times, and placing in sterile seawater for cultivation; (2) Place the cleaned Porphyra yezoensis thallus in a container, add sterile seawater, and shake with a shaker; (3) diluting the shaken seawater and applying it on 2216E solid culture medium for cultivation to obtain a monoclonal strain of seawater bacteria; (4) Isolate and obtain a monoclonal strain of marine bacteria, expand the culture, centrifuge to obtain a supernatant, and add it to the PES culture medium of the Porphyra yezoensis thallus obtained in step (1) for continuous culture for 1-4 weeks.

2. The method for promoting the rapid reproduction and attachment of Porphyra yezoensis by utilizing wastewater from seawater bacterial culture according to claim 1, characterized in that: The used marine bacteria monoclonal strains are one or both of Sulfitobacter sp. and Tenacibaculum sp.

3. The method for promoting the rapid reproduction and attachment of Porphyra yezoensis by utilizing wastewater from seawater bacterial culture according to claim 1, characterized in that: Step (1) The sterile seawater completely immerses the Porphyra yezoensis thallus.

4. The method for promoting the rapid reproduction and attachment of Porphyra yezoensis by utilizing wastewater from seawater bacterial culture according to claim 1, characterized in that: The sterile seawater culture medium in step (2) is PES culture medium, and the culture conditions are: temperature 15-20°C, illumination 1000-2000 Lux, and photoperiod 12 / 12-14 / 10.

5. The method for promoting the rapid reproduction and attachment of Porphyra yezoensis by utilizing wastewater from seawater bacterial culture according to claim 1, characterized in that: The shaking time in step (2) is 1-2 min.

6. The method for promoting the rapid reproduction and attachment of Porphyra yezoensis by utilizing wastewater from seawater bacterial culture according to claim 1, characterized in that: Step (3) is diluted with sterile seawater to 8000-10000 times the original volume. The isolated strain needs to be expanded and cultured in 2216E liquid culture medium at a temperature of 28-29° C. for 12-24 hours to obtain a single colony.

7. The method for promoting the rapid reproduction and attachment of Porphyra yezoensis by utilizing wastewater from seawater bacterial culture according to claim 1, characterized in that: The seawater bacteria Sulfitobacter sp. or Tenacibaculum sp. used for obtaining the waste liquid of seawater bacteria culture is isolated from bacteria attached to the surface of the thallus of Porphyra yezoensis.

8. The method for promoting the rapid reproduction and attachment of Porphyra yezoensis by utilizing wastewater from seawater bacterial culture according to claim 1, characterized in that: The supernatant obtained in step (4) needs to be added to the PES culture medium of Porphyra yezoensis thallus at a ratio of 1:600-800; the culture time is 2-4 weeks, the required temperature is 15°C, the required light is 1000 Lux, and the photoperiod is 12 / 12.

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