Nitrate reducing bacterium, separation and purification method thereof and application of nitrate reducing bacterium in aspect of improving heat resistance of coral larvae

Abstract

The invention discloses a nitrate reducing bacterium, a separation and purification method of the nitrate reducing bacterium and application of the nitrate reducing bacterium in the aspect of improving heat resistance of coral larvae, the nitrate reducing bacterium WH36-1 is preserved in Guangdong Microbiological Culture Collection Center, the preservation number is GDMCC No.65560, the preservation date is November 28, 2024, and the suggested classification name is the nitrate reducing bacterium. By inoculating nitrate reducing bacteria WH36-1, the survival rate of the antler cup type coral larvae in a high-temperature environment can be remarkably improved in a short time, and the adhesion rate of the larvae is not interfered. The whole process is easy to operate, low in cost, safe and free of pollution, the coral supplement amount is increased by improving the heat resistance of the larvae, and restoration and protection of a coral reef ecological system are assisted.

Description

Technical Field

[0001] The invention belongs to the technical field of strain separation and application, and specifically relates to a nitrate-reducing bacterium and a separation and purification method thereof, and an application thereof in improving the heat resistance of coral larvae. Background Art

[0002] The coral reef ecosystem is one of the most important marine ecosystems. It is home to up to a quarter of the world's marine life and is known as the "tropical rainforest in the ocean". It provides food, ecology, socio-economic and cultural services to humans. Reef-building corals are the backbone of coral reef ecosystems. They adapt to the strong light and oligotrophic reef environment by symbiosis with microorganisms such as zooxanthellae and bacteria. Therefore, maintaining a healthy microbial community structure is particularly important for the health of reef-building corals.

[0003] However, global warming-induced marine heat waves and rising surface sea temperatures have led to frequent large-scale bleaching events of reef-building corals, causing the gradual degradation of coral reef ecosystems, seriously threatening the ecological balance of the ocean and the sustainable development of human society. Existing studies have shown that some symbiotic bacteria are closely related to the carbon / nitrogen / sulfur element cycle in the symbiosis of reef-building corals, while some can reduce the negative impact of pathogens on corals through the synthesis of antimicrobial compounds, spatial and nutrient competition. In addition, environmental changes can cause dynamic changes in the symbiotic bacterial community in reef-building corals. Given the important role played by symbiotic bacteria in coral symbiosis and their flexibility and operability, reorganizing the symbiotic bacterial community of reef-building corals is considered a potential means to improve the thermal tolerance of corals.

[0004] At present, the development of probiotic resources for improving the heat tolerance of corals is mainly focused on adult corals. However, high temperatures can also cause DNA damage, metabolic disorders, developmental deformities and high mortality in coral larvae. Given the important role of coral larvae in coral population renewal and maintenance of coral reef ecosystems, it is crucial to improve their heat tolerance. Therefore, there is an urgent need to screen and develop probiotic resources and explore their application in improving the heat tolerance of coral larvae to promote the restoration and protection of coral reef ecosystems. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the shortcomings of the prior art and provide nitrate-reducing bacteria (Nitratireductoraquimarinus) WH36-1, which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No.65560, a deposit date of November 28, 2024, a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and a suggested classification name of Nitratireductor aquimarinus.

[0008] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for separating and purifying nitrate-reducing bacteria.

[0009] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0010] Cut off the Acropora cup coral fragments in seawater, rinse the surface with filtered seawater and grind, dilute the grinding solution in a gradient manner, place it in 2216E liquid culture medium for culture, and finally transfer the culture solution to 2216E solid culture medium for culture, and pick a single colony for purification.

[0011] As a preferred embodiment of the method for separating and purifying nitrate-reducing bacteria of the present invention, the gradient dilution is 10 -4 , 10 -5 and 10 -6 Serial dilutions.

[0012] As a preferred embodiment of the method for separating and purifying nitrate-reducing bacteria of the present invention, the 2216E liquid culture medium is cultured at a temperature of 27.5-28.5°C for 48 hours; the 2216E solid culture medium is cultured at a temperature of 27.5-28.5°C for 48 hours.

[0013] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of nitrate-reducing bacteria in improving the heat resistance of coral larvae.

[0014] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0015] Pick a single colony from the seed tube, streak it on a 2216E solid culture medium plate, invert and culture to obtain activated nitrate-reducing bacteria, pick a single colony of activated nitrate-reducing bacteria and culture it in a 2216E liquid culture medium to obtain a seed solution;

[0016] The seed liquid is inoculated into a new 2216E liquid medium for fermentation, the supernatant is discarded by centrifugation, the bacterial cells are collected, seawater is added to resuspend the bacterial cells, and finally sterile seawater is added to resuspend the bacterial cells to obtain a nitrate-reducing bacterial suspension;

[0017] Cut off the Acropora cup coral branches of different parents in seawater, fix them on cement bases, and place them in water for cultivation; collect the Acropora cup coral larvae of different parents within 24 hours, mix them evenly and place them in a water tank, and then place the collected Acropora cup coral larvae in a container filled with filtered seawater;

[0018] The nitrate-reducing bacteria suspension was added to the container containing the larvae to infect the larvae, and then the larvae were transferred to a new container containing filtered seawater to recover.

[0019] As a preferred embodiment of the application of nitrate-reducing bacteria in the present invention in improving the heat resistance of coral larvae, the inverted culture time is 3 to 7 days; the liquid culture medium culture temperature is 27.5 to 28.5°C, and the culture time is 48 to 50 hours.

[0020] As a preferred solution for the application of nitrate-reducing bacteria in the present invention in improving the heat resistance of coral larvae, the amount of seed liquid inoculation is 8-12% (v / v); the fermentation temperature is 27.5-28.5°C and the fermentation time is 48h.

[0021] As a preferred embodiment of the application of nitrate-reducing bacteria in the present invention in improving the heat resistance of coral larvae, the temperature of the water body for cultivation is 25.7-26.3°C, the salinity is 34.7-35.3‰, and the light intensity is 190-210 μE·m -2 ·s -1 , the photoperiod is 12L:12D.

[0022] As a preferred embodiment of the application of nitrate-reducing bacteria in the present invention in improving the heat resistance of coral larvae, the infection time of the larvae is 24 hours; and the recovery time of the larvae after being transferred to a new container containing filtered seawater is 24 hours.

[0023] Beneficial effects of the present invention:

[0024] (1) The present invention can significantly improve the survival rate of Acropora hornba larvae in a high temperature environment in a short period of time by inoculating nitrate-reducing bacteria (Nitratireductora quimarinus) WH36-1, and does not interfere with the attachment rate of the larvae;

[0025] (2) The operation is simple, low-cost, safe and pollution-free. It aims to increase the amount of coral replenishment by improving the heat tolerance of larvae, thereby helping to restore and protect the coral reef ecosystem. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0027] Figure 1 This is a morphological identification diagram of the bacterial colony prepared in Example 1 of the present invention;

[0028] Figure 2 The phylogenetic tree of the strain constructed in Example 1 of the present invention;

[0029] Figure 3 This is a graph showing the survival rate and attachment rate of Acropora hornba larvae at high temperature after inoculation with nitrate-reducing bacteria in Example 2 of the present invention. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0031] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0032] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0033] The raw materials used in the present invention are commercially available unless otherwise specified.

[0034] The 2216E solid culture medium and 2216E liquid culture medium used in the present invention were purchased from Haibo Biotechnology.

[0035] The components of 2216E solid culture medium include 5.0 g / L peptone, 1.0 g / L yeast powder, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L sodium nitrate, 0.008 g / L sodium dihydrogen phosphate, 15.0 g / L agar, and pH is 7.6±0.2.

[0036] The components of 2216E liquid culture medium include 5.0 g / L peptone, 1.0 g / L yeast powder, 0.1 g / L ferric citrate, 19.45 g / L sodium chloride, 5.98 g / L magnesium chloride, 3.24 g / L sodium sulfate, 1.8 g / L calcium chloride, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 0.08 g / L potassium bromide, 0.034 g / L strontium chloride, 0.022 g / L boric acid, 0.004 g / L sodium silicate, 0.0024 g / L sodium fluoride, 0.0016 g / L sodium nitrate, 0.008 g / L sodium dihydrogen phosphate, and pH 7.6±0.2.

[0037] Example 1: Isolation and purification method of nitrate-reducing bacteria

[0038] Isolation and purification method of strains: In the Sanya Xidao Coral Reef Nature Reserve, at a depth of 3m, cut off a 3cm Acropora cup coral fragment, rinse its surface with filtered seawater 5 times and grind it thoroughly, and then dilute the grinding solution in a gradient manner to obtain 10 -4 , 10 -5 and 10 -6 The diluted grinding solution was placed in 2216E liquid medium, and after culturing at 28°C and 180 rpm for 48 h, 200 μL of the culture solution was evenly spread on 2216E solid medium and cultured for 48 h. A single colony was picked for purification.

[0039] Identification method of strains:

[0040] ① Morphological identification: Use an optical microscope to observe the colony morphology of the strains isolated and purified above, such as Figure 1 As shown;

[0041] ② Molecular identification: The whole genome of the pure culture of the strain was extracted using a bacterial whole genome rapid extraction kit, and PCR amplification was performed using 16s rDNA universal primers 27F and 1492R;

[0042] Amplification system: 2.5 μL 10×PCRBuffer (Mg 2+plus), 2 μL dNTP (2.5 mmol·L -1 ), 1 μL of forward primer (10 μmol·L -1 ), 1 μL of reverse primer (10 μmol·L -1 ), 1 μL DNA template, 0.15 μL rTaq, 17.35 μL DEPC water;

[0043] Amplification conditions: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 35 cycles, 72°C extension for 10 min, and finally storage at 4°C;

[0044] After the PCR amplification products were detected by 1% agarose gel electrophoresis, the target fragments were recovered and purified using the SanPrep column DNA gel recovery kit, and then sequenced. The phylogenetic tree of the strain was constructed using BLAST comparison analysis and MEGA software, as shown in Figure 2. Figure 2 As shown;

[0045] Figure 1 The colony morphology obtained for identification was characterized as follows: the colonies were smooth, with regular edges, easily formed into strips, and milky white in color; Figure 2 Phylogenetic tree of the constructed strains.

[0046] The sequencing results of the strain are shown in the sequence table. The bacteria were identified as nitrate-reducing bacteria and deposited in Guangdong Microbial Culture Collection Center with the deposit number GDMCC No.65560, deposit date: November 28, 2024, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and the proposed classification name: Nitrate-reducing bacteria Nitratireductoraquimarinus

[0047] Example 2: Application of nitrate-reducing bacteria WH36-1 to improve the heat resistance of Acropora hornbeam coral larvae

[0048] (1) Activation of nitrate-reducing bacteria strain WH36-1: A single colony was picked from the seed tube of nitrate-reducing bacteria prepared in Example 1, streaked on the prepared 2216E solid culture medium plate, and inverted for about 7 days to complete the activation;

[0049] (2) Preparation of nitrate-reducing bacteria WH36-1 bacterial suspension: First, pick a single colony of activated nitrate-reducing bacteria and put it into 50 mL of 2216E liquid culture medium, and culture it on a shaker at 28°C and 150 rpm for 48 hours to obtain a seed solution. Inoculate it into a new 2216E liquid culture medium at a 10% (v / v) inoculation amount, ferment it for 48 hours under the same conditions, centrifuge it at 4000 rpm for 5 minutes, discard the supernatant, collect the bacteria, add an appropriate amount of sterilized seawater to resuspend the bacteria, repeat twice, and finally add sterile seawater to resuspend the bacteria to obtain WH36-1 bacterial suspension;

[0050] (3) Collection of Acropora cup coral larvae: Six 6-cm coral fragments from different parents were collected from the Sanya Xidao Coral Reef Nature Reserve at a depth of 3 m. They were fixed on a circular cement base and cultured in 100 L of water. The culture conditions were: water temperature 26 °C, salinity 35‰, and light intensity 200 μE·m -2 ·s -1 , the photoperiod was 12L:12D; larvae from the six parent strains were collected within 24 h and mixed in a plastic water tank with a volume of about 10 L, and then the collected larvae were randomly distributed into 15 plastic boxes containing 500 ml of filtered seawater, each containing 80 Acropora hornii cup coral larvae, where the filtered seawater was obtained by filtering through filter cotton with a pore size of 1 μm;

[0051] (4) Infection of Acropora larvae with nitrate-reducing bacteria WH36-1: Freshly prepared nitrate-reducing bacteria WH36-1 suspension was randomly added into 5 plastic boxes containing larvae and marked (water volume was 500 mL, final concentration of bacteria was 1×10 6 CFU·mL -1 ), and 2 mL of filtered seawater was added to the remaining 10 plastic boxes containing larvae. Among them, after WH36-1 was infected with Acropora hornbeam coral larvae for 24 h, the larvae in the plastic box were transferred to a new plastic box containing filtered seawater to recover for 24 h; during the experiment, the water temperature was maintained at 26 °C and the light intensity was maintained at 200 μE·m -2 ·s -1 , photoperiod 12L:12D.

[0052] (5) Nitrate-reducing bacteria WH36-1 help Acropora cup coral larvae resist high temperatures: 10 plastic boxes containing larvae that were not inoculated with WH36-1 were randomly divided into two groups. One group was the control group (Control), which did not receive any treatment; the other group was the high temperature group (Heat), which was heated in a water bath. At the same time, the larvae that were previously inoculated with WH36-1 were subjected to high temperature treatment (WH36-1+Heat). During this experiment, the initial temperature was 26°C, and the heating rate was 1°C every 12 hours. After the temperature reached 31°C, it was maintained for 15 hours, and the survival rate and attachment rate of the larvae were counted. During the experiment, the light intensity was 200 μE m -2 ·s -1 , photoperiod of 12L:12D, and larvae were transferred daily to plastic boxes containing fresh filtered seawater.

[0053] Figure 3 The figure shows the effect of inoculation with nitrate-reducing bacteria WH36-1 on the survival rate and attachment rate of Acropora hornensis larvae, where (a) is the survival rate and (b) is the attachment rate. It can be seen that the survival rate of Acropora hornensis larvae in the control group was 97.40%, compared with which, the survival rate of larvae in the high temperature group was significantly reduced to 87.90%. However, the survival rate of Acropora hornensis larvae inoculated with WH36-1 after high temperature treatment (WH36-1+Heat) was 97.17%, which was significantly higher than that of the high temperature treatment group. In addition, the attachment rate of Acropora hornensis larvae did not change significantly in the above three groups.

[0054] In summary, the present invention proposes a nitrate-reducing bacteria and its isolation and purification method and its application in improving the heat resistance of coral larvae. The nitrate-reducing bacteria WH36-1 is obtained by isolating and purifying the Acropora cup coral fragments, and the bacteria are inoculated into the Acropora cup coral larvae, thereby improving the heat resistance of the coral larvae without interfering with the attachment rate of the larvae. The process is simple to operate, low in cost, safe and pollution-free. By improving the heat resistance of the larvae, the amount of coral supplementation is increased, which is beneficial to the restoration and protection of the coral reef ecosystem.

[0055] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Nitrate-reducing bacteria (Nitratireductoraquimarinus) WH36-1, deposited in Guangdong Microbial Culture Collection Center, with the deposit number GDMCC No.65560, deposit date: November 28, 2024, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, suggested classification name: Nitrate-reducing bacteria Nitratireductoraquimarinus.

2. The method for separating and purifying nitrate-reducing bacteria according to claim 1, wherein: include, Cut off the Acropora cup coral fragments in seawater, rinse the surface with filtered seawater and grind, dilute the grinding solution in a gradient manner, place it in 2216E liquid culture medium for culture, and finally transfer the culture solution to 2216E solid culture medium for culture, and pick a single colony for purification.

3. The method for separating and purifying nitrate-reducing bacteria according to claim 2, wherein: The gradient dilution is 10 -4 , 10 -5 and 10 -6 Serial dilutions.

4. The method for separating and purifying nitrate-reducing bacteria according to claim 2, wherein: The culture temperature of the 2216E liquid culture medium is 27.5-28.5°C; the culture temperature of the 2216E solid culture medium is 27.5-28.5°C.

5. Use of the nitrate-reducing bacteria as claimed in claim 1 in improving the heat resistance of coral larvae.

6. The use of nitrate-reducing bacteria as claimed in claim 5 in improving the heat resistance of coral larvae, characterized in that: include, Pick a single colony from the seed tube, streak it on a 2216E solid culture medium plate, invert and culture to obtain activated nitrate-reducing bacteria, pick a single colony of activated nitrate-reducing bacteria and culture it in a 2216E liquid culture medium to obtain a seed solution; The seed liquid is inoculated into a new 2216E liquid medium for fermentation, the supernatant is discarded by centrifugation, the bacterial cells are collected, seawater is added to resuspend the bacterial cells, and finally sterile seawater is added to resuspend the bacterial cells to obtain a nitrate-reducing bacterial suspension; Cut off the Acropora cup coral branches of different parents in seawater, fix them on cement bases, and place them in water for cultivation; collect the Acropora cup coral larvae of different parents within 24 hours, mix them evenly and place them in a water tank, and then place the collected Acropora cup coral larvae in a container filled with filtered seawater; The nitrate-reducing bacteria suspension was added to the container containing the larvae to infect the larvae, and then the larvae were transferred to a new container containing filtered seawater to recover.

7. The use of nitrate-reducing bacteria as claimed in claim 6 in improving the heat resistance of coral larvae, characterized in that: The inverted culture time is 3 to 7 days; the liquid culture medium culture temperature is 27.5 to 28.5° C., and the culture time is 48 to 50 hours.

8. The use of nitrate-reducing bacteria in improving the heat resistance of coral larvae as claimed in claim 6, characterized in that: The inoculation amount of the seed liquid is 8-12% (v / v); the fermentation temperature is 27.5-28.5°C.

9. The use of nitrate-reducing bacteria as claimed in claim 6 in improving the heat resistance of coral larvae, characterized in that: The temperature of the water body for culturing is 25.7-26.3°C, the salinity is 34.7-35.3‰, and the light intensity is 190-210 μE·m -2 ·s -1 , the photoperiod is 12L:12D.

10. The use of nitrate-reducing bacteria in improving the heat resistance of coral larvae as claimed in claim 6, characterized in that: The infection time for the larvae is 24 hours; the recovery time for the larvae after transfer to a new container containing filtered seawater is 24 hours.

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