Probiotic algae bacteria and method for culturing isochrysis galbana by using same

Abstract

The invention provides a beneficial algae bacterium and a method for culturing isochrysis galbana by using the beneficial algae bacterium, the beneficial algae bacterium separated from an isochrysis galbana solution is added to effectively improve the breeding density of the isochrysis galbana and inhibit harmful vibrio at the same time, the specific algae strain is isochrysis galbana 3018 (Isochrysis galbana) NMB-Ig-3018, and the preservation number is CCTCC (China Center For Type Culture Collection) 20241471. One specific bacterial strain is pseudoalteromonas aureus NMB-Ig-Pf-0001, and the preservation number of the pseudoalteromonas aureus NMB-Ig-Pf-0001 is CCTCC (China Center For Type Culture Collection) CM20241074. The invention further discloses a preparation method of the pseudoalteromonas aureus NMB-Ig-Pf-0001. According to the method disclosed by the invention, the breeding density of the isochrysis galbana is far higher than that of the isochrysis galbana cultured by a conventional Ningda No.3 culture medium, more microalgae can be supplied under the same water body condition, and the method is very beneficial to microalgae quantity control and production management in the actual large-scale shellfish production process. In addition, the effect of inhibiting vibrios is achieved while the density of the algae is increased, and poison of the vibrios in aquaculture water is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture bait microalgae cultivation, and in particular to an algae-beneficial bacterium and a method for culturing Isochrysis galbana by using the same. Background Art

[0002] Disease is one of the main reasons affecting the development of aquaculture, among which Vibrio disease is one of the most common bacterial diseases in aquaculture. In order to prevent and control diseases, people overuse antibiotics in aquaculture, resulting in drug residues in aquatic products, which has attracted great attention. In order to improve the safety and quality of aquaculture, bacteria with Vibrio antagonism are increasingly becoming a good alternative to antibiotics. In addition, the biomass yield of bait microalgae cannot fully meet the needs of shellfish seed breeding and rapid growth, which has become one of the main limiting factors facing the current shellfish aquaculture industry.

[0003] Isochrysis galbana is a single-celled individual. The cells are naked and have a variety of shapes, but most of them are oval. The young cells have a slightly flat dorsal and ventral surface, so the side view is oblong or rectangular. Two equal-length tail whip-type flagella grow at the front of the cell. The flagella are smooth and have no attachments or swelling bodies. Their length is 1-2 times that of the cell. Isochrysis galbana mainly reproduces by asexual binary fission. It belongs to the class of coccolithophytes and the family of Isochrysis galbana. It is a single-celled motile individual with a variety of shapes, an oval shape, no cell wall, and two equal-length flagella. There are two large and lateral chromatophores in the cell. There is one nucleus, located near the center of the cell, which is golden brown. It mainly reproduces asexually, but also sexually. Asexual reproduction is carried out by longitudinal cell fission, and a special endospore can also be produced. When the environment is bad, it can form a gelatinous group phase, which is a sexual isogamous combination mode. It is widely used to feed mussels, oysters, mud clams, etc., and is considered to be an ideal bait for the larvae of marine bivalves.

[0004] Currently, in terms of cultivating Isochrysis galbana, the reproduction density of Isochrysis galbana cultivated by conventional cultivation methods is low, and the shellfish fed with microalgae grow more harmful Vibrio during the cultivation process, which cannot meet the actual use needs.

[0005] Therefore, screening and obtaining bacteria that can both promote the growth of microalgae and have antagonistic effects on Vibrio is of great significance for further improving the biomass yield of microalgae and promoting the healthy breeding of shellfish seedlings. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a beneficial algae bacterium, which is used to cultivate Isochrysis galbana, so as to solve the problem that the Isochrysis galbana obtained by the existing cultivation method has a low reproduction density during the cultivation process and cannot meet the actual use needs. A beneficial algae bacterium is provided that can not only promote the growth of microalgae but also have an antagonistic effect on Vibrio, and the beneficial algae bacterium can increase the biomass of microalgae and promote the healthy breeding of shellfish seedlings.

[0007] The invention provides a beneficial algae bacterium, the preservation number of the beneficial algae bacterium is CCTCC NO: M20241074, and the beneficial algae bacterium is used for culturing Isochrysis galbana.

[0008] The invention can improve the culture density of Isochrysis galbana and reduce the growth of harmful Vibrio in the shellfish culture process by co-culturing the beneficial algae bacteria and Isochrysis galbana.

[0009] In a possible embodiment, the beneficial algae bacteria are Pseudoalteromonas aureus separated from the algae liquid of Isochrysis galbana.

[0010] In a possible implementation manner, the Isochrysis galbana has a preservation number of CCTCC NO: M20241471, and has strong environmental adaptability and the same nutritional value as that of common Isochrysis galbana.

[0011] In a possible embodiment, the algae strain of Isochrysis galbana is isolated from Isochrysis galbana 3018 in shellfish aquaculture water in Ningde, Fujian.

[0012] Through the above possible implementation modes, the present invention can improve the culture density of Isochrysis galbana and reduce the growth of harmful Vibrio in the shellfish culture process by co-culturing the beneficial algae bacteria of the present invention with Isochrysis galbana. The present invention provides a beneficial algae bacteria that improves the reproduction density of Isochrysis galbana and inhibits Vibrio. By adding a beneficial algae bacteria Pseudoalteromonas aureus separated from the algae liquid of Isochrysis galbana, the reproduction density of Isochrysis galbana is effectively improved, and the growth of harmful Vibrio is inhibited, thereby making up for the deficiencies of the prior art.

[0013] Another technical problem to be solved by the present invention is to provide a method for culturing Isochrysis galbana with the beneficial algae bacteria, so as to solve the problem that the reproduction density of Isochrysis galbana cultivated by the conventional cultivation method in the prior art is low, and the shellfish fed with microalgae as bait grow more harmful Vibrio during the cultivation process, which cannot meet the actual use needs.

[0014] In order to solve the above problems, the present invention provides a method for culturing Isochrysis globosum with the beneficial algae bacteria, comprising the following steps:

[0015] S1: preparing a culture medium, wherein the formula of the culture medium includes: KNO3: 80-120 mg / L, KH2PO4: 8-12 mg / L, MnSO4·H2O: 2-3 mg / L, FeSO4·7H2O: 2-3 mg / L, EDTA-Na2: 8-12 mg / L and vitamin B;

[0016] S2: inoculating Isochrysis globosum into the culture medium prepared in step S1 for culturing;

[0017] S3: When the Isochrysis galbana in step S2 is cultured to the exponential phase, the beneficial algae bacteria are inoculated into the culture medium for culture, and the cell density ratio of the beneficial algae bacteria to the Isochrysis galbana is 1:(1-50).

[0018] The above-mentioned cultivation method of the present invention makes the reproduction density of Isochrysis galbana much higher than that of microalgae cultivated in conventional culture medium cultivation methods, and can provide more microalgae supply under the same water conditions, which is very beneficial to the control of microalgae quantity and production management in the actual large-scale production of shellfish. In addition, a method of cultivating Isochrysis galbana with beneficial algae bacteria in the present invention can increase the algae density while inhibiting Vibrio, thereby reducing the toxicity of Vibrio in the aquaculture water.

[0019] In one possible embodiment, the formula of the culture medium includes: KNO3: 100 mg / L, KH2PO4: 10 mg / L, MnSO4·H2O: 2.5 mg / L, FeSO4·7H2O: 2.5 mg / L, EDTA-Na2: 10 mg / L and vitamin B.

[0020] In one possible embodiment, the vitamin B includes 6 μg / L of vitamin B1 and 0.05 μg / L of vitamin B 12 .

[0021] The present invention further optimizes the formula of the culture medium and selects vitamin B1 and vitamin B 12 As a vitamin, it further increases the reproduction density of the culture.

[0022] The present invention also provides an application of beneficial algae bacteria and Isochrysis galbana, which includes using the culture medium after the culture in step S3 for shellfish culture, and feeding the beneficial algae bacteria and Isochrysis galbana in the culture medium to shellfish, thereby promoting the breeding of shellfish seedlings.

[0023] By using the beneficial algae bacteria and Isochrysis galbana in the culture medium of the present invention in the cultivation of shellfish, the breeding density of shellfish can be increased and the healthy breeding of shellfish can be promoted. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a comparison chart of the effects of different culture methods on the growth of Isochrysis galbana 3018;

[0025] Figure 2 This is a comparison chart of the effects of different beneficial algae bacteria on the growth of Vibrio;

[0026] Figure 3 This graph shows the effect of co-culture of bacteria and Isochrysis galbana 3018 on the growth of Vibrio. DETAILED DESCRIPTION

[0027] First, those skilled in the art should understand that these implementations are only used to explain the technical principles of the embodiments of the present application, and are not intended to limit the protection scope of the embodiments of the present application. Those skilled in the art can make adjustments to them as needed to adapt to specific application scenarios.

[0028] The invention provides a beneficial algae bacterium, the preservation number of the beneficial algae bacterium is CCTCC NO: M20241074, and the beneficial algae bacterium is used for culturing Isochrysis galbana.

[0029] As a preferred solution, the beneficial algae bacteria are Pseudoalteromonas aureus separated from the algae liquid of Isochrysis galbana.

[0030] As a preferred solution, the Isochrysis galbana has a preservation number of CCTCC NO: M20241471, and has strong environmental adaptability and the same nutritional value as that of common Isochrysis galbana.

[0031] As a preferred embodiment, the algae strain of Isochrysis galbana is isolated from Isochrysis galbana 3018 in shellfish aquaculture water in Ningde, Fujian.

[0032] The present invention also provides a method for culturing Isochrysis globosum using beneficial algae bacteria, comprising the following steps:

[0033] S1: preparing a culture medium, wherein the formula of the culture medium includes: KNO3: 80-120 mg / L, KH2PO4: 8-12 mg / L, MnSO4·H2O: 2-3 mg / L, FeSO4·7H2O: 2-3 mg / L, EDTA-Na2: 8-12 mg / L and vitamin B;

[0034] S2: inoculating Isochrysis globosum into the culture medium prepared in step S1 for culturing;

[0035] S3: When the Isochrysis galbana in step S2 is cultured to the exponential phase, the beneficial algae bacteria are inoculated into the culture medium for culture, and the cell density ratio of the beneficial algae bacteria to the Isochrysis galbana is 1:(1-50).

[0036] As a preferred solution, the formula of the culture medium includes: KNO3: 100 mg / L, KH2PO4: 10 mg / L, MnSO4·H2O: 2.5 mg / L, FeSO4·7H2O: 2.5 mg / L, EDTA-Na2: 10 mg / L and vitamin B.

[0037] As a preferred solution, the vitamin B includes 6 μg / L of vitamin B1 and 0.05 μg / L of vitamin B 12 .

[0038] As a preferred solution, the cell density ratio of the beneficial algae bacteria to the Isochrysis galbana is 1:50.

[0039] The following provides an embodiment combined with specific numerical values ​​to further expand the above technical solution of the present invention:

[0040] The present application is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] Embodiment 1:

[0042] This embodiment provides a beneficial algae bacterium and a method for culturing Isochrysis globosum using the same:

[0043] The beneficial algae bacteria are specifically strains of Pseudoalteromonas flavipulchra NMB-Ig-Pf-0001 separated from the algae liquid of Isochrysis galbana 3018, and its preservation number is CCTCC NO: M20241074.

[0044] The galbana Isochrysis, specifically the algae strain, is Isochrysis galbana 3018 (Isochrysis galbana) NMB-Ig-3018 separated from the shellfish aquaculture water body of Ningde, Fujian. The algae strain has strong environmental adaptability and the same nutritional value as ordinary Isochrysis galbana. Its preservation number is CCTCC NO: M20241471.

[0045] The bait microalgae cultivation technology provided by the present invention is as follows:

[0046] The selected culture medium was Ningda No. 3 medium, which was used as the culture medium of Isochrysis galbana 3018. Ningda No. 3 medium consisted of KNO3 (100 mg / L), KH2PO4 (10 mg / L), MnSO4·H2O (2.5 mg / L), FeSO4·7H2O (2.5 mg / L), EDTA-Na2 (10 mg / L), vitamin B1 (6 μg / L), vitamin B 12 (0.05 μg / L), and when the microalgae are cultured to the exponential phase, the bacteria are inoculated into the golden algae culture medium for co-cultivation.

[0047] The method of using it to cultivate Isochrysis globosum includes:

[0048] S1: prepare the above-mentioned Ningda No. 3 culture medium;

[0049] S2: inoculating Isochrysis globosum into the culture medium prepared in step S1 for culturing;

[0050] S3: When the Isochrysis galbana in step S2 is cultured to the exponential phase, the beneficial algae bacteria are inoculated into the culture medium for culture, and the cell density ratio of the beneficial algae bacteria to the Isochrysis galbana is 1:50.

[0051] Embodiment 2:

[0052] This embodiment provides a beneficial algae bacterium and a method for culturing Isochrysis globosum using the same:

[0053] The beneficial algae bacteria are specifically strains of Pseudoalteromonas flavipulchra NMB-Ig-Pf-0001 separated from the algae liquid of Isochrysis galbana 3018, and its preservation number is CCTCC NO: M20241074.

[0054] The galbana Isochrysis, specifically the algae strain, is Isochrysis galbana 3018 (Isochrysis galbana) NMB-Ig-3018 separated from the shellfish aquaculture water body of Ningde, Fujian. The algae strain has strong environmental adaptability and the same nutritional value as ordinary Isochrysis galbana. Its preservation number is CCTCC NO: M20241471.

[0055] The bait microalgae cultivation technology provided by the present invention is as follows:

[0056] The selected culture medium was Ningda No. 3 medium, which was used as the culture medium of Isochrysis galbana 3018. Ningda No. 3 medium consisted of KNO3 (100 mg / L), KH2PO4 (10 mg / L), MnSO4·H2O (2.5 mg / L), FeSO4·7H2O (2.5 mg / L), EDTA-Na2 (10 mg / L), vitamin B1 (6 μg / L), vitamin B 12 (0.05 μg / L), and when the microalgae are cultured to the exponential phase, the bacteria are inoculated into the golden algae culture medium for co-cultivation.

[0057] The method of using it to cultivate Isochrysis globosum includes:

[0058] S1: prepare the above-mentioned Ningda No. 3 culture medium;

[0059] S2: inoculating Isochrysis globosum into the culture medium prepared in step S1 for culturing;

[0060] S3: When the Isochrysis galbana in step S2 is cultured to the exponential phase, the beneficial algae bacteria are inoculated into the culture medium for culture, and the cell density ratio of the beneficial algae bacteria to the Isochrysis galbana is 1:1.

[0061] Embodiment 3:

[0062] This embodiment provides a beneficial algae bacterium and a method for culturing Isochrysis globosum using the same:

[0063] The beneficial algae bacteria are specifically strains of Pseudoalteromonas flavipulchra NMB-Ig-Pf-0001 separated from the algae liquid of Isochrysis galbana 3018, and its preservation number is CCTCC NO: M20241074.

[0064] The galbana Isochrysis, specifically the algae strain, is Isochrysis galbana 3018 (Isochrysis galbana) NMB-Ig-3018 separated from the shellfish aquaculture water body of Ningde, Fujian. The algae strain has strong environmental adaptability and the same nutritional value as ordinary Isochrysis galbana. Its preservation number is CCTCC NO: M20241471.

[0065] The bait microalgae cultivation technology provided by the present invention is as follows:

[0066] The selected culture medium was Ningda No. 3 medium, which was used as the culture medium of Isochrysis galbana 3018. Ningda No. 3 medium consisted of KNO3 (100 mg / L), KH2PO4 (10 mg / L), MnSO4·H2O (2.5 mg / L), FeSO4·7H2O (2.5 mg / L), EDTA-Na2 (10 mg / L), vitamin B1 (6 μg / L), vitamin B 12 (0.05 μg / L), and when the microalgae are cultured to the exponential phase, the bacteria are inoculated into the golden algae culture medium for co-cultivation.

[0067] The method of using it to cultivate Isochrysis globosum includes:

[0068] S1: prepare the above-mentioned Ningda No. 3 culture medium;

[0069] S2: inoculating Isochrysis globosum into the culture medium prepared in step S1 for culturing;

[0070] S3: When the Isochrysis galbana in step S2 is cultured to the exponential phase, the beneficial algae bacteria are inoculated into the culture medium for culture, and the cell density ratio of the beneficial algae bacteria to the Isochrysis galbana is 1:10.

[0071] The above-mentioned Examples 1-3 of the present invention (i.e., the examples in which Pseudoalteromonas aureus was co-cultured with Isochrysis galbana 3018 at different algae-bacterial cell density ratios (1:1, 1:10 and 1:50)) and Isochrysis galbana 3018 cultured in conventional Ningda No. 3 culture medium without adding bacteria were used as controls to compare the algae cell densities of Isochrysis galbana 3018 under different culture methods. The comparison results are as follows: Figure 1As shown, the results show that compared with the cultivation of microalgae in conventional Ningda No. 3 medium, Pseudoalteromonas aureus has a significant promoting effect on the growth of Isochrysis globosum 3018, and the promoting effect is most obvious when the algae-bacteria density ratio of the embodiment is 1:50 ( Figure 1 ).

[0072] At the same time, in order to verify the effect of beneficial algae bacteria on the growth of Vibrio, the present invention preliminarily screened out 7 strains of beneficial algae bacteria that have a promoting effect on the growth of Isochrysis globosum 3018 in the early stage, including: Bacillus, Oceanic stipes, Digitaria roseobacter, Alteromonas, Marinobacter, Alteromonas mackeri and Pseudomonas aureus used in the present invention, and analyzed the effect of these beneficial algae bacteria on the growth of Vibrio. These 7 strains of beneficial algae bacteria were co-cultured with Vibrio vulnificus, Vibrio harveyi, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio anguillarum and Vibrio alginolyticus, and the test results were as follows: Figure 2 As shown in the figure, the results showed that among the 7 strains of beneficial algae, only Pseudoalteromonas aureus had a significant inhibitory effect on the growth of Vibrio ( Figure 2 ), which further verifies that the present invention solves several technical problems existing in the background technology by adding the above-mentioned golden yellow Pseudoalteromonas as the beneficial algae bacteria of the present invention and further processing through a specially formulated culture medium.

[0073] The present invention also analyzes the effect of co-culture of bacteria and Isochrysis galbana 3018 on the growth of Vibrio: Pseudoalteromonas aureus has a significant antagonistic effect on Vibrio when cultured alone. In order to determine whether this antagonistic effect still exists when co-cultured with Isochrysis galbana 3018, the antagonistic effect of Pseudoalteromonas aureus on Vibrio when co-cultured with Isochrysis galbana 3018 is further analyzed. The results of the analysis are as follows: Figure 3 As shown in the results, the co-culture of Pseudoalteromonas aureus and Isochrysis galbana has a significant inhibitory effect on the growth of six strains of Vibrio including Vibrio vulnificus ( Figure 3 ). Therefore, the co-culture of Pseudoalteromonas aureus and I. galbana 3018 not only increased the density of algae, but also inhibited Vibrio.

[0074] The present invention selects Pseudoalteromonas aureus as the beneficial algae bacteria, combines it with Isochrysis galbana for co-cultivation, and adopts the Ningda No. 3 culture medium selected and prepared in the present invention. The three act synergistically, thereby achieving an increase in algae density and also inhibiting Vibrio, thereby solving the problem that the Isochrysis galbana obtained by the conventional cultivation method in the prior art has a low reproduction density, and that more harmful Vibrio grow during the cultivation of shellfish using microalgae as bait, which cannot meet actual use needs.

[0075] In the description of the embodiments of the present application, it should be noted that in the description of the present application, terms such as "inside" and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the drawings. This is only for the convenience of description, and does not indicate or imply that the device or component must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present application.

[0076] In the description of the present application, the description with reference to the terms "one embodiment", "some embodiments", "in the present embodiment", "specific example", or "some examples" etc. means that the specific features, mechanisms, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, mechanisms, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0077] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A beneficial algae bacterium, characterized in that: The preservation number of the beneficial algae bacteria is CCTCC NO: M20241074, and the beneficial algae bacteria is used for culturing Isochrysis galbana.

2. The beneficial algae bacteria according to claim 1, characterized in that The beneficial algae bacteria are Pseudoalteromonas aureus separated from the algae liquid of Isochrysis galbana.

3. The beneficial algae bacteria according to claim 1, characterized in that The preservation number of the Isochrysis galbana is CCTCCNO: M20241471, and the Isochrysis galbana has strong environmental adaptability and the same nutritional value as that of the common Isochrysis galbana.

4. The beneficial algae bacteria according to claim 3, characterized in that The algae strain of Isochrysis galbana was isolated from Isochrysis galbana 3018 in the shellfish breeding water body of Ningde, Fujian.

5. A method for culturing Isochrysis globosum using the beneficial algae bacteria according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1: preparing a culture medium, wherein the formula of the culture medium includes: KNO3: 80-120 mg / L, KH2PO4: 8-12 mg / L, MnSO4·H2O: 2-3 mg / L, FeSO4·7H2O: 2-3 mg / L, EDTA-Na2: 8-12 mg / L and vitamin B; S2: inoculating Isochrysis globosum into the culture medium prepared in step S1 for culturing; S3: When the Isochrysis galbana in step S2 is cultured to the exponential phase, the beneficial algae bacteria are inoculated into the culture medium for culture, and the cell density ratio of the beneficial algae bacteria to the Isochrysis galbana is 1:(1-50).

6. The method for culturing Isochrysis globosum with beneficial algae bacteria according to claim 5, characterized in that: The formula of the culture medium includes: KNO3: 100 mg / L, KH2PO4: 10 mg / L, MnSO4·H2O: 2.5 mg / L, FeSO4·7H2O: 2.5 mg / L, EDTA-Na2: 10 mg / L and vitamin B.

7. The method for culturing Isochrysis globosum with beneficial algae bacteria according to claim 5, characterized in that: The vitamin B includes 6 μg / L of vitamin B1 and 0.05 μg / L of vitamin B 12 .

8. The method for culturing Isochrysis globosum with beneficial algae bacteria according to claim 5, characterized in that: The cell density ratio of the beneficial algae bacteria to the Isochrysis galbana is 1:

50.

9. An application of beneficial algae bacteria and Isochrysis galbana, characterized in that: The application includes using the culture medium after the cultivation in step S3 of claim 5 for shellfish cultivation, feeding the beneficial algae bacteria and Isochrysis galbana in the culture medium to shellfish, thereby promoting the breeding of shellfish seed.

Images