Alteromonas strongly-strong ZHV10-EB2410164 with nitrogen fixation activity and application of alteromonas strongly-strong ZHV10-EB2410164

Abstract

The invention provides an alteromonas strongly-strong strain ZHV10-EB2410164 with nitrogen fixation activity, and belongs to the technical field of marine microorganisms. The alteromonas sp.ZHV10-EB2410164 disclosed by the invention is preserved in the Guangdong Microbial Culture Collection Center, the preservation number is GDMCC No: 65175, the preservation date is September 23, 2024, and the preservation address is the 5th building, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou. The strong alteromonas sp. Disclosed by the invention has relatively good nitrogenase activity, and the nitrogenase activity is 105.54 U / mL. The coculture of the strong alteromonas gelatinae and the eucheuma gelatinae can significantly improve the absorption and utilization of ammonia nitrogen in a culture solution for algae, can significantly promote the growth of the eucheuma gelatinae and improve the stress resistance of the eucheuma gelatinae, has very high application value, and is suitable for large-scale popularization and application.

Description

Technical Field

[0001] The invention relates to the technical field of marine microorganisms, and in particular to a strain of Alteromonas robustus ZHV10-EB2410164 with nitrogen fixation activity and an application thereof. Background Art

[0002] Betaphycus gelatinum is a red algae edible seaweed, widely cultivated and eaten in Southeast Asia. It has a crispy taste and high nutritional value. It contains a variety of trace elements required by the human body and rich proteins, vitamins and carbohydrates. Now, with the increasing demand for ecological and healthy food, the cultivation and sales of Betaphycus gelatinum are attracting more and more attention.

[0003] Eucheuma truncatum is a macroalgae that requires a large amount of nitrogen during its growth. The nitrogen form that Eucheuma truncatum can use is NH 4 + and NO 3- However, if seawater lacks NH 4 + and NO 3- , which will limit the growth of Eucheuma chinensis. Prokaryotes use nitrogenase complexes to convert atmospheric N 2 The catalysis of nitrogen into biologically active ammonia is called biological nitrogen fixation (BNF), but biological nitrogen-fixing bacteria are mostly found in the soil and are difficult to survive in the marine environment. Therefore, finding nitrogen-fixing bacteria that are adapted to the marine environment can help large seaweeds resist the malnutrition environment and is of great significance to the growth of large seaweeds. Summary of the invention

[0004] The purpose of the present invention is to provide a robust Alteromonas ZHV10-EB2410164 with nitrogen-fixing activity and its application. The robust Alteromonas of the present invention can survive in the marine environment, play a nitrogen-fixing role, and thus promote the growth of Eucheuma truncatula.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a strain of Alteromonas robustus ZHV10-EB2410164 with nitrogen fixation activity, which is deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC No: 65175, a deposit date of September 23, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The present invention also provides a method for preparing the bacterial liquid of the Alteromonas robusta ZHV10-EB2410164, wherein the Alteromonas robusta ZHV10-EB2410164 is inoculated into a fermentation medium for fermentation culture to obtain a bacterial liquid.

[0008] Preferably, the fermentation temperature is 25-28° C., the fermentation speed is 140-160 rpm, and the fermentation time is 23-25 ​​h.

[0009] The present invention also provides the use of the Alteromonas robustus ZHV10-EB2410164 in all or part of the following (1) to (5):

[0010] (1) Application in the preparation of nitrogenase;

[0011] (2) Application in promoting the growth of Eucheuma chinensis;

[0012] (3) Application in improving the stress resistance of Eucheuma truncatula;

[0013] (4) Application in the preparation of a bacterial agent for promoting the growth of Eucheuma chinensis;

[0014] (5) Application in the preparation of a bacterial agent to improve the stress resistance of Eucheuma truncatula.

[0015] The present invention also provides a method for promoting the growth of Eucheuma chinensis, wherein Eucheuma chinensis and the Alteromonas robustus ZHV10-EB2410164 are co-cultured.

[0016] Preferably, the bacterial liquid and culture solution of Eucheuma australis and Alteromonas robusta ZHV10-EB2410164 are mixed according to 5 g: 0.8-1.2 mL: 800-1200 mL, placed in the culture solution, and co-cultured.

[0017] Preferably, the culture conditions for the co-culture are as follows: the light quantum flux density is 40 to 100 μmol / (m2·s), the temperature is 25 to 28°C, and the light culture time is 10 to 14 h / d.

[0018] By adopting the above technical scheme, the present invention has the following beneficial effects: the robust Alteromonas ZHV10-EB2410164 of the present invention has good nitrogenase activity, and the nitrogenase activity is 105.54U / mL. The co-cultivation of the robust Alteromonas and Eucheuma agaricus of the present invention can significantly increase the ammonia nitrogen in the culture solution for algae to absorb and utilize, can significantly promote the growth of Eucheuma agaricus and improve its stress resistance, has high application value, and is suitable for large-scale promotion and application.

[0019] Biological Deposit Description

[0020] The strong alteromonas (Alteromonasfortis) ZHV10-EB2410164 of the present invention is deposited in the Guangdong Provincial Microbiological Culture Collection Center, with a deposit number of GDMCC No: 65175, a deposit date of September 23, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The growth of the strain on Ashuber's nitrogen-free medium.

[0022] Figure 2 This is the change of ammonia nitrogen in the culture solution during 1 to 3 days of co-cultivation.

[0023] Figure 3 These are the growth indicators of Eucheuma in each group after 21 days of co-cultivation.

[0024] Figure 4 These are the stress resistance indicators of Eucheuma in each group after 21 days of co-cultivation.

[0025] Figure 5 This is a photo of Eucheuma japonicum. DETAILED DESCRIPTION

[0026] The present invention provides a strain of Alteromonas robustus ZHV10-EB2410164 with nitrogen fixation activity, which is deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC No: 65175, a deposit date of September 23, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0027] The present invention also provides a method for preparing the bacterial solution of Alteromonas robustus ZHV10-EB2410164, wherein the Alteromonas robustus ZHV10-EB2410164 is inoculated into a fermentation medium for fermentation culture to obtain a bacterial solution. The fermentation culture temperature is 25 to 28°C, more preferably 26 to 28°C, and more preferably 28°C; the fermentation culture speed is 140 to 160 rpm, and the fermentation culture time is 23 to 25 hours.

[0028] The present invention also provides the use of the Alteromonas robustus ZHV10-EB2410164 in all or part of the following (1) to (5):

[0029] (1) Application in the preparation of nitrogenase;

[0030] (2) Application in promoting the growth of Eucheuma chinensis;

[0031] (3) Application in improving the stress resistance of Eucheuma truncatula;

[0032] (4) Application in the preparation of a bacterial agent for promoting the growth of Eucheuma chinensis;

[0033] (5) Application in the preparation of a bacterial agent to improve the stress resistance of Eucheuma truncatula.

[0034] The present invention also provides a method for promoting the growth of Eucheuma chinensis, wherein Eucheuma chinensis is co-cultured with the bacterial solution of Alteromonas robustus ZHV10-EB2410164.

[0035] In the present invention, firstly, Eucheuma chinensis is taken, attachments on the surface of the algae are removed, and the algae are soaked in sterile seawater and then ultrasonically cleaned. Then, the Eucheuma chinensis is immersed in sterile seawater containing a mixed antibiotic solution to obtain sterile Eucheuma chinensis. The present invention preferably selects Eucheuma chinensis with vigorous growth, and then removes attachments on the surface of the algae, wherein the attachments on the surface of the algae include epiphytic microorganisms and sand particles loosely attached to the surface of the algae. After removing the attachments on the surface of the algae, the algae are soaked in sterile seawater, and the soaking time is 3 to 5 hours, more preferably 3.5 hours to 4.5 hours, and more preferably 4 hours. After soaking, ultrasonic cleaning is performed, and the power of the ultrasonic cleaning is 40 to 60 W, more preferably 45 to 55 hours, and more preferably 50 hours. The time of the ultrasonic cleaning is 8 to 12 minutes, more preferably 9 to 11 minutes, and more preferably 10 minutes. After ultrasonic cleaning, the Eucheuma truncatum is immersed in sterile seawater containing a mixed antibiotic solution, wherein the mixed antibiotics include ampicillin, streptomycin sulfate and kanamycin sulfate, and the final concentrations of ampicillin, streptomycin sulfate and kanamycin sulfate in sterile seawater are independently 0.4-0.6 g / L, further preferably 0.45-0.55 g / L, and more preferably 0.5 g / L; the immersion time is 23-25 ​​h, further preferably 23.5-24.5 h, and more preferably 24 h.

[0036] Then, Eucheuma agaricus was co-cultured with a bacterial solution of Alteromonas robustus ZHV10-EB2410164, wherein the spore concentration in the bacterial solution of Alteromonas robustus ZHV10-EB2410164 was 2-3×10 8 CFU / mL, more preferably 2.2 to 2.8×10 8 CFU / mL, more preferably 2.5×10 8CFU / mL; the mixing ratio of the bacterial solution of Eucheuma agaricus and Alteromonas robustus ZHV10-EB2410164 and the culture solution is 5g:0.8~1.2mL:800~1200mL, more preferably 5g:0.9~1.1mL:900~1100mL, more preferably 5g:1mL:1000mL. The culture solution is a mixture of seawater and F / 2 culture medium, and the salinity of the culture solution is 28~32‰, more preferably 29~32‰, and more preferably 31‰. The co-culture condition is a light quantum flux density of 40~100μmol / (m 2 ·s), more preferably 70 to 90 μmol / (m 2 ·s), more preferably 80 μmol / (m 2 ·s); the temperature is 25-28°C, more preferably 26-28°C, and more preferably 27°C; the culture includes light culture and dark culture, and the light culture time is 10-14h / d, more preferably 11-13h, and more preferably 12h.

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] Example 1 Isolation and Identification of Alteromonas robustus ZHV10-EB2410164

[0039] Eucheuma chinensis was collected from Haiwei Town, Changjiang City, Hainan Province. The collected Eucheuma chinensis was washed three times with sterile seawater to remove the epiphytic microorganisms, sand and other attachments loosely attached to the algae surface. The epiphytic bacteria firmly attached to the algae were repeatedly wiped with a sterile cotton swab, and then the cotton swab was directly applied to the Asubei nitrogen-free medium plate for bacterial isolation and purification. The strain grew well on the nitrogen-free medium after three streakings ( Figure 1 The transparent small droplet-shaped substance in the solution is the colony), and the strain is numbered as ZHV10-EB240164.

[0040] The genomic DNA of Alteromonas robustus ZHV10-EB2410164 was extracted using a bacterial DNA extraction kit (Guangzhou Meiji Biotechnology Co., Ltd.), and the genomic DNA of the strain was used as a template. The 27F / 1492R universal primers upstream primer 27F: 5'-AGAGTTTGATCA( / C)TGGCTCAG-3' (SEQ ID NO.1) and downstream primer 1492R: 5'-TACGGC( / T)TACCTTGTTACGACTT-3' (SEQ ID NO.2) were used for amplification. The nucleotide sequence of the obtained 16S rDNA (1492R unidirectional sequencing) was a fragment as shown in SEQ ID NO.3:

[0041]

[0042] The size of the amplified fragment was 1116bp. The 16S rDNA sequence of the strain was most similar to that of Alteromonas fortis, with a homology of 96.68%. The strain had a close relationship and the same taxonomic status. Therefore, it can be determined that the strain belongs to the phylum Proteobacteria, class Gammaproteobacteria, orders Alteromonadales, family Alteromonadaceae, genus Alteromonas, and Alteromonas fortis.

[0043] Example 2 Determination of nitrogenase activity of Alteromonas robusta ZHV10-EB2410164 The nitrogenase activity was determined using a microbial nitrogenase (NITS) ELISA assay kit (Jiangsu Enzyme-Free Biotechnology Co., Ltd.). The results showed that the nitrogenase activity of Alteromonas robusta ZHV10-EB2410164 was 105.54 U / mL.

[0044] Example 3 Nitrogen fixation performance of Alteromonas robustus ZHV10-EB2410164 and its growth-promoting effect on Eucheuma truncatula

[0045] Take 5g of vigorously growing Eucheuma chinensis, remove the epiphytic microorganisms, sand and other attachments loosely attached to the surface of the algae, soak it in 1L sterile seawater for 4h, and perform ultrasonic cleaning for 10min (50W, 5s / 5s). Then, soak the Eucheuma chinensis in 1L sterile seawater containing a mixed antibiotic solution (final concentrations of ampicillin, streptomycin sulfate, and kanamycin sulfate were 0.5g / L, respectively) for 24h.

[0046] Alteromonas robustus ZHV10-EB2410164 was inoculated into liquid 2216E medium and cultured at 28°C and 150 rpm until the spore concentration of the strain reached 2.5×10 8 CFU / mL.

[0047] Eucheuma agaricus and Micrococcus luteus ZCP35-EB240159 were mixed and co-cultured. The co-culture culture medium was prepared by adding sterile seawater to F / 2 culture medium. Culture conditions: light quantum flux density 80 μmol / (m 2·s), temperature 27℃, salinity 31‰, pH=8.2, photoperiod: light culture 12h / dark culture 12h, relevant indicators were detected after 21d of culture, and the experiment was divided into 3 groups G1-G3. The control bacteria in group G3 was Micrococcus luteus ZCP35-EB240159 in the patent with patent number "202411617701X"; 4 parallel experiments were set in each group. The experimental settings are shown in Table 1.

[0048] Table 1 Experimental settings

[0049]

[0050] The ammonia nitrogen value in the culture medium was detected on the 1st to 3rd day of co-cultivation. The ammonia nitrogen value was measured using a Lovibond SpectroDirect multi-parameter water quality spectrophotometer and an ammonia nitrogen consumption reagent (purchased from the Lovibond official website, item number 535600). Figure 2 shown. Figure 2 The results showed that the ammonia nitrogen value of group G2 was significantly higher than that of the other groups starting from the second day, proving that Alteromonas robusta ZHV10-EB2410164 can effectively convert nitrogen in the air into ammonia nitrogen for absorption and utilization by algae.

[0051] After 21 days of co-cultivation, the contents of chlorophyll, carotenoids, phycocyanin, phycoerythrin, and related growth indicators such as weight gain rate and specific growth rate of Eucheuma aconitifolia were measured. The results are shown in Table 3. The results showed that there were no significant differences in chlorophyll a, carotenoids, phycocyanin, and phycoerythrin among the three experimental groups ( Figure 3 a- Figure 3 d), the weight gain rate and specific growth rate of the G2 group after co-culture with Alteromonas robusta ZHV10-EB2410164 for 21 days were significantly higher than those of the G1 group and G3 group without strains ( Figure 3 e in Figure 3 f) in the figure indicates that the co-cultivation of Alteromonas robustus ZHV10-EB2410164 and Eucheuma chinensis can significantly promote the growth of Eucheuma chinensis.

[0052] Fv / Fm is the maximum photochemical efficiency and is an important indicator for measuring the light energy conversion efficiency of the PSII reaction center. When subjected to external environmental stress, the Fv / Fm value will drop significantly, which can directly reflect the degree of stress on the algae. Various antioxidant substances and antioxidant enzymes in plants constitute the total antioxidant capacity (T-AOC). After suffering a certain degree of stress, the body adjusts its own antioxidant system to increase the T-AOC content, thereby enhancing its adaptability to the environment. Superoxide dismutase (SOD) is an important resistance enzyme in plants that can remove free radicals in the body and reduce the damage of free radicals to the body. Low plant malondialdehyde (MDA) content means that the plant has strong antioxidant properties and can better resist external pressure.

[0053] After 21 days of co-cultivation, the Fv / Fm value, T-AOC content, SOD content, MDA content and other related stress resistance indicators of Eucheuma were determined. Figure 4 shown. Figure 4 The results showed that there were no significant differences in Fv / Fm values ​​and T-AOC contents among the three experimental groups. The SOD content in group G2 was significantly higher than that in group G1, while the MDA content was significantly lower than that in group G1, indicating that the addition of Alteromonas robustus ZHV10-EB2410164 was harmless to Eucheuma sinensis and could improve its stress resistance.

[0054] As can be seen from the above examples, the present invention provides a robust Alteromonas ZHV10-EB2410164 with nitrogen-fixing activity and its application. The robust Alteromonas of the present invention has good nitrogenase activity, and the nitrogenase activity is 105.54U / mL. The co-cultivation of the robust Alteromonas and Eucheuma agaricus of the present invention can significantly increase the ammonia nitrogen in the culture solution for algae to absorb and utilize, and can significantly promote the growth of Eucheuma agaricus and improve its stress resistance.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Alteromonas robustus ZHV10-EB2410164 having nitrogen-fixing activity, characterized in that: It is deposited in Guangdong Provincial Microbiological Culture Collection Center, with the deposit number GDMCC No: 65175, the deposit date is September 23, 2024, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The method for preparing the bacterial solution of Alteromonas robusta ZHV10-EB2410164 according to claim 1, characterized in that: The Alteromonas robustus ZHV10-EB2410164 is inoculated into a fermentation medium for fermentation culture to obtain the bacterial liquid.

3. The preparation method according to claim 2, characterized in that: The fermentation temperature is 25-28° C., the fermentation speed is 140-160 rpm, and the fermentation time is 23-25 ​​h.

4. Use of the robust bacteria ZHV10-EB2410164 of claim 1 in all or part of the following (1) to (5): (1) Application in the preparation of nitrogenase; (2) Application in promoting the growth of Eucheuma chinensis; (3) Application in improving the stress resistance of Eucheuma truncatula; (4) Application in the preparation of a bacterial agent for promoting the growth of Eucheuma chinensis; (5) Application in the preparation of a bacterial agent to improve the stress resistance of Eucheuma truncatula.

5. A method for promoting the growth of Eucheuma chinensis, characterized in that: The Eucheuma agaricus and the Alteromonas robusta ZHV10-EB2410164 described in claim 1 are co-cultured.

6. The method according to claim 6, characterized in that: The co-culture culture solution is a mixture of seawater and culture medium, and the salinity of the culture solution is 28-32‰.

7. The method according to claim 6, characterized in that The bacterial liquid and culture solution of Eucheuma australis and Alteromonas robusta ZHV10-EB2410164 were mixed according to 5 g: 0.8-1.2 mL: 800-1200 mL, placed in the culture solution, and co-cultured.

8. The method according to claim 7, characterized in that The bacterial solution concentration is 2 to 3×10 8 CFU / mL.

9. The method according to claim 8, characterized in that The co-culture conditions are as follows: the light quantum flux density is 40-100 μmol / (m 2 ·s), the temperature is 25-28°C, and the light culture time is 10-14h / d.

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