Method for regulating competition among microalgae by utilizing rose kocuria and application of method

Abstract

The invention provides a method for regulating competition among microalgae by using bacterium Kocuria rosea and application of the method. According to the method, through a co-culture experiment and environmental factor research, the key effect of the rose kocuria in regulation of competition of the chlorella and the karenia mikimotoi is disclosed, and an effective means for improving productivity and controlling harmful algal blooms is provided for aquaculture.

Description

Technical Field

[0001] The present invention relates to the fields of marine ecology, aquaculture and biotechnology, and particularly to a method for regulating the competition between microalgae by using the strain of Kocuria rosea, and the application of this method in promoting the growth of beneficial microalgae and inhibiting harmful algae in aquaculture. Background Art

[0002] The statements in this section merely provide background technical information related to the present invention and do not necessarily constitute prior art.

[0003] In the marine ecosystem, heterotrophic bacteria and microalgae coexist as the main members of the microbial community. Specific bacterial strains can regulate the growth and metabolic processes of microalgae, but the mechanism of the change in the ecological competitiveness between green algae driven by bacteria and other algae is not yet clear. Chlorella ( Chlorella vulgaris ), as a key microalgae genus in aquaculture, has the dual functions of nutritional supplementation and water purification. However, the toxic dinoflagellate Karenia mikimotoi ( Karenia mikimotoi ) can cause harmful algal blooms, posing a threat to aquaculture and environmental health.

[0004] Currently, there are few studies on the mechanism of how bacteria regulate the ecological competitiveness between microalgae, and there is a lack of effective methods to selectively promote the growth of beneficial microalgae and inhibit harmful algae. Therefore, it is necessary to develop a new method to solve this problem. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for regulating the competition between microalgae by using Kocuria rosea, and the application of this method in aquaculture, so as to selectively promote the growth of Chlorella and inhibit Karenia mikimotoi.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions: The first aspect of the present invention is to provide a method for regulating the competition between microalgae by using Kocuria rosea, including the following steps: Co-culturing Chlorella and Karenia mikimotoi with Kocuria rosea. After cultivation, the growth of Chlorella is promoted while the growth of Karenia mikimotoi is inhibited.

[0007] First, the initial inoculation concentrations of Chlorella and Karenia mikimotoi are 3.5 - 4.5×10 4 cells mL -1 and 2.0 - 3.0×10 4 cells mL -1 .

[0008] Furthermore, the strain concentration of Kocuria rosea is 10 6 - 10 10 CFU mL-1 , further, the strain concentration is 10 9 CFU mL -1 .

[0009] Further, the conditions for co-culture are: the temperature is 20 - 40 °C, the pH is 5.0 - 8.0, the light condition is full-day light or light for 12 hours and darkness for 12 hours, and the culture time is 5 days.

[0010] Furthermore, the conditions for co-culture are: the temperature is 35 - 40 °C, the pH is 7.0 - 7.5, the light condition is full-day light, and the culture time is 5 days.

[0011] The second aspect of the present invention lies in providing the application of the method for regulating the competition among microalgae by using the marine bacterium Ruegeria rosea in aquaculture, and using Ruegeria rosea to selectively promote the growth of Chlorella vulgaris and inhibit Karenia mikimotoi.

[0012] Beneficial effects (1) The method of the present invention improves aquaculture productivity: by promoting the growth of the beneficial microalga Chlorella vulgaris, it provides a rich nutrient source for aquaculture and improves productivity.

[0013] (2) The method of the present invention controls harmful algal blooms: effectively inhibits the growth of the harmful alga Karenia mikimotoi, reduces the occurrence of algal blooms, and protects the aquaculture environment.

[0014] (3) The method of the present invention provides a biotechnological strategy: provides a theoretical basis and practical guidance for improving aquaculture productivity and controlling algal blooms by bacterial inoculation as a biotechnological strategy. Description of the drawings

[0015] Figure 1 shows the change in chlorophyll a concentration under different culture conditions; Figure 2 shows the change in Fv / Fm value under different culture conditions; Figure 3 shows the colony morphology of Ruegeria rosea on the plate ( Kocuria rosea ); Figure 4 shows the normal Karenia mikimotoi algal solution; Figure 5 shows the Karenia mikimotoi algal solution treated with Ruegeria rosea; Figure 6 shows the original Chlorella vulgaris algal solution; Figure 7 shows the Chlorella vulgaris algal solution treated with Ruegeria rosea; Figure 8 shows the cell morphology of Karenia mikimotoi under a thermal field emission scanning electron microscope; Figure 9 The cell morphology of Karenia mikimotoi after being treated with Kocuria rosea under a thermal field emission scanning electron microscope; Figure 10 The cell morphology of Chlorella vulgaris under a thermal field emission scanning electron microscope; Figure 11 The cell morphology of Chlorella vulgaris after being treated with Kocuria rosea under a thermal field emission scanning electron microscope. Specific implementation manners

[0016] The technical solutions of the present invention will be further explained and illustrated through specific embodiments below. All experimental supplies used in the present invention are commercially available.

[0017] Example 1 Algae cultivation: Kocuria rosea was purchased from BeiNa Biotechnology Co., Ltd. ( Kocuria rosea ), and the culture medium was: 2216E medium (5.0 g / L peptone -1 , 1.0 g / L yeast extract -1 , 0.01 g / L ferric phosphate -1 , 1000 mL of aged seawater), and it was cultured by shaking at 30 °C at a rotation speed of 150 rpm; the colony morphology of Kocuria rosea is shown in Figure 3 .

[0018] Cultivation of Chlorella vulgaris and Karenia mikimotoi: Axenic Chlorella vulgaris and Karenia mikimotoi were purchased from Shanghai Guangyu Biotechnology Co., Ltd., and the microalgae were cultured in sterilized f / 2 medium under the following culture conditions: 24 ± 0.5 °C, 12:12 h light-dark cycle, and the light intensity was 80 ± 5 µmol photons·m -2 s -1 . To ensure the aseptic state, plate culture tests were carried out and combined with microscopic examination.

[0019] Example 2 It can be seen from the thermal field emission scanning electron microscope that the normal cell morphology of Karenia mikimotoi is plump and smooth ( Figure 8 ). After adding Kocuria rosea to the normal Karenia mikimotoi algal solution ( Figure 4 ), the algal solution became turbid and then precipitated ( Figure 5 ). Thermal field emission scanning electron microscopy was performed, and the cell morphology of Karenia mikimotoi after being treated with Kocuria rosea was severely deformed and the cells were inactivated ( Figure 9 ).

[0020] It can be seen from the thermal field emission scanning electron microscope that the normal cell morphology of Chlorella vulgaris is plump and complete ( Figure 10 ). After adding Kocuria rosea to the Chlorella vulgaris algal solution ( Figure 6 ), the Chlorella vulgaris algal solution became denser ( Figure 7). Thermo-field emission scanning electron microscopy was performed, and the Chlorella cells remained plump and intact ( Figure 11 ).

[0021] Example 3 Chlorella and Karenia mikimotoi were co-cultured with a certain concentration of Kocuria rhizophila, and the growth of the algae was observed and recorded. By adjusting the temperature, pH value, and light conditions, the effects of environmental factors on the algal competition dynamics were studied.

[0022] (1) The concentration of Kocuria rhizophila strain was set to 10 6 , 10 7 , 10 8 , 10 9 and 10 10 CFU mL -1 , and the above different doses were added to the Chlorella-Karenia mikimotoi mixed algal solution. The initial inoculation concentrations of Chlorella and Karenia mikimotoi were set to 4.2×10 4 cells mL -1 and 2.5×10 4 cells mL -1 . Taking the mixed algal solution added with sterile 2216E medium as the control group, counting was performed every 24 hours by the hemocytometer method under an electron microscope, and three parallels were set. The results showed that the promotion / inhibition rates of the five doses of Kocuria rhizophila bacterial solution on Chlorella and Karenia mikimotoi were 10.2 / 20.4%, 15.5 / 25.7%, 46.8 / 44.2%, 50.1 / 77.8%, 45.2 / 70.2% respectively, proving that the Kocuria rhizophila bacterial solution with a concentration of 10 9 CFU mL -1 could exert the maximum effect, promoting the growth of 50.1% of Chlorella and inhibiting the growth of 77.8% of Karenia mikimotoi. In the control group, Chlorella and Karenia mikimotoi grew from 4.2×10 4 cells mL -1 and 2.5×10 4 cells mL -1 on the 0th day to 6.1×10 4 cells mL -1 and 1.2×10 5 cells mL -1 on the 5th day. In the mixed culture system with natural growth without bacteria, the growth and reproduction of Chlorella were completely suppressed by Karenia mikimotoi, and this situation was completely changed and reversed under the treatment of the Kocuria rhizophila bacterial solution with a concentration of 10 9 CFU mL -1 .

[0023] (2)During the cultivation for 0 - 5 days, detect the chlorophyll a content: Calculate the chlorophyll a concentration using the following formula: Ca (mgL −1 )= 12.21A 663nm − 2.81A 645nm , where A λ represents the absorbance at wavelength λ (nm).

[0024] It can be seen from Figure 1 that Rhodococcus rhodochrous significantly increased the chlorophyll a content of Chlorella vulgaris while decreasing the pigment content of Karenia mikimotoi. This result indicates that Rhodococcus rhodochrous promoted the synthesis of photosynthetic pigments in Chlorella vulgaris and inhibited the synthesis of photosynthetic pigments in Karenia mikimotoi.

[0025] (3)During the cultivation for 0 - 5 days, calculate the Fv / Fm value: Through pulse amplitude modulation (PAM) technology, use a dedicated instrument (Walz, Germany) to quantitatively analyze the chlorophyll fluorescence characteristics of Chlorella vulgaris and Karenia mikimotoi to obtain the Fv and Fm values.

[0026] The Fv / Fm value is an indicator of the efficiency of cell photosynthesis. It can be seen from Figure 2 that Rhodococcus rhodochrous significantly increased the Fv / Fm value of Chlorella vulgaris while decreasing the Fv / Fm value of Karenia mikimotoi. This result indicates that Rhodococcus rhodochrous promoted the photosynthesis of Chlorella vulgaris, improved the photosynthetic efficiency, and inhibited the photosynthetic efficiency of Karenia mikimotoi.

[0027] (4)Optimize the promoting / inhibiting effects of Rhodococcus rhodochrous on Chlorella vulgaris and Karenia mikimotoi by optimizing environmental conditions such as temperature, pH, and light conditions to improve the performance of Rhodococcus rhodochrous. It is shown that the efficacy of Rhodococcus rhodochrous in regulating the interspecific competition ability of Chlorella vulgaris and Karenia mikimotoi is optimal under the conditions of temperature 35 - 40 °C, pH 7.0 - 7.5, and full - day light illumination.

[0028] Table 1 shows the influence of temperature Table 2 shows the influence of pH Table 3 shows the influence of light conditions Example 4 Separate the cell - free filtrate and bacterial cells of Rhodococcus rhodochrous, and study their effects on the growth of Chlorella vulgaris and Karenia mikimotoi respectively. Through comparative experiments, verify the role of extracellular compounds in regulating algal competition.

[0029] The fermentation broth of *Kocuria rhizophila* cultured for 3 days was centrifuged at high speed by a centrifuge and filtered through a 0.22-μm ultra-microporous filter membrane to obtain a filtered solution after sterilization. The precipitate was washed repeatedly 3 times with an equal volume of f / 2 nutrient solution to obtain pure bacteria. The sterilized filtrate and the resuspended solution of pure bacteria were added to the mixed algal solution of *Chlorella vulgaris* and *Karenia mikimotoi* at an equal ratio (3%). The initial inoculation concentrations of *Chlorella vulgaris* and *Karenia mikimotoi* were 4.2×10 4 cells mL -1 and 2.5×10 4 cells mL -1 , respectively. The results showed that the promotion / inhibition rates of the pure bacteria of *Kocuria rhizophila* on *Chlorella vulgaris* and *Karenia mikimotoi* were 1.2% and 1.8%, respectively, and the promotion / inhibition rates of the sterilized filtrate on *Chlorella vulgaris* and *Karenia mikimotoi* were 50.2% and 78.9%, respectively. It was shown that *Kocuria rhizophila* indirectly affected the interspecific competition relationship between *Chlorella vulgaris* and *Karenia mikimotoi* by secreting extracellular substances.

[0030] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, various modifications and changes can be made to the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for regulating competition among microalgae using Kocuria rosea, characterized in that, Including the following steps: Co-culturing Chlorella vulgaris and Karenia mikimotoi with Kocuria rosea. After culturing, it promotes the growth of Chlorella vulgaris while inhibiting the growth of Karenia mikimotoi.

2. The method for regulating the competition among microalgae by using Kocuria rosea according to claim 1, characterized in that, The initial concentrations of Chlorella vulgaris and Karenia mikimotoi are 3.5 - 4.5×10 4 cells mL -1 and 2.0 - 3.0×10 4 cellsmL -1 .

3. A method for regulating competition among microalgae using Kocuria rosea according to claim 1, characterized in that, The strain concentration of Kocuria rosea is 10 6 -10 10 CFU mL -1 .

4. The method for regulating competition among microalgae by using Kocuria rosea according to claim 3, characterized in that The strain concentration is 10 9 CFU mL -1 .

5. A method for regulating competition among microalgae using Kocuria rosea according to claim 1, characterized in that, The conditions for co-culturing are as follows: the temperature is 20 - 40 °C, the pH is 5.0 - 8.0, the light condition is full-day light or 12 hours of light and 12 hours of darkness, and the culture time is 5 days.

6. The method for regulating competition among microalgae by using Kocuria rosea according to claim 5, wherein The conditions for co-culturing are as follows: the temperature is 35 - 40 °C, the pH is 7.0 - 7.5, the light condition is full-day light, and the culture time is 5 days.

7. A method for regulating competition among microalgae by using Kocuria rosea according to claim 1, characterized in that, Kocuria rosea includes Kocuria rosea, isolated bacterial cells or cell-free filtrates.

8. Use of the method for regulating competition among microalgae by using the marine bacterium *Kocuria rosea* described in claim 1 in aquaculture, characterized in that, Using Kocuria rosea to selectively promote the growth of Chlorella vulgaris and inhibit Karenia mikimotoi.

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