Bacterial-algal symbiotic system applied to freshwater aquaculture pond and construction method of bacterial-algal symbiotic system

Abstract

The invention relates to the technical field of ecological environment protection and aquaculture, in particular to a bacteria-algae symbiotic system applied to a freshwater aquaculture pond and a construction method of the bacteria-algae symbiotic system, and the construction method comprises the following steps: (1) respectively culturing chlorella pyrenoidosa and bacillus subtilis to a logarithmic phase; and (2) taking chlorella pyrenoidosa and bacillus subtilis cultured to the logarithmic phase in the step (1), adjusting the concentration, adding the chlorella pyrenoidosa and bacillus subtilis into a culture medium, and culturing in a culture room to obtain the algal-bacterial symbiotic system. According to the present invention, ammonia nitrogen, nitrite and other harmful substances in water can be efficiently and stably removed, the stable and efficient purification mechanism is provided, the water quality can be adjusted by replacing the addition of chemicals, the secondary pollution and the negative influence on aquatic organisms are avoided, and the water quality is improved. And the adaptability of the system to environmental changes such as temperature and illumination and the quick response capability to sudden pollution events are also improved.

Description

Technical Field

[0001] The invention belongs to the technical field of ecological environment protection and aquaculture, and specifically relates to a bacteria-algae symbiotic system applied to a freshwater aquaculture pond and a construction method thereof. Background Art

[0002] In the field of freshwater aquaculture, water quality management is one of the key factors for the success of aquaculture. With the development of intensive aquaculture models, how to effectively control and improve the aquaculture environment while ensuring the quality and safety of aquatic products has become a hot topic of research. Biological treatment technology, especially the symbiotic system using microorganisms and algae, has received more and more attention due to its environmental protection and sustainable characteristics. Microorganisms such as nitrifying bacteria and denitrifying bacteria can convert ammonia nitrogen into nitrite and nitrate respectively, and then into nitrogen gas, thereby reducing the toxicity of ammonia nitrogen and nitrite in the water body. Algae release oxygen through photosynthesis to improve water quality, and can also be used as natural bait to promote the growth of aquatic animals. The bacterial-algal symbiotic system is currently mainly used in urban sewage, marine aquaculture wastewater, and livestock aquaculture wastewater. However, in the field of freshwater aquaculture, the research on bacterial-algal regulation is only at the "combination" level, and there is no research on "symbiosis".

[0003] In addition, the existing technology has some problems and shortcomings in the actual application process: Difficulty in maintaining ecological balance: Under natural conditions, the ratio of bacteria and algae is difficult to control, and it is easy for one side to over-breed, resulting in system imbalance. Strong dependence on environmental conditions: Factors such as temperature, light, and pH have a great influence on the growth of bacteria and algae, and the slightest carelessness may cause the system to collapse. Slow response speed and treatment efficiency: The response speed and treatment efficiency of biological treatment are slower than those of physical and chemical methods, especially in the face of sudden large-scale pollution, the treatment capacity is limited. Therefore, it is urgent to provide a bacteria-algae symbiotic system that can overcome the above problems and apply it to water quality management in the field of freshwater aquaculture. Summary of the invention

[0004] The purpose of the present invention is to provide a bacteria-algae symbiotic system for use in freshwater aquaculture ponds and a construction method thereof. The bacteria-algae symbiotic system optimizes the pond environmental conditions by scientifically configuring and controlling the bacteria-algae ratio, thereby achieving continuous improvement of water quality and stable maintenance of ecological balance, enhancing the adaptability and stability of the system, and improving aquaculture efficiency and ecological sustainability.

[0005] The objective of the present invention is achieved through the following technical solutions:

[0006] The present invention provides a method for constructing a bacteria-algae symbiotic system applied to a freshwater aquaculture pond, comprising the following steps:

[0007] (1) Cultivating Chlorella pyrenoidosa and Bacillus subtilis to logarithmic phase respectively;

[0008] (2) Take the Chlorella pyrenoidosa and Bacillus subtilis cultured to the logarithmic phase in step (1), adjust the concentration and add them to the culture medium, place them in the culture room for cultivation, and obtain the bacteria-algae symbiotic system.

[0009] Furthermore, in step (1), the specific method for culturing the Chlorella pyrenoidosa is: using BG-11 culture medium at 25-30° C., 2000-4000 lux, and a photoperiod of more than 12 hours.

[0010] Furthermore, in step (1), the specific culture method of the Bacillus subtilis is: using LB medium to culture under the conditions of 25-30° C., 2000-4000 lux, 12 hours light / 12 hours dark.

[0011] Furthermore, in step (2), the concentration is adjusted to 1×10 6 -1×10 8 CFU·mL -1 .

[0012] Furthermore, in step (2), the adding into the culture medium refers to adding the Bacillus subtilis and the Chlorella pyrenoidosa into the culture medium so that the Bacillus subtilis and the Chlorella pyrenoidosa each account for 5% of the culture medium components.

[0013] Furthermore, in step (2), the specific parameters of the culture in the culture chamber are culturing for 3-7 days under the conditions of 25-30° C., 2000-4000 lux and a photoperiod of more than 12 hours.

[0014] The present invention also provides a bacteria-algae symbiotic system constructed by using the construction method.

[0015] The present invention also provides an application of the bacteria-algae symbiotic system in water quality management in the field of freshwater aquaculture.

[0016] The beneficial effects of the present invention are:

[0017] The present invention improves biological efficiency: by constructing and optimizing the cultivation method of the bacteria-algae symbiotic system, the biological efficiency of the bacteria-algae symbiotic system is significantly improved. The synergistic effect between bacteria and algae enhances the growth rate and yield of algae, providing new possibilities for bioenergy production.

[0018] The present invention enhances stability: the optimized bacteria-algae symbiotic system has high stability and can maintain a good growth state under different environmental conditions. This stability is an advantage that traditional methods do not have and is of great significance for long-term application and large-scale production.

[0019] The present invention improves the removal rate of nitrogen and phosphorus nutrients: compared with single algae and single bacteria systems, the bacteria-algae symbiotic system demonstrates a better ability to remove nitrogen and phosphorus nutrients from aquaculture water. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 paying creative work.

[0021] Figure 1 The figure is a diagram showing the dynamic changes of chlorophyll and cell dry weight in the bacterial-algal symbiotic system of Chlorella pyrenoidosa and Bacillus subtilis at different ratios in the present invention, wherein the left figure shows the chlorophyll concentration and the right figure shows the cell dry weight;

[0022] Figure 2 The photos are real photos observed by naked eyes of a single algae system containing only Chlorella pyrenoidosa and a bacteria-algae symbiotic system with a bacteria-algae ratio of 1:1, wherein the left photo is a single algae system and the right photo is a bacteria-algae symbiotic system;

[0023] Figure 3 The scanning electron microscope images of the single algae system containing only Chlorella pyrenoidosa and the bacteria-algae symbiotic system with a bacteria-algae ratio of 1:1 in the present invention, wherein the left image is the single algae system and the right image is the bacteria-algae symbiotic system;

[0024] Figure 4 This is a diagram showing the dynamic changes in the concentration of water quality indicators in the bacteria-algae symbiotic system with a bacteria-algae ratio of 1:1 in the present invention. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] In the following examples, Bacillus subtilis and Chlorella pyrenoidosa were purchased from China General Microbiological Culture Collection Center (1.7740) and Institute of Hydrobiology, Chinese Academy of Sciences (FACHB-9).

[0031] The method for constructing the bacteria-algae symbiotic system used in the freshwater aquaculture pond of the present invention is as follows:

[0032] (1) Strain selection:

[0033] Selecting bacterial strains that can coexist with algae, wherein the algae is preferably Chlorella pyrenoidosa, and the bacterial strain is preferably Bacillus subtilis, Bacillus subtilis and Chlorella pyrenoidosa have good affinity and synergistic effect, and these two bacteria and algae are often used in aquaculture to regulate water quality;

[0034] (2) Expansion of bacteria and algae:

[0035] Chlorella vulgaris was cultured using BG-11 medium (Macklin, Shanghai, China) at 25-30°C, 2000-4000 lux, and a photoperiod of more than 12 hours; Bacillus subtilis was cultured using LB medium (OXOID, Shanghai, China) at 25-30°C, 2000-4000 lux, and 12 hours of light / 12 hours of darkness.

[0036] (3) Construction of symbiotic system:

[0037] Take the Bacillus subtilis and Chlorella pyrenoidosa cultured to the logarithmic phase in step (2) and adjust the concentration of bacteria and algae to 1×10 6 -1×10 8 CFU·mL -1 and 1×10 6 -1×10 8 Cell·mL -1 After adjusting the ratio, add bacteria and algae into the new BG-11 culture medium so that bacteria and algae each account for 5% in the culture medium. Culture in a culture room at 25-30℃, 2000-4000lux, and a photoperiod of more than 12 hours for 3-7 days to obtain a bacteria-algae symbiotic system.

[0038] The present invention is specifically described below by way of examples.

[0039] Example 1

[0040] The method for constructing the bacteria-algae symbiotic system applied to freshwater aquaculture ponds in this embodiment 1 comprises the following steps:

[0041] (1) using BG-11 medium at 28°C, 3000 lux, and a photoperiod of 15 hours to culture Chlorella vulgaris, and using LB medium at 28°C, 3000 lux, and 12 hours of light / 12 hours of darkness to culture Bacillus subtilis;

[0042] (2) Take the Chlorella pyrenoidosa and Bacillus subtilis cultured to the logarithmic phase in step (1), and adjust the concentration of Bacillus subtilis to 1×10 6 -1×10 8 CFU·mL -1 The concentration of Chlorella pyrenoidosa was 1×10 6 -1×10 8 Cell·mL -1 , and add 5% of Chlorella and Bacillus subtilis to the culture medium according to the above concentrations, and culture it in a culture room at 28°C, 3000 lux, and a photoperiod of 15 hours for 5 days to obtain a bacteria-algae symbiotic system.

[0043] Test Example 1

[0044] The biological performance of the bacteria-algae symbiotic system for freshwater aquaculture ponds prepared in Example 1 of the present invention was evaluated by observing the color of Chlorella and the bacteria-algae symbiotic system with the naked eye and observing Chlorella and the bacteria-algae symbiotic system with a scanning electron microscope, and determining the growth rate, yield, chlorophyll content and other indicators of the bacteria-algae symbiotic system to evaluate the biological performance and advantages of the symbiotic system.

[0045] The dynamic changes of chlorophyll and cell dry weight in the bacterial-algal symbiotic system with different ratios of Chlorella pyrenoidosa and Bacillus subtilis are shown in the figure. Figure 1 As shown in the figure, the actual visual observation of the single algae system containing only Chlorella pyrenoidosa and the bacterial-algal symbiotic system with a bacterial-algal ratio of 1:1 is shown in the figure. Figure 2 As shown in the electron microscope image Figure 3 shown.

[0046] Depend on Figure 1 It can be seen that when the ratio of Chlorella pyrenoidosa and Bacillus subtilis in the constructed bacteria-algae symbiotic system is 1:1, the values ​​of chlorophyll and cell dry weight are the highest, which not only shows that compared with the single algae system, the bacteria-algae symbiotic system can significantly increase the chlorophyll concentration and cell dry weight in the system, but also proves that the 1:1 bacteria-algae ratio can significantly increase the growth rate of algae. Figure 2 It can be seen that the bacteria-algae symbiotic system with a ratio of 1:1 constructed by the present invention is darker in color than the single algae system containing only Chlorella pyrenoidosa, which obviously shows that the algae content in the bacteria-algae symbiotic system with a ratio of 1:1 in the present invention is higher, proving that the bacteria-algae symbiotic system designed by the present invention can significantly increase the growth rate of algae. Figure 3 It can be seen that in the bacteria-algae symbiotic system with a bacteria-algae ratio of 1:1 in the present invention, the bacterial cells are firmly adhered to the surface of the algae cells, the bacterial adhesion will not change or destroy the shape of the algae cells, and there is no phagocytic effect between the bacteria and the algae, which proves that the bacteria-algae symbiotic system has high stability and can maintain a good growth state under different environmental conditions.

[0047] Test Example 2

[0048] The application effect of the bacteria-algae symbiotic system for freshwater aquaculture ponds prepared in Example 1 of the present invention in aquaculture pond water samples is evaluated, specifically comprising the following steps:

[0049] The 5-point sampling method was used to collect the water sample of the breeding pond, and 5 liters were collected after mixing. After filtering through 0.22 μm, 30-50 ml of the bacteria-algae symbiotic system prepared in Example 1 of the present invention was added, and the water was placed in a culture room at 28°C, 3000 lux, and a photoperiod of 15 hours for 7 days. 500 mL was collected on the 0th, 1st, 3rd, 5th, and 7th days, respectively, and after filtering through 0.22 μm, Nessler's reagent spectrophotometry was used to detect ammonia nitrogen, ultraviolet spectrophotometry was used to detect nitrite, zinc-cadmium reduction was used to detect nitrate, alkaline potassium persulfate digestion ultraviolet spectrophotometry was used to detect total nitrogen, molybdenum antimony anti-spectrophotometry was used to detect phosphate, ammonium molybdate spectrophotometry was used to detect total phosphorus, potassium permanganate-sodium oxalate titration was used to detect chemical oxygen demand, and ultraviolet photocatalytic oxidation was used to detect soluble organic carbon.

[0050] The final result is as follows Figure 4 As shown by Figure 4It can be seen that compared with single bacteria and single algae systems, the bacteria-algae symbiotic system can significantly reduce total nitrogen, ammonia nitrogen, nitrite, nitrate, total phosphorus, phosphate, soluble organic carbon and chemical oxygen demand in freshwater aquaculture water, and the removal efficiency is increased by at least 50%, demonstrating a better ability to remove nitrogen and phosphorus nutrients from aquaculture water.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution 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 solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A method for constructing a bacteria-algae symbiotic system for freshwater aquaculture ponds, characterized in that: The following steps are involved: (1) Cultivating Chlorella pyrenoidosa and Bacillus subtilis to logarithmic phase respectively; (2) Taking the Chlorella pyrenoidosa and Bacillus subtilis cultured to the logarithmic phase in step (1), adjusting the concentration and adding them to the culture medium, placing them in a culture room for cultivation, thereby obtaining the bacteria-algae symbiotic system.

2. The method for constructing a bacteria-algae symbiotic system for freshwater aquaculture ponds according to claim 1, characterized in that: In step (1), the specific method for culturing the Chlorella pyrenoidosa is: using BG-11 culture medium at 25-30° C., 2000-4000 lux, and a photoperiod of more than 12 hours.

3. The method for constructing a bacteria-algae symbiotic system for freshwater aquaculture ponds according to claim 1, characterized in that: In step (1), the specific method for culturing the Bacillus subtilis is: using LB medium to culture under the conditions of 25-30° C., 2000-4000 lux, 12 hours light / 12 hours dark.

4. The method for constructing a bacteria-algae symbiotic system for freshwater aquaculture ponds according to claim 1, characterized in that: In step (2), the concentration adjustment is to adjust the concentrations of the Bacillus subtilis and the Chlorella pyrenoidosa to 1×10 6 -1×10 8 CFU·mL -1 .

5. The method for constructing a bacteria-algae symbiotic system for freshwater aquaculture ponds according to claim 1, characterized in that: In step (2), the adding into the culture medium refers to adding the Bacillus subtilis and the Chlorella pyrenoidosa into the culture medium so that the Bacillus subtilis and the Chlorella pyrenoidosa each account for 5% of the culture medium components.

6. The method for constructing a bacteria-algae symbiotic system for freshwater aquaculture ponds according to claim 1, characterized in that: In step (2), the specific parameters of culturing in the culture chamber are culturing for 3-7 days under the conditions of 25-30° C., 2000-4000 lux and a photoperiod of more than 12 hours.

7. A bacteria-algae symbiotic system constructed by the construction method according to any one of claims 1 to 6.

8. Application of the bacteria-algae symbiotic system as claimed in claim 7 in water quality management in the field of freshwater aquaculture.

Images