Cultivation circulating water quality regulation and control method based on bacteria-algae synergy

Abstract

The invention relates to the technical field of water quality purification, in particular to a bacteria-algae synergy-based culture circulating water quality regulation and control method, which comprises a settleable diatom screening and culture method, a bacteria screening and culture method and a bacteria-algae biological membrane construction and culture method. Through separation, purification, culture and symbiosis of algae species and bacteria, the formed system can degrade harmful substances in water and inhibit harmful bacteria, so that the water quality of circulating water can be stabilized, the stability of a bacterial-algae biological membrane system of the circulating water can be improved, and the water quality of the circulating water can be improved. The problem that the normal operation of the circulating water system function is influenced by system damage caused by long-time culture of the system is avoided, and the problems of long-term instability of the algal-bacterial symbiotic structure and slow growth of the algal-bacterial symbiotic system during large-scale practical application of the algal-bacterial symbiotic system are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of water quality regulation, in particular to a breeding circulating water quality regulation method based on bacteria-algae cooperation. BACKGROUND

[0002] Microalgal-bacterial consortium (MBC) is an emerging wastewater treatment technology that has gradually become a research hotspot in the field of wastewater treatment due to its unique advantages of low-carbon resource recovery and high-value biomass production. MBC, through the interaction between microalgae and bacteria, acts on pollutants in wastewater, converting them into renewable energy and valuable biomass products, achieving the dual goals of wastewater treatment and resource recovery. This process is in line with the green and environmentally friendly development concept and provides a feasible technical path for sustainable development. Although MBC technology has shown significant advantages in wastewater treatment and resource recovery, its actual application still faces certain challenges. The physicochemical properties and biological activity of MBC are influenced by various factors, especially the fluctuations in wastewater quality and changes in environmental factors such as temperature, light intensity, and pH. These factors can lead to unstable treatment performance of the MBC system, limiting its promotion effect and practical application value in large-scale applications.

[0003] Currently, there are many studies on the influencing factors and treatment effects of MBC, most of which focus on wastewater treatment. However, the scale-up of MBC is affected by pollution in open systems, and how to maintain the long-term stability of the MBC structure in circulating water and how to quickly start the MBC system are major problems. Based on this, a breeding circulating water quality regulation method based on bacteria-algae cooperation is proposed. SUMMARY

[0004] To overcome the deficiencies of the prior art, the present application provides a breeding circulating water quality regulation method based on bacteria-algae cooperation, which solves the problems of long-term instability of the MBC structure and slow growth of the MBC system in large-scale practical applications.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a breeding circulating water quality regulation method based on bacteria-algae cooperation, comprising the following steps, (1) Separation and purification of settleable diatoms: collect samples and filter out large particle impurities, then coat them on agar plates after gradient dilution, and cultivate under suitable conditions for 7-14 days, then select well-grown single colonies as algae; (2) Cultivation of settleable diatoms: inoculate the algae into a photobioreactor, add the prepared culture medium, and cultivate under suitable conditions to obtain a microalgae solution; (3) Isolation and purification of bacteria: after pretreatment of the sample, gradient dilution is performed, and then the sample is coated on solid culture medium. After culture, single colonies with good growth are selected as bacterial strains; (4) Bacterial culture: the bacterial strains are inoculated into a first liquid culture medium for culture to obtain a bacterial liquid; (5) Establishment of a bacteria-algae symbiotic system: the microalgae liquid and the bacterial liquid are inoculated into a second liquid culture medium for mixed culture to obtain a bacteria-algae mixed liquid; (6) Establishment of a circulating water bacteria-algae biofilm system: the bacteria-algae mixed liquid and a nutrient liquid are added to a sedimentation module of a circulating water system for final culture to form a bacteria-algae biofilm.

[0006] Preferably, in step (1), the algal species is selected from species that can secrete extracellular substances and benthic diatoms.

[0007] Preferably, in step (1), the agar plate is prepared by adding neomycin and streptomycin to a BG-11 agar plate; and the addition amount of neomycin and streptomycin is 50 mg / L.

[0008] Preferably, in step (2), the culture conditions are as follows: temperature is 25°C, pH is 8.0±0.2, light is trichromatic light, light intensity is 4000-8000 LUX, and light / dark ratio is 8:16; the culture medium formula is as follows: Na2SiO3-0.35 mg / L, urea-75 mg / L, KH2PO4-7.5 mg / L, vitamin B1-0.2 mg / L, vitamin B12-0.1 mg / L, EDTA-3.25 mg / L, FeCl3-1.03 mg / L, H3BO3-2 mg / L, MnSO4-4 mg / L, ZnSO4-0.5 mg / L, CoCl2-0.25 mg / L; and the microalgae liquid concentration is 1×10 8 cells / mL.

[0009] Preferably, in step (4), the first liquid culture medium formula is as follows: yeast extract-10 g / L, biological leaching powder-5 g / L, EDTA-3.25 mg / L, FeCl3-1.03 mg / L, H3BO3-2 mg / L, MnSO4-4 mg / L, ZnSO4-0.5 mg / L, CoCl2-0.25 mg / L; and the culture conditions are as follows: the bacterial culture shaking frequency is 150 times / min, and the light intensity is 2000 LUX; and the bacterial liquid concentration is 1×10 9 cells / mL.

[0010] Preferably, in step (5), the inoculation ratio of the bacterial liquid and the microalgae liquid mixed culture is 1:4; the mixed culture conditions are as follows: the temperature is 28 DEG C, the pH is 7.5+ / -0.2, the light is three-color light, the light intensity is 4000-8000LUX, the light-dark ratio is 16:8, the culture time is 14d; the aeration amount is 0.15VVM, and 5% CO2 is mixed in proportion.

[0011] Preferably, in step (5), the second liquid culture medium formula is as follows: molasses-2g / L, Na2SiO3-0.7mg / L, urea-75mg / L, KH2PO4-15mg / L, vitamin B1-0.2mg / L, vitamin B12-0.1mg / L, EDTA-3.25mg / L, FeCl3-1.03mg / L, H3BO3-2mg / L, MnSO4-4mg / L, ZnSO4-0.5mg / L, CoCl2-0.25mg / L.

[0012] Preferably, in step (6), the bacteria-algae mixed liquid is added every 3 days for a total of 20L for 15 days; and the nutrient solution is added twice during the period.

[0013] Preferably, in step (6), the nutrient solution formula is as follows: Na2SiO3-0.7mg / L, urea-75mg / L, KH2PO4-15mg / L, EDTA-3.25mg / L, FeCl3-1.03mg / L, H3BO3-2mg / L, MnSO4-4mg / L, ZnSO4-0.5mg / L, CoCl2-0.25mg / L; the light intensity of the final culture is 2000LUX, no aeration is performed in the week before the final culture, and aeration is performed from the bottom after one week of culture, and the aeration amount is 0.25VVM.

[0014] Preferably, in step (3), the solid culture medium formula is as follows: yeast paste-10g / L, biological leaching powder-5g / L, NaCl-15g, purified agar powder-15g, and distilled water 1000mL.

[0015] EDTA is ethylenediaminetetraacetic acid; FeCl3 is ferric chloride; H3BO3 is boric acid; MnSO4 is manganese sulfate; ZnSO4 is zinc sulfate; CoCl2 is cobalt chloride; Na2SiO3 is sodium silicate; and KH2PO4 is potassium dihydrogen phosphate.

[0016] The application provides a breeding circulating water quality regulation method based on bacteria-algae cooperation, and has the following beneficial effects compared with the prior art: The application can realize degradation of harmful substances in water and inhibition of harmful bacteria by forming a system through separation, purification, culture and symbiosis of algae and bacteria, can not only stabilize the water quality of circulating water, but also improve the stability of the circulating water system, avoid damage of the system caused by long-term breeding, and affect the normal operation of the circulating water system; and solve the problems of long-term instability of the bacteria-algae symbiotic structure and slow growth of the bacteria-algae symbiotic system in large-scale practical application.

[0017] The application utilizes the system formed by the sedimentable diatom and bacteria to treat the circulating water through the bacteria-algae symbiotic synergistic effect, is more efficient than the traditional circulating water treatment, and has more excellent treatment effect.

[0018] The structure in the application is relatively stable, has a longer survival time in the regulation process of the circulating water, reduces the replacement frequency, and reduces the cost. BRIEF DESCRIPTION OF DRAWINGS

[0019] The drawings described herein are used to provide further understanding of the present application, constitute a part of the present application, and the illustrative embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitations on the present application. In the drawings: Figure 1 It is a pre-stage diagram of the bacteria-algae biofilm in Example 1 of the present application; Figure 2 It is a pre-middle stage diagram of the bacteria-algae biofilm in Example 1 of the present application; Figure 3 It is a post-stage diagram of the bacteria-algae biofilm in Example 1 of the present application. DETAILED DESCRIPTION

[0020] The following examples are used to illustrate the embodiments of the present application, so that the realization process of how to apply technical means to solve technical problems and achieve technical effects of the present application can be fully understood and implemented.

[0021] Example 1

[0022] A breeding circulating water quality regulation method based on bacteria-algae cooperation, comprising the following steps, (1) Separation and purification of sedimentable diatoms: collect samples and filter to remove large particle impurities, coat on agar plates after gradient dilution under suitable conditions (light intensity is 8000LUX, temperature is 25℃, and light is three-color light) for 14d, and select well-grown single colonies as algae; The agar plate is prepared by adding neomycin and streptomycin to the BG-11 agar plate; wherein the addition amount of neomycin and streptomycin is 50mg / L.

[0023] (2) Cultivation of Settleable Diatom: inoculate the algal species (benthic diatom) into the photobioreactor, add the prepared culture medium, and cultivate under suitable conditions, i.e. control the temperature at 25℃, pH at 8.0±0.2, light at three-color light, light intensity at 8000LUX, light-dark ratio at 8:16, to obtain the microalgae liquid, and the concentration of the microalgae liquid is 1×10 8 cells / mL.

[0024] The above-mentioned culture medium formula: Na2SiO3-0.35mg / L, urea-75mg / L, KH2PO4-7.5mg / L, vitamin B1-0.2mg / L, vitamin B12-0.1mg / L, EDTA-3.25mg / L, FeCl3-1.03mg / L, H3BO3-2mg / L, MnSO4-4mg / L, ZnSO4-0.5mg / L, CoCl2-0.25mg / L.

[0025] (3) Isolation and purification of bacteria: after collecting the sample and pretreating (shake well after vortex oscillation, take the supernatant after standing, and collect the bacterial cells by high-speed centrifugation), gradient dilution is performed, and then the bacteria are inoculated on the solid culture medium, and after cultivation, the single colonies with good growth are selected as the bacterial species; The above-mentioned solid culture medium formula: yeast extract-10g / L, biological immersion powder-5g / L, NaCl-15g, purified agar powder-15g, distilled water 1000mL.

[0026] (4) Cultivation of bacteria: inoculate the bacterial species into the first liquid culture medium for cultivation, i.e. control the bacterial culture shaking frequency at 150 times / min, and the light intensity at 2000LUX, to obtain the bacterial liquid, and the concentration of the bacterial liquid is 1×10 9 cells / mL; The above-mentioned first liquid culture medium formula: yeast extract-10g / L, biological immersion powder-5g / L, EDTA-3.25mg / L, FeCl3-1.03mg / L, H3BO3-2mg / L, MnSO4-4mg / L, ZnSO4-0.5mg / L, CoCl2-0.25mg / L.

[0027] (5) Establishment of bacteria-algae symbiotic system: inoculate the microalgae liquid and the bacterial liquid into the second liquid culture medium for mixed cultivation, i.e. control the temperature at 28℃, pH at 7.5±0.2, light at three-color light, light intensity at 8000LUX, light-dark ratio at 16:8, ventilation volume at 0.15VVM, mix 5% CO2 in proportion, and the cultivation time is 14d, to obtain the bacteria-algae mixed liquid; The inoculation ratio of the mixed cultivation of the bacterial liquid and the microalgae liquid is 1:4, the concentration of the microalgae is 4×10 6 cells / mL, and the concentration of the bacteria is 1×10 6cells / mL; The above-mentioned second liquid culture medium formula: molasses-2 g / L, Na2SiO3-0.7 mg / L, urea-75 mg / L, KH2PO4-15 mg / L, vitamin B1-0.2 mg / L, vitamin B12-0.1 mg / L, EDTA-3.25 mg / L, FeCl3-1.03 mg / L, H3BO3-2 mg / L, MnSO4-4 mg / L, ZnSO4-0.5 mg / L, CoCl2-0.25 mg / L.

[0028] (6) Establish a circulating water bacteria-algae biofilm system: add bacteria-algae mixed solution and nutrient solution to the sedimentation module of the circulating water system (disclosed in the patent with application number 202322920390.1) for final cultivation, form bacteria-algae biofilm, the bacteria-algae mixed solution is added every 3 days for 20 L, a total of 15 days, add nutrient solution twice during the period, and control the light intensity to be 2000 LUX, do not aerate for one week before the final cultivation, aerate from the bottom after one week of cultivation, and the aeration amount is 0.25 VVM; The above-mentioned nutrient solution formula is: Na2SiO3-0.7 mg / L, urea-75 mg / L, KH2PO4-15 mg / L, EDTA-3.25 mg / L, FeCl3-1.03 mg / L, H3BO3-2 mg / L, MnSO4-4 mg / L, ZnSO4-0.5 mg / L, CoCl2-0.25 mg / L.

[0029] Example 2

[0030] A breeding circulating water quality regulation method based on bacteria-algae cooperation, which is basically the same as example 1, the difference is that the benthic diatom is replaced by a bacterium that can secrete extracellular substances (needle-shaped algae).

[0031] Comparative example 1 A breeding circulating water quality regulation method based on bacteria-algae cooperation, which is basically the same as example 1, the difference is that the benthic diatom is replaced by a bacterium that can secrete extracellular substances (needle-shaped algae).

[0032] Comparative example 2 A breeding circulating water quality regulation method based on bacteria-algae cooperation, which is basically the same as example 1, the difference is that the benthic diatom is replaced by a bacterium that can secrete extracellular substances (needle-shaped algae).

[0033] Comparative example 3 A breeding circulating water quality regulation method based on bacteria-algae cooperation, which is basically the same as example 1, the difference is that the benthic diatom is replaced by a bacterium that can secrete extracellular substances (needle-shaped algae).

[0034] Comparative example 4 A kind of cultivation circulating water quality regulation method based on bacteria-algae cooperation, substantially same with embodiment 1, difference lies in: replace benthic diatom with chlorella.

[0035] Performance verification 1, using the regulation method in embodiment 1-2 and comparative example 1-4 repeats 3 groups of tests to verify the survival time of the bacteria-algae biofilm prepared. The specific results are shown in the following table.

[0036]

[0037] From the above table, it can be seen that the survival time of the bacteria-algae biofilm in the examples is much longer than that in the comparative examples. The use of surface-settling diatoms and bacteria greatly improves the life of the bacteria-algae biofilm.

[0038] 2, using the regulation method in embodiment 1-2 repeats 3 groups of tests to verify the regulation effect on water quality. The specific results are shown in the following table.

[0039]

[0040] From the above table, it can be seen that at different time stages of the bacteria-algae biofilm, its influence on water quality is also different. Usually, the treatment is stable after 30 days, and the water treatment efficiency is the highest.

[0041] Although the embodiments of the present application have been shown and described, it can be understood by those skilled in the art that various changes, modifications, replacements and modifications can be made to these embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for regulating the quality of aquaculture circulating water based on the collaboration of bacteria and algae, characterized in that: The following steps are included: (1) Isolation and purification of sedimentable diatoms: Collect samples and filter to remove large particles of impurities, spread them on agar plates after gradient dilution, culture them under appropriate conditions, and select well-grown single colonies as algae species; (2) Cultivation of settleable diatoms: inoculate the algae seeds into a photobioreactor, add the prepared culture medium, and cultivate under appropriate conditions to obtain microalgae liquid; (3) Isolation and purification of bacteria: After pretreatment of the collected sample, it is spread on a solid culture medium through gradient dilution, and after cultivation, a single colony with good growth is selected as the strain; (4) Cultivating bacteria: inoculating the bacterial strain into the first liquid culture medium to obtain a bacterial liquid; (5) Establishing a bacterial-algal symbiotic system: inoculating the microalgae solution and the bacterial solution into the second liquid culture medium for mixed culture to obtain a bacterial-algal mixed solution; (6) Establishing a circulating water bacteria-algae biofilm system: Adding bacteria-algae mixed solution and nutrient solution into the sedimentation module of the circulating water system for final cultivation to form a bacteria-algae biofilm.

2. The method for controlling the water quality of aquaculture circulating water based on bacterial and algal collaboration according to claim 1, characterized in that: In step (1), the algae species are selected from algae that can secrete extracellular substances and benthic diatoms.

3. The method for controlling the water quality of aquaculture circulating water based on bacterial and algal collaboration according to claim 1, characterized in that: In step (1), the agar plate is prepared by adding neomycin and streptomycin to the BG-11 agar plate; wherein the added amount of neomycin and streptomycin is 50 mg / L.

4. The method for controlling the water quality of aquaculture circulating water based on bacterial and algal collaboration according to claim 1, characterized in that: In step (2), the culture conditions are as follows: temperature of 25°C, pH of 8.0±0.2, trichromatic light, light intensity of 4000-8000LUX, and light-dark ratio of 8:16; culture medium formula: Na2SiO3-0.35mg / L, urea-75mg / L, KH2PO4-7.5mg / L, vitamin B1-0.2mg / L, vitamin B12-0.1mg / L, EDTA-3.25mg / L, FeCl3-1.03mg / L, H3BO3-2mg / L, MnSO4-4mg / L, ZnSO4-0.5mg / L, CoCl2-0.25mg / L; microalgae solution concentration is 1×10 8 cells / mL.

5. The method for controlling the quality of aquaculture circulating water based on bacterial and algal collaboration according to claim 1, characterized in that: In step (4), the first liquid culture medium formula is: yeast extract-10g / L, biological extract powder-5g / L, EDTA-3.25mg / L, FeCl3-1.03mg / L, H3BO3-2mg / L, MnSO4-4mg / L, ZnSO4-0.5mg / L, CoCl2-0.25mg / L; the culture conditions are: the bacterial culture shaking frequency is 150 times / min, the light intensity is 2000LUX; the bacterial liquid concentration is 1×10 9 cells / mL.

6. The method for controlling the quality of aquaculture circulating water based on bacterial and algal collaboration according to claim 1, characterized in that: In step (5), the inoculation ratio of the mixed culture of bacterial liquid and microalgae liquid is 1:4; the mixed culture conditions are: temperature of 28°C, pH of 7.5±0.2, three-color light, light intensity of 4000-8000LUX, light-dark ratio of 16:8, culture time of 14 days; ventilation volume of 0.15VVM, 5% CO2 mixed in proportion.

7. The method for controlling the quality of aquaculture circulating water based on bacterial and algal collaboration according to claim 1, characterized in that: In step (5), the second liquid culture medium formula is: molasses-2g / L, Na2SiO3-0.7mg / L, urea-75mg / L, KH2PO4-15mg / L, vitamin B1-0.2mg / L, vitamin B12-0.1mg / L, EDTA-3.25mg / L, FeCl3-1.03mg / L, H3BO3-2mg / L, MnSO4-4mg / L, ZnSO4-0.5mg / L, CoCl2-0.25mg / L.

8. The method for controlling the quality of aquaculture circulating water based on bacterial and algal collaboration according to claim 1, characterized in that: In step (6), 20 L of the bacterial-algal mixture was added every 3 days for a total of 15 days; nutrient solution was added twice during this period.

9. The method for controlling the quality of aquaculture circulating water based on bacterial and algal collaboration according to claim 1, characterized in that: In step (6), the formula of the nutrient solution is: Na2SiO3-0.7mg / L, urea-75mg / L, KH2PO4-15mg / L, EDTA-3.25mg / L, FeCl3-1.03mg / L, H3BO3-2mg / L, MnSO4-4mg / L, ZnSO4-0.5mg / L, CoCl2-0.25mg / L; the light intensity of the final culture is 2000LUX, no ventilation is performed one week before the final culture, and ventilation is performed from the bottom after one week of culture, with a ventilation volume of 0.25VVM.

10. The method for controlling the quality of aquaculture circulating water based on bacterial and algal collaboration according to claim 1, characterized in that: In step (3), the solid culture medium formula is: yeast extract-10g / L, biological extract powder-5g / L, NaCl-15g, purified agar powder-15g, and distilled water 1000mL.

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