Gp24-regulated triphasic algal-bacterial symbiosis for pig wastewater treatment and construction method thereof

Abstract

This invention belongs to the field of wastewater biological treatment technology, specifically a GR24-regulated triphasic algae-bacterial symbiotic and its construction method for treating pig farm wastewater. This invention uses *Chlorella vulgaris*-endophytic bacteria S395-2-*Polyspora spiralis* as the dominant bacterial species, inoculating them into a co-culture system at a certain cell number ratio, and adding 10... ‑9 The synthetic strigolactone analog GR24 was cultured under specific temperature, light intensity, and light-dark ratio conditions to construct a triphasic algal-bacterial symbiotic with uniform spherical diameter and strong stress resistance. This triphasic algal-bacterial symbiotic was used for the pretreatment of pig farm wastewater. This invention is simple to operate, low in cost, and can significantly improve the stability of the symbiotic in high-stress wastewater, providing reliable materials for subsequent antibiotic resistance gene (ARG) reduction, making it suitable for industrial-scale promotion.

Description

Technical Field

[0001] This invention belongs to the field of wastewater biological treatment technology, specifically relating to a GR24-regulated triphase algae-bacteria symbiotic for pig farm wastewater treatment and its construction method. Background Technology

[0002] Against the backdrop of the rapid development of the environmental protection industry, biological wastewater treatment technologies have become one of the mainstream directions due to their low cost and environmental friendliness. Algae-bacterial symbionts, as important biological carriers, directly affect wastewater treatment effectiveness. In particular, there is an urgent need to develop efficient pretreatment technologies to address the serious problem of antibiotic resistance gene (ARG) pollution in pig farm wastewater.

[0003] Existing triphasic algae-bacteria-fungi symbionts (microalgae-bacteria-fungi) face key challenges in practical applications, including long pellet formation time, uneven size (0.2-1.0 cm), and poor stress resistance. They are also unable to withstand the stress of heavy metals and residual antibiotics in swine wastewater, limiting their large-scale application. Current research focuses primarily on optimizing culture media and light conditions, with limited effectiveness in improving pellet formation stability and stress resistance. Although the strigolactone analog GR24 has shown potential in regulating plant and algal growth, a standardized GR24 regulation scheme for constructing triphasic algae-bacteria symbionts is currently lacking. This makes it difficult to systematically address the core issue of poor stability, thus limiting the practical effectiveness of this technology in the pretreatment of swine wastewater. Summary of the Invention

[0004] The purpose of this invention is to provide a GR24-regulated triphase algae-bacterial symbiotic with high sphericity and good stress resistance for pig farm wastewater treatment, and its construction method.

[0005] The method for constructing a GR24-regulated triphasic algae-bacteria symbiotic relationship provided by this invention comprises the following steps:

[0006] (a) Preparation of microbial strains

[0007] (1) Chlorella cultivation: Chlorella, which has high photosynthetic efficiency and strong pollutant absorption capacity, was selected as the dominant algal species and purchased from an environmental science and technology engineering center in Shandong Province. The initial concentration was strictly controlled at (2.0 × 10⁻⁶). 6 cell·mL -1This concentration, verified through extensive preliminary experiments, ensures that Chlorella can maintain its own growth vitality while forming a favorable symbiotic relationship with bacteria and fungi. Activation culture was performed using BG11 medium, a classic medium specifically designed for culturing cyanobacteria and green algae. BG11 is rich in nitrogen, phosphorus, potassium, and various trace elements, meeting the nutritional needs of Chlorella. Culture conditions were set at a temperature of 25±2 ℃ (preferably 25±1 ℃), a temperature range close to the optimal growth temperature for Chlorella, maximizing its photosynthetic efficiency and growth rate; light intensity was controlled at (180-200 μmol·m⁻¹). -2 ·s -1 The light intensity was set to ensure sufficient photosynthesis for Chlorella while avoiding photoinhibition caused by excessive light. The light-dark ratio was set to (8 h - 14 h): (8 h - 14 h) (preferably 12 h: 12 h), simulating the day-night cycle in the natural environment and conforming to the growth rhythm of Chlorella. Under the above culture conditions, Chlorella reached the required initial concentration and maintained good physiological activity after 5 days of cultivation.

[0008] (2) Culture of endophytic bacteria S395-2: Endophytic bacteria S395-2 was deposited at the China General Microbiological Culture Collection Center (CGMCC) under the number CGMCC No. 202401. Endophytic bacteria S395-2 possesses strong organic pollutant degradation capabilities and synergistic symbiotic abilities with microalgae, making it one of the key strains for constructing a triphasic algae-bacteria symbiotic system. Its initial concentration was controlled at (2.0 × 10⁻⁶). 7 CFU·mL -1 This concentration ensures a sufficient number of bacteria in the co-culture system to rapidly decompose organic pollutants in the wastewater. LB medium was used for activation culture. LB medium is rich in nutrients, containing tryptone, yeast extract, and sodium chloride, providing ample carbon, nitrogen, and inorganic salts for bacterial growth. The culture conditions were 37 ℃ and 180 rpm shaking for 24 h. 37 ℃ is the optimal growth temperature for most bacteria, and the 180 rpm shaking speed ensured sufficient oxygen supply in the medium, promoting aerobic respiration and rapid reproduction of the bacteria. After 24 h of culture, the endophytic bacteria S395-2 reached the required initial concentration and were in an active metabolic state.

[0009] (3) Culture of *Aspergillus spiralis*: Deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 202402. *Aspergillus spiralis* possesses strong structural support and tolerance to complex pollutants, which can enhance the stability and stress resistance of the triphasic algae-bacteria symbiotic relationship. Activation culture was performed using PDA medium, a commonly used fungal culture medium containing potato extract, glucose, and other components, which can provide abundant nutrients for fungal growth. The culture conditions were 25 ℃ for 7 days. 25 ℃ is suitable for the growth of *Aspergillus spiralis*, and the 7-day culture time ensures that the fungus forms sufficient mycelium, preparing it for subsequent symbiosis with microalgae and bacteria.

[0010] (ii) Setting up the GR24 concentration gradient

[0011] The synthetic strigolactone analog GR24 was added to the co-culture system, with a concentration gradient of 10⁻⁶. -7 M, 10 -9 M, 10 -11 M was used as a control group, while 0 M was used as a control group. Multiple concentration gradients were set up to screen for the optimal GR24 concentration for the ball-forming performance and stress resistance of the triphasic algae-bacteria symbiotic system. The initial algae-bacteria biomass of the co-culture system was strictly controlled at 85-110 mg / L. -1 (Preferred 90 mg L) -1 This initial biomass was determined based on extensive prior experiments, ensuring a balanced ratio among the various bacterial species in the co-culture system and providing a stable foundation for subsequent symbiosis construction and performance optimization.

[0012] (III) Symbiotic Culture and Optimization

[0013] Activated Chlorella, endophytic bacteria S395-2, and Polyspora spiralis were inoculated into a co-culture system containing different concentrations of GR24 at a cell ratio of Chlorella:endophytic bacteria:S395-2:Polyspora spiralis = (1-20):(1-10):1 (preferably 10:4:1). This ratio, after repeated experimental screening and optimization, enables the three microorganisms to form an optimal synergistic symbiotic relationship and fully utilize their respective functional advantages. The culture was carried out at 25±2 ℃ (preferably 25±1 ℃) and a light intensity of (180-200 μmol·m⁻¹). -2 ·s -1 The cultures were cultivated under light-dark ratios of (8 h - 14 h): (8 h - 14 h) (preferably 12 h: 12 h). These culture conditions are consistent with those for the activation culture of the strains, which can ensure that each strain maintains good growth and metabolic activity during co-culture.

[0014] During the cultivation process, the pellet formation process was observed in real time using a laser scanning confocal microscope (CLSM). The CLSM is characterized by high resolution and high sensitivity, enabling clear observation of the algal pellet formation process, internal structure, and distribution of various bacterial species. Pellet formation time and pellet diameter were recorded every 5-6 hours. By comparing and analyzing pellet formation data at different GR24 concentrations, the optimal GR24 concentration was determined to be 10. -7 M--10 -11 M (preferred 10) -9 M). At this concentration, the triphasic algae-bacterial symbionts cultured at this concentration exhibited the shortest spheroidization time, the most uniform algal-bacterial spheroid diameter, and the strongest stress resistance.

[0015] The above steps yielded a GR24-regulated triphase algae-bacterial symbiotic. This symbiotic exhibited the shortest spheroidization time, the most uniform algae-bacterial spheroid diameter, and the strongest stress resistance. This triphase algae-bacterial symbiotic can be used for pig farm wastewater treatment, specifically:

[0016] The optimal GR24 concentration (10) -9 A three-phase algae-bacterial symbiotic constructed by M) was introduced into pig farm wastewater; the treatment temperature was controlled to be consistent with the symbiotic culture temperature to avoid adverse effects of temperature changes on symbiotic performance; dissolved oxygen was controlled at 2-4 mg / L. -1 This dissolved oxygen range can meet the needs of bacterial aerobic metabolism while avoiding energy waste and inhibition of microalgae photosynthesis caused by excessive dissolved oxygen.

[0017] In the process of treating pig farm wastewater, the specific growth rate and superoxide dismutase (SOD) activity of the symbiont were measured to verify its resistance to heavy metals (Zn). 2+ Cu 2+ The ability of microorganisms to withstand antibiotic (tetracycline, sulfonamide) stress. Specific growth rate is an important indicator reflecting the growth status of microorganisms and can directly reflect the growth vitality of symbionts under stress conditions; SOD is an important antioxidant enzyme, and its activity level can reflect the tolerance of symbionts to oxidative stress, thus reflecting the strength of their stress resistance.

[0018] This invention focuses on a triphasic algae-endophytic bacteria-fungus symbiosis. By optimizing the concentration of the artificially synthesized strigolactone analog GR24, it constructs a symbiotic system to address the problem of pig farm wastewater treatment, aiming to achieve compliant discharge of pig farm wastewater and effectively control environmental risks. Specifically:

[0019] (1) First, the pig farm wastewater is pretreated, that is, the COD in the collected pig farm wastewater is adjusted to 1500 mg / L. -1 Ammonia nitrogen was adjusted to 200 mg / L. -1 Add Zn2+ Up to 5 mg L -1 Cu 2+ Up to 3 mg L -1 .

[0020] The tetracycline antibiotic concentration in the collected pig farm wastewater was adjusted to 3 mg / L. -1 Sulfonamide antibiotics up to 2 mg / L -1 Adjust the pH to 7.0 ± 0.2.

[0021] (2) Then, the triphasic algae-bacteria symbiotic was added to the pretreated pig farm wastewater at a ratio of 10% (v / v) and at a temperature of 25±1 ℃ and dissolved oxygen of 2-4 mg L. -1 Aeration treatment for 5-8 days under the specified conditions;

[0022] During the treatment period, the specific growth rate of the symbiont was measured periodically (≥0.22 d). -1 The activity of Zn and SOD (≥110U / mgprot), as well as the levels of heavy metals and antibiotic residues in the wastewater, are monitored to ensure Zn 2+ Removal rate ≥60%, Cu 2+ Removal rate ≥65%, tetracycline antibiotic removal rate ≥70%, sulfonamide antibiotic removal rate ≥75%.

[0023] The aeration treatment uses porous aeration heads, and the aeration rate is controlled at 0.5-1.0 L (L·h). -1 Dissolved oxygen concentration is monitored in real time using a portable dissolved oxygen meter to ensure that dissolved oxygen remains stable within the set range during the reaction process.

[0024] The tetracycline antibiotics include tetracycline, oxytetracycline, and chlortetracycline, and the sulfonamide antibiotics include sulfadiazine, sulfadiazine, and sulfamethoxazole.

[0025] In this invention, the inoculation ratio of microalgae-endophytic bacteria-fungi is calculated based on the number of cells.

[0026] Preferably:

[0027] In this invention, the essential components of the BG-11 culture medium include: NaNO3 1.5 g L. -1 K2HPO4·3H2O 0.04 g L -1 MgSO4·7H2O 0.075 g L -1 CaCl2·2H2O 0.036 g L -1 Citric acid 0.006 g / L -1 Ferric ammonium citrate 0.006 g / L -1 EDTA 0.001 g / L -1Na2CO3 0.02 g L -1 1 mL of trace element solution -1 The trace element solution contains 2.86 g / L H3BO3. -1 MnCl2·4H2O 1.81 g L -1 ZnSO4·7H2O 0.222 g L -1 Na₂MoO₄·2H₂O 0.39 g L -1 CuSO4·5H2O 0.079 g L -1 Co(NO3)2·6H2O 0.049 g L -1 .

[0028] In this invention, the Chlorella is cultured at a temperature of 25 °C and a light intensity of 200 μmol·m⁻². -2 ·s -1 They were cultured in a constant temperature and light incubator with a light-dark ratio of 12h:12h.

[0029] In this invention, after continuous culture of Chlorella for 5 days, the cell concentration of Chlorella was measured under a microscope using a hemocytometer, and the cell concentration reached (2.0 × 10⁻⁶). 6 cell·mL -1 When the value is 0, it indicates that Chlorella is in the logarithmic growth phase, has strong physiological activity, and is suitable for subsequent co-culture experiments.

[0030] In this invention, the essential components of the endophytic bacterial LB culture medium include: 10 g / L tryptone. -1 5 g L of yeast extract -1 NaCl 10 g L -1 .

[0031] In this invention, during the cultivation of the endophytic bacteria, the pH is adjusted to 7.0-7.2 using NaOH solution. The inoculated culture medium is then placed in a shaker at 37 ℃ and 180 rpm for shaking culture to ensure uniform distribution of nutrients and provide sufficient oxygen for the bacteria.

[0032] In this invention, during the cultivation of the endophytic bacteria, samples were taken every 6 hours, and the concentration of the bacteria was determined using the plate count method. After 24 hours of cultivation, the concentration of the endophytic bacteria S395-2 reached (2.0 × 10⁻⁶). 7 CFU·mL -1 At this time, the bacteria are in an active metabolic state and have a strong ability to degrade organic pollutants.

[0033] In this invention, the essential components and concentrations of the *Polyspora spiralis* PDA culture medium include: 200 g potato, 20 g sucrose, 15-20 g agar, 1000 mL distilled water, sterilized at 121°C for 20 min to ensure that the culture medium is free from contamination by other microorganisms.

[0034] In this invention, when the PDA medium is cooled to 45-50 ℃, it is poured into a sterile petri dish to make a plate, inoculated with Polyspora spiralis, and placed in a constant temperature incubator at 25 ℃ for continuous culture for 7 days. After a large number of mycelia are formed, they are collected for later use.

[0035] In this invention, the ratio of the three-phase symbiotic organisms is based on the cell number ratio of Chlorella: Endophytic bacteria: S395-2: Polyspora spiralis = 10:4:1, and the inoculation amount of each bacterial species is precisely calculated.

[0036] In this invention, the co-culture system ensures an initial biomass of 90 mg / L. -1 The accurate inoculation volume needs to be calculated based on the concentration and dry weight of each bacterial species. For example, the dry weight of Chlorella cells is approximately 1.0 × 10⁻⁶. -12 g / cell, the dry weight of endophytic bacteria S395-2 is approximately 1.0 × 10 g / cell. -13 g / CFU, the dry weight of the mycelium of *Polyspora spiralis* was determined based on its growth.

[0037] In this invention, the GR24 solution is added to the co-culture system to achieve a final concentration of GR24 of 10-1 in each system. -7 M, 10 -9 M, 10 -11 M, and a control group without GR24 were also set up. Three parallel samples were set up for each experiment to ensure the reliability and repeatability of the experimental results.

[0038] In this invention, a 405 nm laser was used for excitation, and the formation process of algal balls was observed and the time was recorded under a 63× oil immersion microscope.

[0039] In this invention, the biomass of the triphase algae-bacteria symbiotic organism is determined by centrifugation. 10 mL of sample is centrifuged at 8000 rpm for 10 min. After discarding the supernatant, the precipitate is dried in an 80 ℃ oven until constant weight, and the biomass per unit volume (mg / L) is obtained.

[0040] In this invention, the photosynthetic biomass parameter (Fv / F) of the triphasic algae-bacteria symbiotic organism is... M The maximum quantum yield of photosystem II was measured using a pulse-modulated chlorophyll fluorometer (model PAM-2500). Before measurement, the samples were dark-adapted for 30 min. 650 nm red light was used as the measurement light, and the saturation pulse intensity was set to 3000 μmol·m⁻¹. -2 ·s-1 Record minimum fluorescence (Fo) and maximum fluorescence (Fo). M According to the formula Fv / F M =(F M -Fo) / F M calculate.

[0041] In this invention, the SOD activity of the triphasic algae-bacterial symbiont was determined using the xanthine oxidase method. A 5 mL symbiont sample was centrifuged at 8000 rpm for 10 min, the precipitate was collected, and 5 mL of phosphate buffer (0.05 mol / L) was added. -1 The enzyme was sonicated for 5 min (pH 7.8), then centrifuged at 4 ℃ and 12000 rpm for 20 min. The supernatant was used as the enzyme extraction solution. The absorbance at 550 nm was measured according to the instructions of the SOD kit (Nanjing Jiancheng Bioengineering Institute), and the SOD activity (U / mgprot) was calculated.

[0042] In this invention, the dissolved oxygen in the GR24-regulated triphasic algae-bacteria symbiotic organism is controlled at 2-4 mg / L during the pretreatment of pig farm wastewater. -1 This dissolved oxygen range can meet the needs of bacterial aerobic metabolism while avoiding energy waste and inhibition of microalgae photosynthesis caused by excessive dissolved oxygen.

[0043] In this invention, the heavy metal (Zn) 2+ Cu 2+ The measurements were performed using an inductively coupled plasma mass spectrometer (ICP-MS, model NexION 350X).

[0044] In this invention, the antibiotics (tetracycline, sulfadiazine) were determined using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS, Agilent 1290-6460). The chromatographic column was a C18 column (2.1 × 150 mm, 3.5 μm), the mobile phase was methanol-0.1% formic acid aqueous solution (gradient elution), and the mass spectrometry was performed using an electrospray ionization (ESI) source in positive ion mode.

[0045] This invention effectively shortens the pelleting time of triphasic algae-bacteria symbionts, significantly improves pelleting uniformity and stress resistance, thus providing a stable and efficient symbiotic material for the pretreatment of pig farm wastewater. By constructing high-performance triphasic algae-bacteria symbionts, a solid foundation is laid for the efficient reduction of antibiotic resistance genes (ARGs) in subsequent pig farm wastewater treatment, ultimately achieving compliant discharge of pig farm wastewater and effective control of environmental risks, promoting the green and sustainable development of the pig farming industry.

[0046] Compared with the prior art, the beneficial effects of this invention are as follows:

[0047] By adding 10 -9 M's GR24 reduced the pelleting time of the triphase algae-bacterial symbiont from 72 hours to 54 hours, and stabilized the pellet diameter at 0.5±0.05 cm, significantly improving pelleting speed and uniformity, and ensuring the stable operation of the treatment system; in Zn-containing... 2+ Under tetracycline stress, the symbiont's specific growth rate increased by 31.7% to 0.25 days. -1 The method increases SOD activity by 28.9% to 120 U / mgprot, significantly enhances stress resistance, and can maintain stable metabolic activity in actual pig farm wastewater. The method is simple to operate, requires a small amount of GR24 and is low in cost. It does not require complex equipment and is easy to scale up. It provides an efficient, stable solution with good industrialization prospects for the pretreatment of pig farm wastewater. Detailed Implementation

[0048] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0049] In the following examples, the proportion of each bacterial species added in the GR24-regulated triadic algae-bacteria symbiotic construction method was calculated based on the ratio of cell numbers.

[0050] Example 1: Control group and pretreatment of pig farm wastewater

[0051] The blank control group (without symbiotic and GR24) took 500 mL of pretreated pig farm wastewater (COD 1500 mg / L). -1 Ammonia nitrogen 200 mg / L -1 Zn 2+ 5 mg L -1 Cu 2+ 3 mg L -1 Tetracycline 3 mg L -1 2 mg / L sulfadiazine -1 The dissolved oxygen (pH 7.0) was placed in a 1 L glass reactor, and the temperature was controlled at 25±1℃ using a constant temperature water bath. The aeration rate was adjusted by a small aeration pump (ACO-002 type) with a gas flow meter to maintain the dissolved oxygen concentration at 2-4 mg / L. -1 (Dissolved oxygen was monitored using a YSI Pro20 meter). No symbiotics or GR24 were added to the reaction system, and each group was tested in triplicate. Samples were taken at days 0, 3, and 7 after treatment, filtered through a 0.45 μm filter, and analyzed for heavy metals (Zn) using the following methods. 2+ Cu 2+The Zn group was determined using inductively coupled plasma mass spectrometry (NexION 350X); antibiotics (tetracycline and sulfadiazine) were detected using high-performance liquid chromatography-tandem mass spectrometry (Agilent 1290-6460, C18 column, methanol-0.1% formic acid-water gradient elution, ESI positive ion mode). Results showed that after 7 days of treatment, Zn... 2+ The residual concentration was 4.92 mg / L. -1 (Removal rate 1.6%) Cu 2+ 2.94 mg L -1 (Removal rate 2.0%), tetracycline was 2.91 mg / L. -1 (Removal rate 3.0%), sulfadiazine was 1.96 mg / L. -1 (Removal rate 2.0%), see Table 1.

[0052] Therefore, under the same design conditions, the pretreatment effect of the blank group without the addition of symbiotic and GR24 on pig farm wastewater was much lower than that of the control reaction group.

[0053] Example 2: Control group and pretreatment of pig farm wastewater

[0054] The control group (with symbiont without GR24) constructed a triphasic algae-bacterial symbiont without GR24 (Chlorella: Endophytic bacteria S395-2: Polyspora spiralis = 10:1:4, initial biomass 90 mg / L). -1 The same pig farm wastewater as in Example 1 was added at 10% (v / v), and the reactor configuration and environmental control conditions were the same as in Example 1. Samples were taken every 24 hours during the cultivation period. Biomass was determined by centrifugation: 10 mL of sample was centrifuged at 8000 rpm for 10 min, and the precipitate was dried at 80 ℃ to constant weight and then weighed. SOD activity was determined by xanthine oxidase method: 5 mL of sample was centrifuged, the precipitate was collected, phosphate buffer (0.05 mol / L, pH 7.8) was added, and the mixture was sonicated. The supernatant was collected by centrifugation, and the enzyme activity was calculated by measuring the absorbance at 550 nm according to the SOD kit (Nanjing Jiancheng) instructions. Results after 7 days showed that the symbiotic specific growth rate was 0.19 d. -1 The SOD activity was 93.1 U / mgprot; the residual pollutant concentration was Zn. 2+ 3.21 mg L -1 (Removal rate 35.8%) Cu 2+ 1.98 mg L -1 (Removal rate 34.0%), Tetracycline 1.56 mg L -1 (Removal rate 48.0%), sulfadiazine 1.12 mg L -1(Removal rate 44.0%), see Table 1, indicating that the lack of GR24 resulted in limited stress resistance and degradation capacity of the symbiont.

[0055] Therefore, under the same design conditions, although this implementation showed improved degradation capacity compared to the control group, it did not achieve a more significant benefit.

[0056] Example 3: Pretreatment of pig farm wastewater based on GR24-regulated triphasic algae-bacteria symbiosis.

[0057] In the system of this invention (with added GR24-enhanced symbiont), the concentration of GR24 was 10. -7 M, 10 -9 M, 10 -11 M, a three-phase algae-bacterial symbiotic constructed (strain ratio and initial biomass same as in Example 2), was added at 10% (v / v) to 500 mL of the same pig farm wastewater. The reaction conditions and detection methods were the same as in Example 2. During the cultivation process, it was observed that adding 10... -9 The GR24 symbiont of M formed uniform algal spheres with a diameter of 0.50±0.03 cm at 54 h, which was 25% shorter than the control group (spheres formed at 72 h, with a diameter of 0.52±0.15 cm), and the uniformity was significantly improved. After 7 days of treatment, the symbiont biomass reached 185 mg / L. -1 Fv / F M The value was 0.78 (measured using a PAM-2500 chlorophyll fluorometer; Fo and F were recorded after 30 min of dark adaptation). M ); specific growth rate was 0.25 d. -1 The SOD activity was 120.0 U / mgprot, representing increases of 31.7% and 28.9% compared to the control group, respectively; the pollutant removal effect was significantly optimized, and Zn 2+ The residue decreased to 1.85 mg L. -1 (Removal rate 63.0%) Cu 2+ Decreased to 1.02 mg L -1 (Removal rate 66.0%), tetracycline decreased to 0.68 mg / L. -1 (Removal rate 77.3%), sulfadiazine decreased to 0.45 mg / L. -1 (Removal rate 77.5%), see Table 1; demonstrating that when the GR24 concentration in the symbiotic system is 10... -9 M can effectively regulate and enhance the stress resistance and pollutant degradation efficiency of the symbiont.

[0058] Therefore, under the same design conditions, this embodiment achieves a GR24 concentration of 10 in the system. -9 The M-time adjustment resulted in more significant benefits in the growth efficiency of the triphasic algae-bacteria symbiotic system and the removal of pollutants from pig farm wastewater.

[0059] Furthermore, it should be understood that after reading the above description of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0060] Table 1. Comparison of the effects of the blank group, control group, and the pretreatment of pig farm wastewater according to the present invention (treatment for 7 consecutive days).

[0061] .

Claims

1. A method for constructing a GR24-regulated triphasic algal-bacterial symbiotic, characterized in that, The specific steps include: (a) Preparation of microbial strains (1) Chlorella cultivation: Chlorella, which has high photosynthetic efficiency and strong pollutant absorption capacity, was selected as the dominant algal species; the initial concentration was strictly controlled at 2.0×10⁻⁶. 6 cell·mL -1 Activation culture was performed using BG11 medium; culture conditions were set as follows: temperature 25±2 ℃; light intensity controlled at 180-200 μmol·m⁻¹ -2 ·s -1 The light-dark ratio was set to (8 h - 14 h): (8 h - 14 h) to simulate the day-night cycle in the natural environment, in order to conform to the growth rhythm of Chlorella. Under the above culture conditions, Chlorella reached the required initial concentration and maintained good physiological activity after 5 days of culture. (2) Culture of endophytic bacteria S395-2: The initial concentration was controlled at 2.0 × 10⁻⁶. 7 CFU·mL -1 ; LB medium was used for activation culture; the culture conditions were 37 ℃ and 180 rpm shaking for 24 h to promote aerobic respiration and rapid reproduction of bacteria. After 24 h of culture, the endophytic bacteria S395-2 reached the required initial concentration and were in an active metabolic state. (3) Culture of Polyspora spiralis: PDA medium was used for activation culture, and the culture conditions were 25 ℃ for 7 days to prepare for the subsequent formation of symbiotic relationship with microalgae and bacteria; (ii) Setting up the GR24 concentration gradient The synthetic strigolactone analog GR24 was added to the co-culture system, with a concentration gradient of 10⁻⁶. -7 M, 10 -9 M, 10 -11 M was used as a control group, while 0 M was used to screen for the optimal GR24 concentration for the ball-forming performance and stress resistance of the triphasic algae-bacteria symbiotic system; the initial algae-bacteria biomass of the co-culture system was controlled at 80-100 mg / L. -1 This ensures a balanced ratio among the various microbial species in the co-culture system, providing a stable foundation for subsequent symbiont construction and performance optimization. (III) Symbiotic Culture and Optimization Activated Chlorella, endophytic bacteria S395-2, and Polyspora spiralis were inoculated into co-culture systems containing different concentrations of GR24 at a cell ratio of Chlorella:endophytic bacteria:S395-2:Polyspora spiralis = (1-20):(1-10):1; and cultured at 25±2 ℃ under light intensity of 180-200 μmol·m -2 ·s -1 The cultures were cultivated under a light-dark ratio of (8 h - 14 h): (10 h - 12 h) to ensure that each strain maintained good growth and metabolic activity during the co-culture process. During the cultivation process, the pellet formation process was observed in real time using a laser scanning confocal microscope (CLSM). Pellet formation time and pellet diameter were recorded every 5-6 hours. By comparing and analyzing pellet formation data at different GR24 concentrations, the optimal GR24 concentration was ultimately determined to be 10⁻⁻⁶. 7 M--10⁻ 11 M; At this concentration, the triphase algae-bacterial symbiosis has the shortest spheroidization time, the most uniform algae-bacterial spheroid diameter, and the strongest stress resistance.

2. The method for constructing a GR24-regulated triphasic algal-bacterial symbiotic according to claim 1, characterized in that, Chlorella was cultured using BG11 medium, with the following components: NaNO3 1.5 g / L. -1 K2HPO4·3H2O 0.04 g L -1 MgSO4·7H2O 0.075g L -1 CaCl2·2H2O 0.036 g L -1 Citric acid 0.006 g / L -1 Ferric ammonium citrate 0.006 g / L -1 EDTA 0.001 g L -1 Na2CO3 0.02 g L -1 1 mL of trace element solution -1 The trace element solution contains 2.86 g / L H3BO3. -1 MnCl2·4H2O 1.81 g L -1 ZnSO4·7H2O 0.222 g L -1 Na₂MoO₄·2H₂O 0.39 g L -1 CuSO4·5H2O 0.079 g L -1 Co(NO3)2·6H2O 0.049 g L -1 .

3. The method for constructing a GR24-regulated triphasic algal-bacterial symbiotic relationship according to claim 1, characterized in that, Endophytic bacteria S395-2 were cultured using LB medium, the composition of which was: NaCl 10 g, peptone 10 g, yeast extract 5 g, agar 15-20 g, distilled water 1000 mL, pH 7, sterilized at 121 ℃ for 20 min.

4. The method for constructing a triphasic algal-bacterial symbiotic based on GR24 regulation according to claim 1, characterized in that, The PDA medium used for culturing Polyspora spiralis consists of: 200 g potato, 20 g sucrose, 15–20 g agar, 1000 mL distilled water, and sterilized at 121 °C for 20 min.

5. A triphasic algae-bacteria symbiotic obtained by the construction method according to any one of claims 1-4.

6. The application of the triphasic algae-bacteria symbiosis as described in claim 5 in the treatment of pig farm wastewater, characterized in that, GR24 concentration 10 -9 A three-phase algae-bacteria symbiotic constructed by M was introduced into pig farm wastewater; the treatment temperature was controlled to be consistent with the symbiotic culture temperature to avoid adverse effects of temperature changes on symbiotic performance; dissolved oxygen was controlled at 2-4 mg / L. -1 .

7. The application as described in claim 6, characterized in that, In the process of treating pig wastewater, the specific growth rate and superoxide dismutase activity of the symbiont were measured to verify its ability to resist heavy metal and antibiotic stress.

8. The application as described in claim 7, characterized in that, The specific process is as follows: (1) First, the pig farm wastewater is pretreated, that is, the COD of the collected pig farm wastewater is adjusted to 1000-2000 mg / L. -1 Ammonia nitrogen should be adjusted to 100-400 mg / L. -1 Add Zn 2+ Up to 1-10 mg / L -1 Cu 2+ Up to 1-5 mg / L -1 Tetracycline antibiotics adjusted to 2-5 mg / L -1 Sulfonamide antibiotics up to 0.3-6 mg / L -1 Adjust the pH to 7.0 ± 0.2; (2) Then, the triphasic algae-bacteria symbiotic was added to the pretreated pig farm wastewater at a ratio of 10% (v / v) and at a temperature of 25±1 ℃ and dissolved oxygen of 2-4 mg L. -1 Aeration treatment for 5-8 days under the specified conditions; During treatment, the specific growth rate of the symbiont and SOD activity, as well as the levels of heavy metals and antibiotic residues in the wastewater, were measured regularly to ensure the control of Zn. 2+ Removal rate ≥60%, Cu 2+ Removal rate ≥65%, tetracycline antibiotic removal rate ≥70%, sulfonamide antibiotic removal rate ≥75%.

9. The application as described in claim 8, characterized in that, The aeration treatment uses a porous aeration head, and the aeration rate is controlled at 0.5-1.0 L (L·h). -1 Dissolved oxygen concentration is monitored in real time using a portable dissolved oxygen meter to ensure that dissolved oxygen remains stable within the set range during the reaction process.

10. The application as described in claim 8, characterized in that, The tetracycline antibiotics are selected from tetracycline, oxytetracycline, and chlortetracycline, and the sulfonamide antibiotics are selected from sulfadiazine, sulfadiazine, and sulfamethoxazole.