Complex microbial inoculant and biological polysaccharide for flocculent precipitation of aquaculture wastewater and application of complex microbial inoculant and biological polysaccharide

Abstract

The invention discloses a complex microbial inoculant and biological polysaccharide for flocculent precipitation of aquaculture wastewater and application of the complex microbial inoculant and the biological polysaccharide, and relates to the field of microorganisms, in particular to the complex microbial inoculant and the biological polysaccharide for flocculent precipitation of aquaculture wastewater and application of the biological polysaccharide. The method solves the problems that an existing method for removing suspended solids in aquaculture wastewater is prone to equipment blockage, has no continuous treatment effect and is high in input cost. The complex microbial inoculant disclosed by the invention is prepared from filamentous flocculation fusarium fungi and far-antarctic pseudomonas. The complex microbial inoculant is used for treating aquaculture wastewater, a better flocculation effect is achieved by combining microbial cell structure characteristics and microbial growth and metabolism activities, extracellular polymeric substances generated by denitrifying bacteria adsorb suspended particles and fungal hyphae in water to be directly connected with particulate matter, a larger and more stable flocculating constituent is formed, the flocculation advantages of the two bacteria are played, and the flocculation effect of the two bacteria is improved. The turbidity and the suspended matter concentration of sewage can be reduced, and the soluble nitrogen pollutant concentration in the aquaculture wastewater can be removed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms, in particular to a composite microbial agent for flocculating and precipitating aquaculture wastewater, a biological polysaccharide and application thereof. BACKGROUND

[0002] Livestock farming is the main source of water pollutant emissions in agricultural production. The high-concentration, high-ammonia-nitrogen and high-suspended solids (three-high) type of livestock and poultry wastewater generated by large-scale farms is difficult to treat. Livestock and poultry wastewater is mainly composed of animal manure, urine, feed residues and shed flushing water, among which the main components are urine and flushing water. Therefore, the quality and quantity of aquaculture wastewater change greatly, with high suspended solids, high organic matter concentration, high ammonia nitrogen concentration, heavy metals, pathogenic bacteria and foul odor. If not treated, these pollutants will not only pollute the surrounding water, but also penetrate into groundwater and enter the food chain, thereby endangering human health.

[0003] For large-scale farms, the solid-liquid separation process is the first step in the treatment of aquaculture wastewater. By using solid-liquid separation technology, the solid suspended solids in the wastewater can be initially removed, the organic pollutants in the wastewater can be reduced, and the equipment for subsequent treatment can be prevented from being blocked. The method for achieving solid-liquid separation of aquaculture wastewater can be physical, chemical and biological methods. The physical method is mainly screening, which can only initially remove particles with a particle size of 1-1.5 times the size of the screen hole. After treatment, there are still many colloids and small solid suspended solids in the water, and the screen is easily blocked. The chemical method, such as flocculation, requires the addition of a coagulant. However, the addition of the reagent not only increases the cost, but also easily causes secondary pollution and affects the water quality of the subsequent treatment. The biological method has the characteristics of high-efficiency flocculation, degradation, ecological friendliness and resource recovery. The biological flocculant is the core of the biological flocculation technology, which can be a high-molecular-weight compound secreted by microorganisms, including polysaccharides, proteins and lipids. However, the cost of extracting the biological flocculant alone is high, and there is no sustained flocculation effect. SUMMARY

[0004] The present application provides a composite microbial agent for flocculating and precipitating aquaculture wastewater, a biological polysaccharide and application thereof in order to solve the problems of easy blocking of equipment, no sustained treatment effect and high cost of existing methods for removing suspended solids in aquaculture wastewater.

[0005] The composite microbial agent for flocculating and precipitating aquaculture wastewater consists of Fusarium filamenticoagulans and Pseudomonas extremaustralis. In the composite microbial agent for flocculating and precipitating aquaculture wastewater, the Fusarium filamenticoagulans 553A-3 has a preservation number of CGMCC No. 42124 and a preservation date of July 10, 2025, and the Pseudomonas extremaustralis Y39-6 is Pseudomonas extremaustralis Y39-6.

[0006] Further, the preparation method of the composite microbial agent for flocculating and precipitating aquaculture wastewater is carried out according to the following steps:

[0007] I. Fusarium filamenticoagulans 553A-3 is inoculated into a Chodat yeast paste liquid culture medium for culture, and Pseudomonas extremaustralis Y39-6 is inoculated into an LB liquid culture medium for culture; the liquid culture mediums inoculated with Fusarium filamenticoagulans 553A-3 and Pseudomonas extremaustralis Y39-6 are respectively placed in a shaking table with conditions of 140 rpm and 30 DEG C for 72 h of culture, so that Fusarium filamenticoagulans bacterial liquid and Pseudomonas extremaustralis bacterial liquid are obtained; II. The Fusarium filamenticoagulans bacterial liquid and the Pseudomonas extremaustralis bacterial liquid are mixed, so that the composite microbial agent is prepared.

[0008] Further, in step I, the concentration of K2HPO4 in the Chodat yeast paste liquid culture medium is 1.0 g·L -1 , the concentration of yeast powder is 5 g·L -1 , the concentration of sucrose is 30 g·L -1 , the concentration of Chodat concentrated stock solution is 10 mL·L -1 , and the pH is 7; the Chodat concentrated stock solution is composed of 30 g of NaNO3, 5 g of KCl, 0.5 g of MgSO4·7H2O, 0.5 g of FeSO4·7H2O and 100 mL of distilled water; in the LB liquid culture medium, the concentration of tryptone is 10 g·L -1 , the concentration of yeast powder is 5 g·L -1 , the concentration of NaCl is 10 g·L -1 , and the pH is 7.

[0009] Further, in step II, the Fusarium filamenticoagulans bacterial liquid and the Pseudomonas extremaustralis bacterial liquid are mixed according to a liquid volume ratio of 1:4.

[0010] The composite microbial agent is applied to flocculating and precipitating aquaculture wastewater.

[0011] The preparation method of the Pseudomonas extremaustralis biological polysaccharide for flocculating and precipitating aquaculture wastewater is carried out according to the following steps:

[0012] I. Pseudomonas extremaustralis Y39-6 is inoculated into 100 mL of LB culture solution, and seed liquid is obtained by overnight culture at a rotation speed of 140 rpm and 25 DEG C in a shaker; the seed liquid is inoculated into 100 mL of denitrification culture solution at a inoculation amount of 2%, and fermentation liquid is obtained by fermentation at a rotation speed of 140 rpm and 25 DEG C for 72 hours;

[0013] II. The fermentation liquid is placed in a 50 HZ ultrasonic cleaning instrument for ultrasonic treatment for 2 minutes, and 10,000 rpm centrifugation is performed for 10 minutes to remove the bacterial bodies, and the supernatant is collected;

[0014] III. The supernatant is concentrated to 1 / 10 of the original volume by a rotary evaporator, 4 times the volume of absolute ethanol is added, and the precipitate is precipitated at 4 DEG C overnight, and the precipitate is collected by centrifugation at 4 DEG C and 4,000 rpm for 5 minutes, and is dried at 60 DEG C to obtain a crude polysaccharide;

[0015] IV. The crude polysaccharide precipitate is redissolved in deionized water, 80% trichloroacetic acid (TCA) is added to the crude polysaccharide solution, the final concentration is adjusted to 6%, and incubation is performed at 4 DEG C overnight; V. The solution obtained in step IV is centrifuged at 10,000 r / min for 10 min, and the supernatant is collected, thereby obtaining Pseudomonas extremaustralis biological polysaccharide.

[0016] The application of the Pseudomonas extremaustralis biological polysaccharide in treating aquaculture wastewater.

[0017] Further, the application method of the biological polysaccharide in treating aquaculture wastewater is as follows: Pseudomonas extremaustralis Y39-6 biological polysaccharide is added to the aquaculture wastewater to be treated at a weight percentage of 1% to 2% of the aquaculture wastewater to be treated, and then stirring and standing are performed.

[0018] The application of the composite microbial agent and the Pseudomonas extremaustralis biological polysaccharide in treating aquaculture wastewater.

[0019] Further, the application of the composite microbial agent and the Pseudomonas extremaustralis biological polysaccharide is as follows: the composite microbial agent is used to treat the aquaculture wastewater first, and then the biological polysaccharide is used for treatment. That is, the composite microbial agent is used to treat the aquaculture wastewater with a medium concentration, and then the biological polysaccharide is added to achieve direct flocculation.

[0020] The composite microbial agent is applied to treat aquaculture wastewater and other wastewater with relatively large turbidity and good biodegradability. The microbial composite agent is used to remove suspended solids in aquaculture wastewater, reduce the subsequent treatment pressure, and reduce the cost of wastewater treatment through microbial community recycling.

[0021] The present application has the following advantages:

[0022] 1. The microbial compound agent of this invention can adapt to changes in water temperature. The *Pseudomonas antarctica* used in this invention grows at temperatures ranging from 4 to 37°C and maintains its activity even in environments below 8°C. It retains flocculation activity even at low temperatures, achieving solid-liquid separation in wastewater. The filamentous flocculent *Fusarium* fungus used in this invention can adapt to high-stress environments and can be used for high-concentration wastewater treatment. Its filamentous structure not only effectively adsorbs particulate matter from high-concentration wastewater and degrades organic matter in the water, but also acts as a carrier for bacteria, forming larger and more stable bioflocs that enhance flocculation advantages.

[0023] 2. The microbial compound agent of the present invention does not require the addition of additional carbon sources or chemical agents during the sewage treatment process, which can save sewage treatment costs.

[0024] 4. The Pseudomonas extremaustralis Y39-6 biopolysaccharide described in this invention is safe and environmentally friendly, and does not pose a risk of secondary pollution compared to traditional chemical flocculants.

[0025] 5. The biopolysaccharide of Pseudomonas extremaustralis Y39-6 of this invention has high flocculation properties, can adapt to a wide range of pH, and can directly achieve solid-liquid separation of low-concentration aquaculture wastewater within 5 minutes after stirring with the assistance of metal ions.

[0026] 6. This invention combines a composite microbial agent composed of filamentous flocculent Fusarium fungi and Pseudomonas antarctica with a biopolysaccharide prepared by Pseudomonas antarctica. The composite microbial agent is first used to treat aquaculture wastewater, and then the biopolysaccharide is added to achieve direct flocculation. The bioflocculation technology of this invention can treat aquaculture wastewater of varying concentrations; that is, this method can treat aquaculture wastewater, and after the bioreaction of the composite microbial agent, the aquaculture wastewater reaches the limit of polysaccharide treatment. The polysaccharide promotes further flocculation of the aquaculture wastewater after the bioreaction. Solid content and turbidity are key parameters for flocculation characterization. This invention can not only treat high-concentration aquaculture wastewater with the composite microbial agent, but also directly treat low-concentration aquaculture wastewater with the biopolysaccharide produced by Pseudomonas antarctica, achieving a turbidity removal rate of 97-99%.

[0027] The present application aims to provide a compound microbial agent of denitrifying bacteria (Pseudomonas extremaustralis) and fungi (Fusarium filamenticoagulans), which is used for treating aquaculture wastewater, and realizes better flocculation effect by combining the cell structure characteristics of microorganisms and the growth and metabolism activities of microorganisms, the extracellular polymers produced by denitrifying bacteria adsorb suspended particles in water, and the fungal hyphae directly connect the particles to form larger and more stable flocculation bodies, which play the flocculation advantages of the two bacteria, can not only reduce the turbidity and suspended matter concentration of the wastewater, but also remove the soluble nitrogen pollutant concentration in the aquaculture wastewater. In addition, the biological polysaccharide can directly and quickly realize solid-liquid separation, has high-efficiency flocculation, environmental protection and safety. The present application solves the problems of easy equipment blockage, no continuous treatment effect and high investment cost in the existing method for removing suspended matter in aquaculture wastewater, realizes flocculation of aquaculture wastewater by using biological method, saves the aquaculture wastewater treatment cost, promotes resource recycling, and realizes sustainable development of ecological environment.

[0028] Fusarium filamenticoagulans 553A-3, the preservation number is CGMCC No. 42124, and the preservation date is July 10, 2025; the preservation address of Pseudomonas extremaustralis Y39-6 and Fusarium filamenticoagulans 553A-3 is Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is a bacterial body morphology diagram of Fusarium filamenticoagulans 553A-3 in Example 1.

[0030] Figure 2 It is an effect diagram of treating aquaculture wastewater under different temperatures of the compound microbial agent in Example 1.

[0031] Figure 3 It is a flocculation effect diagram at 30°C in Example 1.

[0032] Figure 4 It is an effect diagram of treating aquaculture wastewater under different microbial liquid ratios of the compound microbial agent in Example 1.

[0033] Figure 5 It is an effect diagram of removing other pollutants under the optimal conditions of the compound microbial agent in Example 1.

[0034] Figure 6 It is a self-flocculation effect diagram of the compound microbial agent in Example 2.

[0035] Figure 7The flocculation rate of Pseudomonas subsp. Y39-6 bio-polymer on kaolin suspension under different metal ion species in Example 3;

[0036] Figure 8 The flocculation of Pseudomonas subsp. Y39-6 bio-polymer on kaolin suspension under FeCl3, ZnSO4, CuSO4 solution in Example 3;

[0037] Figure 9 The flocculation rate of Pseudomonas subsp. Y39-6 bio-polymer on kaolin suspension under different metal ion dosage in Example 3;

[0038] Figure 10 The flocculation rate of Pseudomonas subsp. Y39-6 bio-polymer on kaolin suspension under FeCl3 auxiliary in Example 3;

[0039] Figure 11 The flocculation rate of Pseudomonas subsp. Y39-6 bio-polymer on kaolin suspension under different pH in Example 3;

[0040] Figure 12 The turbidity removal rate of Pseudomonas subsp. Y39-6 bio-polymer on aquaculture wastewater under FeCl3 auxiliary in Example 3;

[0041] Figure 13 The flocculation of Pseudomonas subsp. Y39-6 bio-polymer on aquaculture wastewater under FeCl3 auxiliary in Example 3;

[0042] Figure 14 The low-concentration aquaculture wastewater diluted 4 times treated by composite microbial agent in Example 4;

[0043] Figure 15 The aquaculture wastewater treated by bio-polymer in Example 4;

[0044] Figure 16 The flocculation of aquaculture wastewater in Example 2 experimental group and blank group (from left to right are Y39-6, 553A-3, Y39-6 and 553A-3 composite, blank group);

[0045] Figure 17 The turbidity change of aquaculture wastewater in Example 2 each group;

[0046] Figure 18 The sedimentation diagram of Example 5 circulating treatment;

[0047] Figure 19 The pollutant removal diagram of Example 5 circulating treatment. DETAILED DESCRIPTION

[0048] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0049] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0050] Example 1 Preparation of a composite microbial agent for flocculating and precipitating suspended matter in aquaculture wastewater

[0051] 1、The composite microbial agent for flocculating and precipitating suspended matter in aquaculture wastewater is prepared from Fusarium and Pseudomonas extremaustralis. Among them, the Fusarium filamenticoagulans 553A-3 has a preservation number of CGMCC No. 42124 and a preservation date of July 10, 2025; the Pseudomonas extremaustralis Y39-6 has a preservation number of CGMCC No. 16652 and a preservation date of October 29, 2018, and has been disclosed in the invention patent CN201910073221.4.

[0052] Screening of Fusarium filamenticoagulans 553A-3:

[0053] In June 2024, 50 g of mixed soil was taken from a flower bed in the campus of Heilongjiang University, 100 ml of distilled water was added, and after stirring uniformly, it was placed in a shaking bed at 35℃ and 160 rpm, 1 g of chloramphenicol was added daily, and after 15 days, it was taken out and left to stand. The supernatant after standing is the soil leachate. Take 3 portions of 100 μL of soil leachate and add 900 mL of physiological saline for gradient dilution, then perform streaking separation on an inorganic salt culture medium at 35℃, and select typical single colonies for purification culture, and select strain 553A-3.

[0054] The inorganic salt culture medium has the following components: NH4NO31 g, KH2PO40.5 g, K2HPO4·3H2O 1.5 g, NaCl 1.0 g, MgSO4·7H2O 0.2 g, 500 mg / L CAP 20 ml, H2O 1.0 L, pH 7.0, agar 2%, wherein the ammonia source is ammonium nitrate, and agar in the culture medium is used as a carbon source.

[0055] The strain 553A-3 is identified as Fusarium filamenticoagulans, and named as Fusarium filamenticoagulans 553A-3.

[0056] The Fusarium filamenticoagulans 553A-3 is white in initial culture for several days, and the white needle-like hyphae continuously spread outward. The strain is vertically raised as a peak from the culture medium, and the color of the colony center becomes light red after further culture. The color of the colony center becomes deeper at low temperature, and the whole colony gradually becomes red-brown. The suitable growth temperature is 25-37℃, and the suitable growth pH value is 5-8. The Fusarium filamenticoagulans 553A-3 can assimilate and utilize L-threonine, L-proline, L-tryptophan, L-leucine and glycine, but cannot utilize L-methionine. The Fusarium filamenticoagulans 553A-3 can utilize glucose, maltose, starch, sucrose and cellulose, and has strong assimilation ability to starch. Figure 1 FIG. 1 is a morphological diagram of the Fusarium filamenticoagulans 553A-3 strain.

[0057] 2. A method for preparing a composite microbial agent for flocculating and precipitating aquaculture wastewater by using the two strains above:

[0058] I. The Fusarium filamenticoagulans 553A-3 is inoculated into a liquid culture medium of a Chodat's yeast paste for culture, and the Pseudomonas extremaustralis Y39-6 is inoculated into a liquid LB medium for culture. The liquid culture media inoculated with the Fusarium filamenticoagulans 553A-3 and the Pseudomonas extremaustralis Y39-6 are respectively placed in a shaker with conditions of 140 rpm and 30℃ for 72 h of culture, so as to obtain a Fusarium filamenticoagulans liquid and a Pseudomonas extremaustralis liquid. II. The Fusarium filamenticoagulans liquid and the Pseudomonas extremaustralis liquid are mixed to obtain the composite microbial agent. The concentration of K2HPO4 in the liquid culture medium of the Chodat's yeast paste is 1.0 g·L -1 The concentration of yeast powder is 5 g·L -1 The concentration of sucrose is 30 g·L -1 The concentration of Chodat's concentrated stock solution is 10 mL·L -1, pH 7; the Cha's concentrated stock solution is composed of 30 g of NaNO3, 5 g of KCl, 0.5 g of MgSO4·7H2O, 0.5 g of FeSO4·7H2O and 100 mL of distilled water; the concentration of tryptone in the LB liquid medium is 10 g·L -1 , the concentration of yeast powder is 5 g·L -1 , the concentration of NaCl is 10 g·L -1 , pH 7; the volume ratio of the Fusarium filamenticoagulans bacterial solution and the Pseudomonas extremaustralis bacterial solution is 1:4.

[0059] The prepared composite microbial agent can be directly used in aquaculture wastewater.

[0060] 3. The flocculation and precipitation effect of the composite microbial agent of the present embodiment in aquaculture wastewater is verified.

[0061] 3.1 200 mL of aquaculture wastewater was taken, and 160 mL of the composite microbial agent was added according to the aquaculture wastewater volume of 80%, wherein the composite microbial agent was composed of 32 mL of Pseudomonas extremaustralis Y39-6 bacterial solution and 128 mL of Fusarium filamenticoagulans 553A-3 bacterial solution. It was placed in a 140 rpm shaker for 5 days, and the shaker was set at five different temperatures, i.e. 30°C, 25°C, 20°C, 10°C and 5°C.

[0062] Figure 2 The effect of the composite microbial agent of the present embodiment on the treatment of aquaculture wastewater at the above different temperatures was verified from the Figure 2 It can be seen that the removal rate is the highest at 30°C, and the treatment effect is the best, Figure 3 The flocculation effect at 30°C, combined with Figure 2 and Figure 3 It can be seen that the composite microbial agent has the highest removal rate of total solids (ST) in the supernatant of aquaculture wastewater at 30°C, which can reach about 37%; a high removal rate can also be maintained at room temperature, and the actual wastewater treatment can be carried out at room temperature.

[0063] Table 1 Index of aquaculture wastewater used in the experiment

[0064] Turbidity (NTU) Total solids (g / L) COD (mg / L) NO3 - - N (mg / L) NO2 - - N (mg / L) Concentration of each pollutant in aquaculture wastewater 2000~4000 16~22 9000~12000 110~130 0.25~0.5

[0065] 3.2 200 mL of aquaculture wastewater was taken, and 160 mL of the composite microbial agent was added according to the aquaculture wastewater volume of 80%, and the Fusarium filamenticoagulans and Pseudomonas extremaustralis bacterial solutions were mixed according to different volume ratios, i.e. 1:1, 1:2.5, 2.5:1, 1:4 and 4:1, respectively,Figure 4 The effect diagram of the composite microbial agent treating aquaculture wastewater at different ratios of microbial solution.

[0066] From Figure 4 It can be seen that the total solid removal rate of supernatant is similar, but the massive reproduction of filamentous bacteria may cause sludge bulking. The results also show that increasing the ratio of Pseudomonas subantarctica Y39-6 microbial solution can improve the turbidity removal rate. In actual treatment of aquaculture wastewater, filamentous flocculating Fusarium 553A-3 and Pseudomonas subantarctica Y39-6 can be used according to a ratio of 4:1 by volume.

[0067] 3.3 200 mL of aquaculture wastewater was taken, 160 mL of composite microbial agent was added at a proportion of 80%, and filamentous flocculating Fusarium and Pseudomonas subantarctica microbial solution was mixed according to a ratio of 1:4 by volume. The reaction was carried out at 140 rpm, 30°C, and pH 7 for 5 days. The removal effect of other organic pollutants (COD, NO3 - -N, NO2 - -N) in aquaculture wastewater is shown in Figure 5 .

[0068] From Figure 5 It can be seen that the composite microbial agent can not only flocculate and precipitate, but also remove COD and nitrogen pollutants when treating high-concentration aquaculture wastewater under this condition. The composite microbial agent has a certain degree of degradation for removing other organic pollutants.

[0069] Example 2

[0070] Observation of the filamentous flocculating Fusarium 553A-3 and Pseudomonas subantarctica Y39-6 composite microbial agent floc for flocculating and precipitating aquaculture wastewater.

[0071] 1. Two rings of Pseudomonas subantarctica Y39-6 and filamentous flocculating Fusarium 553A-3 were taken with an inoculation ring and placed in a 500 mL Erlenmeyer flask containing 250 mL of denitrification medium (denitrification medium: anhydrous sodium acetate 10 g·L -1 , NaNO30.6 g·L -1 , anhydrous magnesium sulfate 0.489 g·L -1 , NaCl 1 g·L -1 , anhydrous calcium chloride 0.005 g·L -1 , phosphate buffer 7.5 mL / L; phosphate buffer: Na2HPO415 g·L -1 , KH2PO4 4 g·L -1, PH adjusted to about 7, after sterilization, add 5 mL of iron and manganese solution to every 100 mL of medium; iron and manganese solution: 0.14 g ferrous ammonium sulfate, 0.06 g magnesium sulfate, 100 mL distilled water), then place the flask in a constant temperature shaker at a speed of 140 rpm, 30°C for 5 days, Figure 6 Self-flocculation effect of the complex bacterial agent.

[0072] From Figure 6 It can be seen that the culture of the complex bacterial agent can appear self-flocculation, and the mycelium of the filamentous flocculating Fusarium fungus 553A-3 can firmly stick to the bottle wall. Figure 6 The left side is the flocculation of the complex bacterial agent from the outside of the flask, and the right side is the flocculation of the complex bacterial agent from the inside of the flask. Combined with Figure 6 It can be seen that the mycelium of the filamentous flocculating Fusarium fungus 553A-3 can achieve flocculation through winding.

[0073] 2. Experimental settings Pseudomonas subantarctica Y39-6, filamentous flocculating Fusarium fungus 553A-3, Pseudomonas subantarctica Y39-6 and filamentous flocculating Fusarium fungus 553A-3 complex flocculation breeding wastewater, and set up a blank control group. 200 mL of breeding wastewater was taken, and the breeding wastewater pollutant index can be seen from Table 1 (Table 1: Breeding wastewater index used in the experiment). According to the 80% proportion, 160 mL of bacterial agent was added, of which the complex bacterial agent composition was 32 mL of Pseudomonas subantarctica Y39-6 bacterial solution and 128 mL of Fusarium fungus 553A-3 bacterial solution, and the blank group was 360 mL of breeding wastewater. Four groups were placed in a shaker at a speed of 140 rpm, 30°C for 5 days. After 5 days, stand for 20 min to form a precipitate. Figure 16 The flocculation of the experimental group and the blank group (from left to right are Y39-6, 553A-3, Y39-6 and 553A-3 complex, and the blank group) breeding wastewater. Figure 17 The turbidity change of each group of breeding wastewater.

[0074] From Figure 16 It can be seen that compared with the blank group, the experimental group can form a large amount of precipitate, among which the precipitate of the Y39-6 group is granular and the structure is relatively tight, the precipitate of the 553A-3 group is filamentous, and compared with the Y39-6 group, the structure is loose, but the filamentous flocculation body is larger and can capture more particles. The complex bacterial agent combines the flocculation advantages of the two bacteria, so that the precipitate forms larger filamentous bodies and the structure is more compact.

[0075] From Figure 17 It can be seen that the turbidity of the blank group is unstable, while after adding the bacterial agent, the turbidity is obviously reduced, and the turbidity of the complex bacterial agent is reduced to the lowest after 5 days of treatment.

[0076] Example 3

[0077] Preparation of Pseudomonas extremaustralis Y39-6 bio-polymer for flocculating and precipitating suspended solids in aquaculture wastewater.

[0078] 1. A method for preparing Pseudomonas extremaustralis Y39-6 bio-polymer:

[0079] I. Two loops of Pseudomonas extremaustralis Y39-6 (CGMCC No. 16652, deposited on October 29, 2018) were inoculated into a 250 mL flask containing 100 mL LB culture solution, and then the flask was placed in a constant temperature shaker at a speed of 140 rpm and 25℃ overnight to obtain a seed solution; then 2% (v / v) inoculation amount was inoculated into a 250 mL flask containing 100 mL denitrification medium in Example 2, and fermented at a speed of 140 rpm and 25℃ for 72 h; II. The fermentation broth was placed in an ultrasonic cleaning instrument at 50 HZ for 2 min to remove the extracellular polysaccharide from the cell surface; centrifuged at 10000 rpm for 10 min to remove the bacteria, and the supernatant was collected; III. The supernatant was concentrated to 1 / 10 of the original volume by a rotary evaporator, 4 times the volume of absolute ethanol was added, and precipitated at 4℃ overnight; centrifuged at 4000 rpm and 4℃ for 5 min to collect the precipitate, and dried at 60℃ to obtain the crude polysaccharide; the crude polysaccharide precipitate was redissolved in deionized water, 80% (w / v) trichloroacetic acid (TCA) was added to the crude polysaccharide solution, and the final concentration was adjusted to 6% (v / v) to remove protein, and incubated at 4℃ overnight; IV. The resulting solution was centrifuged at 10000 r / min for 10 min, and the supernatant was collected to obtain Pseudomonas extremaustralis Y39-6 bio-polymer.

[0080] 2. The bio-polymer prepared in this example is used to treat aquaculture wastewater, and the flocculation and precipitation effect of the bio-polymer in aquaculture wastewater is verified.

[0081] 2.1 This test is used to determine the flocculation activity of extracellular bio-polymer produced by Pseudomonas extremaustralis Y39-6 for flocculating and precipitating suspended solids in aquaculture wastewater.

[0082] Experiment 1: 48 ml of kaolin suspension (5 g·L -1) and 1 mL of 1% metal ion solution, 1% metal ion solution is KCl, CaCl2, FeCl3, CuSO4, ZnSO4 solution respectively, the control group does not add metal ion solution. First, fast stirring at 200 rpm ~ 250 rpm for 1 min, then slow stirring at 50 ~ 100 rpm for 4 min, standing for 5 min, taking the supernatant to measure the absorbance at 550 nm, and calculating the flocculation rate FR.

[0083] FR= ×100%

[0084] In the formula, B is the absorbance of the control kaolin suspension at 550 nm, and A is the absorbance of the sample system after flocculation at 550 nm.

[0085] Figure 7 The flocculation rate of Pseudomonas extremaustralis Y39-6 bio-polymer on kaolin suspension under different metal ion species. Figure 8 The flocculation of Pseudomonas extremaustralis Y39-6 bio-polymer on kaolin suspension in FeCl3, ZnSO4 and CuSO4 solution, from left to right in the figure are FeCl3, ZnSO4 and CuSO4 solution, combined with Figure 7 Figure 8 It can be seen that Pseudomonas extremaustralis Y39-6 bio-polymer can realize flocculation with the assistance of Fe3 + , Zn2 + , Cu2 + , which indicates that the bio-polymer can remove some heavy metal ions in aquaculture wastewater.

[0086] Test 2: Different from test 1, 0.7 mg of Pseudomonas extremaustralis Y39-6 bio-polymer was added to the kaolin suspension, and different volumes of 1% FeCl3 solution were added, and the volume of FeCl3 was 0.3 mL, 0.5 mL, 0.8 mL, 1 mL, 1.5 mL, 2 mL and 2.2 mL respectively.

[0087] Figure 9 The flocculation rate of Pseudomonas extremaustralis Y39-6 bio-polymer on kaolin suspension under different metal ion dosages, from Figure 9 It can be seen that the presence of Fe 3+ does not inhibit the flocculation effect of Y39-6 bio-polymer.

[0088] Test 3: Different from test 1, different mass of Pseudomonas extremaustralis Y39-6 biosurfactant was added into the kaolin suspension, and 1 mL of 1% FeCl3 solution was added; the control group only added 1 mL of 1% FeCl3 solution, wherein the mass of Pseudomonas extremaustralis Y39-6 biosurfactant was 0.007 mg, 0.014 mg, 0.035 mg, 0.14 mg, 0.35 mg, 0.56 mg, 0.7 mg, 1.4 mg, 2.1 mg, and 2.8 mg, respectively.

[0089] Figure 10 For the flocculation rate of different mass of Pseudomonas extremaustralis Y39-6 biosurfactant to the kaolin suspension under the dosage of 1 mL of 1% FeCl3, from Figure 10 It can be seen that at the beginning, Y39-6 biosurfactant can promote flocculation, but excessive biosurfactant will inhibit flocculation.

[0090] Test 4: Different from test 1, equal mass of Pseudomonas extremaustralis Y39-6 biosurfactant was added into the kaolin suspension, and equal volume of 1% FeCl3 solution was added, and the pH of the solution was changed, the pH was 5, 6, 7, 8, 9, and 10, respectively.

[0091] Figure 11 For the flocculation rate of Pseudomonas extremaustralis Y39-6 under different pH conditions to the kaolin suspension, from Figure 11 It can be seen that Y39-6 biosurfactant can achieve flocculation under a wide range of pH (5-10) with the assistance of FeCl3, and the flocculation effect is better under alkaline conditions.

[0092] Test 5: Pseudomonas extremaustralis Y39-6 biosurfactant was directly used for flocculation of low-concentration aquaculture wastewater. 40 mL of aquaculture wastewater was taken, mixed with 360 mL of distilled water to obtain low-concentration aquaculture wastewater diluted by 10 times, then Y39-6 biosurfactant and 1 mL of 1% FeCl3 solution were continuously added, 1000 rpm high-speed stirring for 1 min, 400 rpm low-speed stirring for 2 min, and standing for 5 min. Figure 12 For the turbidity removal rate of Pseudomonas extremaustralis Y39-6 biosurfactant to aquaculture wastewater with the assistance of FeCl3, Figure 13 For the flocculation of Pseudomonas extremaustralis Y39-6 biosurfactant to aquaculture wastewater with the assistance of FeCl3. The left side of the figure is before treatment, and the right side is after treatment. Combined with Figure 12 and Figure 13 It can be seen that Pseudomonas extremaustralis Y39-6 biosurfactant can achieve good solid-liquid separation effect of aquaculture wastewater in a very short time.

[0093] Example 4

[0094] The complex microbial agent was cultured according to the culture method in Example 1, and then 100 mL of the aquaculture wastewater was mixed with 300 mL of distilled water to obtain low-concentration aquaculture wastewater diluted 4 times. After the aquaculture wastewater was treated with the complex microbial agent for 5 days, 4 mg of biosaccharide and 5 mL of 1% FeCl3 were added, and the mixture was stirred at 1000 rpm for 1 min, at 400 rpm for 2 min, and then left to stand for 5 min.

[0095] In this example, the aquaculture wastewater was first treated with the complex microbial agent, and then the biosaccharide was added. The flocculation effect of the complex microbial agent and the biosaccharide on the suspended solids in the aquaculture wastewater was verified, and Table 2 shows the flocculation results of the aquaculture wastewater, Figure 14 The complex microbial agent was used to treat low-concentration aquaculture wastewater diluted 4 times, Figure 15 The aquaculture wastewater after treatment with the biosaccharide is shown on the right side of the figure. As can be seen from Table 2, the complex microbial agent can maintain a high removal rate for low-concentration aquaculture wastewater, and the secondary treatment with the biosaccharide can make the aquaculture wastewater clear, thereby greatly reducing the pressure on the subsequent treatment process. In combination with Figure 14 and Figure 15 It can be seen that the complex microbial agent and the biosaccharide of Pseudomonas sp. Y39-6 can be used to jointly treat low-concentration aquaculture wastewater. The biosaccharide can promote the re-flocculation of the aquaculture wastewater after the biological reaction.

[0096] Table 2 Flocculation results of aquaculture wastewater

[0097] Before treatment with bacterial agent After treatment with bacterial agent Removal rate After further treatment of polysaccharide Total removal rate Turbidity in water (NTU) 803 72.9 90.92% 4.22 99.47% Total solids in water (g / L) 4.2 3 28.57% 1.2 71.43%

[0098] Example 5 The aquaculture wastewater was treated with the complex microbial agent according to Example 2, and after 5 days, the supernatant was gently poured off, and the sediment was retained. Then, 200 mL of untreated aquaculture wastewater was added to the flask containing the filamentous flocculation sediment. The aquaculture wastewater had the pollutant indexes shown in Table 1 (Table 1 Aquaculture wastewater indexes used in the experiment), and was placed in a shaking bed under the conditions of 140 rpm and 30°C for 5 days.

[0099] Figure 18 The sediment was recycled for treatment, Figure 19 The pollutant removal was recycled for treatment. As can be seen from Figure 18 After the first treatment, the sediment was a large amount of mature flocculation formed by Fusarium sp. 553A-3 and Pseudomonas sp. Y39-6, which could be used to continue treating new aquaculture wastewater and could form obvious solid-liquid separation. As can be seen from Figure 19 It can be seen that the aquaculture wastewater can still maintain the original treatment effect after being treated with the sediment, and even has a slight increase.

Claims

1. A compound microbial agent for flocculation and sedimentation of aquaculture wastewater, characterized in that, The compound microbial agent used for flocculation and sedimentation of aquaculture wastewater consists of filamentous flocculant Fusarium and Pseudomonas extremaustralis. The filamentous flocculant Fusarium (Fusarium filamenticoagulans) is 553A-3, with accession number CGMCC No. 42124 and accession date of July 10, 2025; the Pseudomonas extremaustralis is Y39-6.

2. The method for preparing the composite microbial agent for flocculation and sedimentation of aquaculture wastewater according to claim 1, characterized in that, The preparation method of the compound microbial agent for flocculation and sedimentation of aquaculture wastewater is carried out according to the following steps:

1. Inoculate Fusarium filamenticoagulans 553A-3 into Saccharomyces chalcogenide liquid medium for culture, and inoculate Pseudomonas extremaustralis Y39-6 into LB liquid medium for culture. Place the liquid media inoculated with Fusarium filamenticoagulans 553A-3 and Pseudomonas extremaustralis Y39-6 in a shaker at 140 rpm and 30°C for 72 h to obtain Fusarium filamenticoagulans and Pseudomonas extremaustralis liquids, respectively.

2. Mix the Fusarium filamenticoagulans and Pseudomonas extremaustralis liquids to prepare a compound inoculum.

3. The method for preparing the composite microbial agent for flocculation and sedimentation of aquaculture wastewater according to claim 2, characterized in that, In step one, the concentration of K₂HPO₄ in the Czapekella extract liquid culture medium is 1.0 g·L⁻¹. -1 The concentration of yeast powder is 5 g·L. -1 The concentration of sucrose is 30 g·L. -1 The concentration of the Czapek concentrated stock solution is 10 mL·L⁻¹. -1 The pH is 7; the Czapek's stock solution consists of 30 g NaNO3, 5 g KCl, 0.5 g MgSO4·7H2O, 0.5 g FeSO4·7H2O, and 100 mL distilled water; the concentration of tryptone in the LB liquid medium is 10 g·L⁻¹. -1 The concentration of yeast powder is 5 g·L. -1 The concentration of NaCl is 10 g·L⁻¹ -1 The pH value is 7.

4. The method for preparing the composite microbial agent for flocculation and sedimentation of aquaculture wastewater according to claim 2, characterized in that, In step two, the filamentous flocculent Fusarium fungus solution and the Far Antarctic Pseudomonas aeruginosa solution are prepared at a volume ratio of 1:

4.

5. The application of the compound microbial agent as described in claim 1 in flocculation and sedimentation of aquaculture wastewater.

6. A method for preparing *Pseudomonas antarctica* biopolysaccharide for flocculation and sedimentation of aquaculture wastewater, characterized in that, The preparation method of Pseudomonas far Antarctica polysaccharide is carried out according to the following steps:

1. *Pseudomonas extremaustralis* Y39-6 was inoculated into 100 mL of LB medium and cultured overnight on a shaker at 140 rpm and 25°C to obtain a seed culture. The seed culture was then inoculated into 100 mL of denitrification medium at a 2% inoculation rate and fermented for 72 h at 140 rpm and 25°C to obtain the fermentation broth.

2. Place the fermentation broth in a 50 Hz ultrasonic cleaner and sonicate for 2 min, then centrifuge at 10,000 rpm for 10 min to remove the cells and collect the supernatant.

3. The supernatant was concentrated to 1 / 10 of its original volume using a rotary evaporator. Four times the volume of the supernatant was added with anhydrous ethanol, and the mixture was allowed to precipitate overnight at 4°C. The precipitate was collected by centrifugation at 4000 rpm and 4°C for 5 min and then dried at 60°C to obtain crude polysaccharide.

4. Redissolve the crude polysaccharide precipitate in deionized water, add 80% trichloroacetic acid (TCA) to the crude polysaccharide solution, adjust the final concentration to 6%, and incubate overnight at 4°C; 5. Centrifuge the solution obtained in step 4 at 10,000 r / min for 10 min, collect the supernatant, and obtain the Pseudomonas far Antarctica biopolysaccharide.

7. The application of the biopolysaccharide prepared by the method described in claim 6 in the treatment of aquaculture wastewater.

8. The application of the biopolysaccharide in treating aquaculture wastewater according to claim 7, characterized in that... Add 1% to 2% of the weight of the aquaculture wastewater to be treated with Pseudomonas extremaustralis Y39-6 biopolysaccharide, then stir and let stand.

9. The application of the compound microbial agent as described in claim 1 and the Pseudomonas far Antarctica polysaccharide as described in claim 6 in the treatment of aquaculture wastewater.

10. The application according to claim 9, characterized in that... First, treat the aquaculture wastewater with a compound microbial agent, then add biological polysaccharides for further treatment.

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