Complex microbial inoculant embedded pellet as well as preparation method and application thereof

Abstract

The invention relates to the technical field of sewage treatment, and particularly discloses a complex microbial inoculant embedded pellet and a preparation method and application thereof, the method comprises the following steps: respectively inoculating Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 into a culture medium, culturing at 25-35 DEG C to a stable initial stage, then centrifuging at 4-6 DEG C, and removing supernate to obtain the complex microbial inoculant embedded pellet. Washing and re-suspending for three times by using a buffer solution, and then mixing the pseudomonas stutzeri CICC-10428 and the pseudomonas stutzeri XL-2 to obtain a compound bacterium suspension; the preparation method comprises the following steps: adding polyvinyl alcohol and sodium alginate into water, heating, uniformly mixing, and cooling to room temperature to obtain gel; the preparation method comprises the following steps: adding a gel into a compound bacterium suspension, uniformly mixing to obtain a mixed gel bacterium solution, then sucking the mixed gel bacterium solution, dropwise adding the mixed gel bacterium solution into a cross-linking agent, cross-linking at 4-6 DEG C for 24-48 hours, washing with sterile water for 3 times, and drying to obtain the compound bacterium agent embedded pellets. The denitrification effect of the recirculating aquaculture wastewater reactor can be improved, the addition amount of an extra carbon source is reduced, and the operation cost of a recirculating aquaculture plant can be greatly reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of sewage treatment, relates to circulating aquaculture water treatment, and particularly relates to a composite bacterial agent-embedded pellet and a preparation method and application thereof. Background Art

[0002] As an important part of the food industry, traditional aquaculture has always been the focus of research on improving productivity and optimizing resource utilization efficiency. The process technology that achieves high output and high efficiency in a high-density aquaculture environment has attracted much attention. The factory-based recirculating aquaculture system (RAS) has developed rapidly due to its advantages such as water saving, small land occupation, high aquaculture efficiency and low environmental pollution.

[0003] However, in the system for evaluating the water environment, nitrogen pollutants are an important basic parameter for measuring water quality. Under high-density farming and unscientific feeding methods, nitrogen pollutants in RAS water bodies are more likely to accumulate than in traditional flow-through aquaculture systems. In particular, when the concentration of ammonia nitrogen is too high, it will have serious toxic effects on fish, such as increased mucus on the fish surface, congestion of the skin, decreased body function, and even coma and death in severe cases. Therefore, it is crucial to ensure that the aquaculture water meets the Class III water body standards required by the "Surface Water Environmental Quality Standard (GB3838-2002)".

[0004] Moving bed biofilm reactor (MBBR) is a highly efficient biological treatment reactor, which is often used for biological denitrification in recirculating aquaculture. However, in actual applications, MBBR often faces problems such as increased operating costs or excessive nitrogen pollutant concentration in effluent caused by environmental factors such as oligotrophy, low carbon-nitrogen ratio (low C / N), and high dissolved oxygen. 3 / d circulating water volume, 4.5 kg of glucose needs to be added every day to make 1m 3 The C / N ratio of the water body increases from 6:1 to 24:1, which is convenient for the subsequent rapid and deep denitrification of MBBR. This will undoubtedly increase the operating cost of the MBBR reactor, that is, increase the operating cost of the recirculating aquaculture plant, and the denitrification effect is limited. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a composite bacterial agent embedded pellet and a preparation method and application thereof. The present invention can improve the denitrification effect of the reactor, reduce the amount of additional carbon source added, and significantly reduce the operating cost of the recirculating aquaculture plant.

[0006] The technical solution of the present invention is achieved in this way: A method for preparing composite bacterial agent-embedded beads, specifically comprising the following steps: (1) Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 are inoculated into a culture medium respectively, cultured at 25-35°C until the initial stable stage, then centrifuged at 4-6°C, the supernatant is removed, the culture is washed and resuspended three times with a buffer solution, and then Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 are mixed to obtain a composite bacterial suspension; (2) adding polyvinyl alcohol and sodium alginate into water, heating and mixing them evenly, and then cooling them to room temperature to obtain a gel; (3) Add the gelling agent to the composite bacterial suspension and mix evenly to obtain a mixed gel bacterial solution, then draw up the mixed gel bacterial solution and drop it into the cross-linking agent, then cross-link at 4-6°C for 24-48h, wash with sterile water three times and dry to obtain the composite bacterial agent embedded beads.

[0007] Furthermore, the volume ratio of Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 in the composite bacterial suspension is 1:2~1:5.

[0008] Furthermore, the concentration of polyvinyl alcohol in the gel is 10-15%, and the concentration of sodium alginate is 2-4%.

[0009] Furthermore, the volume fraction of the composite bacterial suspension in the mixed gel bacterial liquid is 10-15%.

[0010] Furthermore, the buffer solution is a phosphate buffer solution.

[0011] Furthermore, the cross-linking agent is a mixture of saturated boric acid and 2-4% by mass of calcium chloride, and the pH is adjusted to 6-7 using sodium carbonate or sodium bicarbonate.

[0012] The present invention also provides a composite bacterial agent embedded small ball, which is prepared by using the above-mentioned preparation method of the composite bacterial agent embedded small ball.

[0013] The present invention also provides the use of the above-mentioned composite bacterial agent-embedded beads in treating circulating aquaculture water.

[0014] Furthermore, the moving bed biofilm reactor is filled with biological fillers, the filling rate of the biological fillers is 15-50%, and the volume of the composite bacterial agent embedded beads in the biological fillers accounts for 1 / 10-1 / 3.

[0015] Furthermore, the dissolved oxygen content in the moving bed biofilm reactor is 4-8 mg / L.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention embeds Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 in small balls to prepare composite bacterial agent embedded small balls, which can effectively increase the biomass, and is also beneficial to improve the biological stability and survival rate. In addition, both Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 have efficient denitrification capabilities, so that the composite bacterial agent embedded small balls have a good denitrification effect.

[0017] In addition, the present invention adopts Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 mixed in a volume ratio of 1:2 to 1:5 as a composite bacterial agent to prepare embedded beads. The two strains of the composite bacterial agent are colonized on the carrier, obtain partial ecological niche competitive advantages, and the richness and diversity of the microbial community are increased. The denitrification effect of the embedded beads can be further improved by coupling the enhanced denitrification enzyme activity of the two.

[0018] 2. Adding the composite bacterial agent-encapsulated pellets and other biological fillers prepared in the present invention to the MBBR reactor to treat low C / N circulating aquaculture wastewater is an economical and efficient way. It not only reduces the addition of carbon sources, but also reduces energy consumption from the production process of carbon sources such as glucose, that is, from the source. It is green, environmentally friendly and sustainable, responding to FAO's "Blue Transformation" and "2030 Agenda for Sustainable Development" to achieve habitat reconstruction to revitalize the ecosystem. Therefore, a small amount of additional carbon source can be added to efficiently remove nitrogen and significantly reduce the operating costs of circulating aquaculture plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 -Macroscopic photograph of the composite bacterial agent-embedded spheres obtained in Example 1.

[0020] Figure 2 - Cross-sectional SEM image of the composite bacterial agent-embedded pellets obtained in Example 1.

[0021] Figure 3 -The removal effect of the embedded beads obtained in Examples 1-2 and Comparative Examples 1-5 on nitrogen pollutants. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0023] Example 1 A method for preparing composite bacterial agent-embedded beads, specifically comprising the following steps: (1) Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 were inoculated into LB medium respectively, cultured at 30°C until the initial stable stage, then centrifuged at 4°C, removed the supernatant, washed and resuspended three times with buffer, and then mixed Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 to obtain a composite bacterial suspension, wherein the volume ratio of Pseudomonas stutzeri CICC-10428 to Pseudomonas stutzeri XL-2 in the composite bacterial suspension was 1:5; (2) adding polyvinyl alcohol and sodium alginate into water and mixing them, then heating them in a water bath to 90°C, cooling them to room temperature after they are completely dissolved, and obtaining a gel, wherein the concentrations of polyvinyl alcohol and sodium alginate in the gel are 10% and 2%, respectively; (3) Add the gelling agent to the composite bacterial suspension and mix evenly to obtain a mixed gel bacterial solution, wherein the volume fraction of the composite bacterial suspension in the mixed gel bacterial solution is 10%; then, the mixed gel bacterial solution is added dropwise to a saturated boric acid solution and a cross-linking agent composed of 2% by mass of calcium chloride (before adding, the pH of the cross-linking agent is adjusted to 6.7 with sodium carbonate), and then cross-linked at 4°C for 48 hours. After washing three times with sterile water and drying, the composite bacterial agent-embedded beads are obtained.

[0024] Example 2 This example is the same as Example 1, except that the volume ratio of Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 in the composite bacterial suspension in this example is 1:2.

[0025] Comparative Example 1 This example is the same as Example 1, except that only Pseudomonas stutzeri CICC-10428 is used in this example.

[0026] Comparative Example 2 This example is the same as Example 1, except that the volume ratio of Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 in the composite bacterial suspension in this example is 5:1.

[0027] Comparative Example 3 This example is the same as Example 1, except that the volume ratio of Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 in the composite bacterial suspension of this example is 2:1.

[0028] Comparative Example 4 This example is the same as Example 1, except that the volume ratio of Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 in the composite bacterial suspension in this example is 1:1.

[0029] Comparative Example 5 This example is the same as Example 1, except that only Pseudomonas stutzeri XL-2 is used in this example.

[0030] 1. The macroscopic photograph of the composite bacterial agent-embedded sphere obtained in Example 1 and the SEM image of the cross section magnified 1000 times are shown as follows: Figure 1 and Figure 2 As shown by Figure 1 It can be seen that the composite bacterial agent embedded balls are milky white and spherical, and the balls are uniform in size. Figure 2 It can be seen that multiple clusters of bacteria are attached to the internal pores of the composite bacterial agent-embedded spheres, and the spheres have a porous and rugged structure, which is suitable for the growth of microorganisms and can effectively prevent the loss of microorganisms.

[0031] 2. Take seven reactors of the same size, and set an aeration ring at the bottom of the reactor. During operation, control the dissolved oxygen in the reactor to 5-6 mg / L. Use the embedded beads obtained in Examples 1-2 and Comparative Examples 1-5 to treat low C / N circulating aquaculture water. The content of each component in the circulating aquaculture water is: glucose concentration is 420 mg / L, NH4 + -N、NO3 - -N concentrations were 20 mg / L and 50 mg / L, respectively, and C / N was controlled at 6:1; a peristaltic pump was used to continuously feed water, and the HRT was 11.5h. The filling rate of the biofiller in the reactor was 33%, and the embedded beads and other biofillers were added to the reactor at a ratio of 1:2.

[0032] After 45 days of operation, the comparative example 1 had a negative effect on NH4 + -N、NO3 - -N removal rates were 83.9% and 92.6%, and NO2 - -N effluent concentration is 2.07 mg / L; Comparative Example 2 for NH4 + -N、NO3 - -N removal rates were 82.3% and 92.4%, and NO2 - -N effluent concentration is 2.16 mg / L; Comparative Example 3 for NH4 + -N、NO3 - -N removal rates were 85.4% and 94.8%, and NO2 - -N effluent concentration is 2.08 mg / L; Comparative Example 4 for NH4 + -N、NO3 - -N removal rates were 89.1% and 93.7%, and NO2 - -N effluent concentration is 2.01 mg / L; Example 2 for NH4 + -N、NO3 - -N removal rates were 88.2% and 96.5%, and NO2 --N effluent concentration is 1.77 mg / L; Example 1 for NH4 + -N, NO3 - -N removal rate is 91.8%, 95%, NO2 - -N effluent concentration is 1.71 mg / L; Comparative Example 7 for NH4 + -N, NO3 - -N removal rates were 86.1% and 93.5%, and NO2 - The -N effluent concentration is 2.32 mg / L.

[0033] It can be seen that the treatment effect of the two bacterial agents on nitrogen pollutants is better than that of a single bacterial agent, indicating that there is a synergistic effect between CICC-10428 and XL-2, but the different matching ratios of the two strains have a significant effect on the degradation efficiency of different nitrogen pollutants, and the effect on NH4 + -N, NO2 - The removal effect of -N showed an overall increasing trend with the increase of Pseudomonas stutzeri XL-2, and the removal of NO3 - -N removal effect did not show a clear trend. Among them, in the current experimental environment, NH4 + The best removal effect of -N is in Example 1, which has a good effect on NO3 - The best removal effect of -N is in Example 2, which has a good effect on NO2 - The best N removal effect is in Example 1. In summary, when the added volume ratio of Pseudomonas stutzeri CICC-10428 to Pseudomonas stutzeri XL-2 is 1:5, the composite bacterial agent embedded beads have the best comprehensive removal rate of nitrogen pollutants.

[0034] 3. Take four reactors of the same size. The bottom of the reactor is equipped with an aeration ring. During operation, the dissolved oxygen in the reactor is controlled to be 5-6 mg / L. The reactor inlet water is simulated aquaculture circulating water. The content of each component in the aquaculture circulating water is: glucose concentration is 420 mg / L, NH4 + -N, NO3 --N concentrations were 20 mg / L and 50 mg / L, respectively, and C / N was controlled at 6:1; a peristaltic pump was used for continuous water inflow, and the HRT was 11.5h. Among them, R1 was the control group, and biological fillers were added, with a filling rate of 33%; R2 was the experimental group, and a biological composite bacterial solution composed of Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 was added for five consecutive days on the basis of R1 (the bacterial solution with the same bacterial biomass as that added to R3 obtained by calculation); R3 was the experimental group, and on the basis of R1, one-third of the volume of the biological filler was replaced with an equal volume of composite bacterial agent embedded beads in Example 1; R4 was the experimental group, and on the basis of R1, one-third of the volume of the biological filler was replaced with a polymethyl ethylene carbonate (PPC) carrier with an equal volume of composite bacterial agent biofilm.

[0035] The pores of polymethylethylene carbonate are evenly distributed, with a volume of 1.5±0.1 cm cube in the dry state and a volume of 2±0.1 cm cube after absorbing water. The average diameter of the composite bacterial agent embedded spheres is 4 mm, and there is no water absorption expansion.

[0036] The entire operation cycle lasted for 90 days. The experimental groups all showed fluctuations within a few days after the addition of the composite microbial agent, but as the experiment progressed, the NH4 + -N, NO2 - -N、NO3 - -N removal showed an increasing trend. From the short-term effect (within 30 days), the three experimental groups showed enhanced denitrification effects compared with the control group, but R2 and R4 had their own advantages and disadvantages. + R4 has the best effect in enhancing the removal of NO2, which increases the removal rate by 5.8%. However, due to the long film formation period of the initial filler, the time cost is greatly increased. - R2 has the best effect on the removal of -N, reducing the effluent concentration by 0.08 mg / L; - R2 has the best effect on the removal of -N, increasing the removal rate by 5.3%, but the rebound phenomenon is the most serious; the experimental cycle and treatment effect of R3 are close to those of R2; from the perspective of long-term operation, R3 has a short preparation cycle, the longest experimental cycle stability, and the most stable denitrification treatment effect, especially in the removal of NO3 - -N treatment is better than R2, so from the perspective of enhanced denitrification effect and long-term stability, the composite bacterial agent embedded beads have obvious advantages.

[0037] Finally, it should be noted that the above embodiments of the present invention are only examples for illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For those skilled in the art, other different forms of changes and modifications can be made based on the above description. It is impossible to list all implementation methods here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for preparing composite bacterial agent embedded beads, characterized in that: The specific steps include: (1) Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 are inoculated into a culture medium respectively, cultured at 25-35°C until the initial stable stage, then centrifuged at 4-6°C, the supernatant is removed, the culture is washed and resuspended three times with a buffer solution, and then Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 are mixed to obtain a composite bacterial suspension; (2) adding polyvinyl alcohol and sodium alginate into water, heating and mixing them evenly, and then cooling them to room temperature to obtain a gel; (3) Add the gelling agent to the composite bacterial suspension and mix evenly to obtain a mixed gel bacterial solution, then draw up the mixed gel bacterial solution and drop it into the cross-linking agent, then cross-link at 4-6°C for 24-48h, wash with sterile water three times and dry to obtain the composite bacterial agent embedded beads.

2. The method for preparing composite bacterial agent-embedded beads according to claim 1, characterized in that: The volume ratio of Pseudomonas stutzeri CICC-10428 and Pseudomonas stutzeri XL-2 in the composite bacterial suspension is 1:2~1:

5.

3. The method for preparing composite bacterial agent-embedded beads according to claim 1, characterized in that: The concentration of polyvinyl alcohol in the gel is 10-15%, and the concentration of sodium alginate is 2-4%.

4. The method for preparing composite bacterial agent-embedded beads according to claim 1, 2 or 3, characterized in that: The volume fraction of the composite bacterial suspension in the mixed gel bacterial solution is 10-15%.

5. The method for preparing composite bacterial agent embedded beads according to claim 1, characterized in that: The buffer solution is a phosphate buffer solution.

6. The method for preparing composite bacterial agent-embedded beads according to claim 1, characterized in that: The cross-linking agent is a mixture of saturated boric acid and 2-4% by mass of calcium chloride, and the pH is adjusted to 6-7 using sodium carbonate or sodium bicarbonate.

7. A composite bacterial agent embedded pellet, characterized in that: The composite bacterial agent embedded pellets are prepared by the method for preparing the composite bacterial agent embedded pellets according to any one of claims 1 to 6.

8. Use of the composite bacterial agent-encapsulated pellets as described in claim 7 in treating circulating aquaculture water.

9. The use of a composite bacterial agent-embedded pellet according to claim 8, characterized in that: The moving bed biofilm reactor is filled with biological fillers, the filling rate of the biological fillers is 15-50%, and the volume of the composite bacterial agent embedded beads in the biological fillers accounts for 1 / 10-1 / 3.

10. The use of a composite bacterial agent-embedded pellet according to claim 8, characterized in that: The dissolved oxygen content in the moving bed biofilm reactor is 4~8 mg / L.

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