Preparation method and application of oyster shell immobilized probiotic biofiller

Abstract

The present invention relates to a preparation method of an oyster shell immobilized probiotic biological filler, comprising the following steps: S1, optimizing and treating oyster shells to prepare modified oyster shell powder; S2, testing the ability of the modified oyster shell powder prepared in step S1 to immobilize microorganisms and preparing filler raw materials; S3, completing the preparation of the biological filler by modifying the oyster shell powder. The advantages of the present invention are: compared with the PE filler used in production, after the oyster shell immobilized probiotic biological filler treated at 400°C and 600°C is run in the biofilter for 18 days, the community diversity of microorganisms on its surface increases, and the relative abundance of the immobilized microorganisms in the biofilm increases. It shows that the oysters treated at 400°C and 600°C are suitable for microbial immobilization materials, and the oyster shell immobilized probiotic biological filler can increase the relative abundance of the immobilized microorganisms at the genus level, has an efficient purification effect on the aquaculture water body, and can be used to improve the aquaculture water environment.

Description

Technical Field

[0001] The invention relates to a method for preparing an oyster shell immobilized probiotic biological filler, and belongs to the field of environmental engineering. Background Art

[0002] The nitrogen and phosphorus emissions from aquaculture account for a considerable proportion of the land-based emissions. Studies have shown that the nitrogen emissions from seawater pond aquaculture in my country reach 450,000 tons, while the total amount of ammonia nitrogen discharged from urban domestic sewage in my country is more than 900,000 tons per year (Dong Shuanglin, High Efficiency and Low Carbon - The Only Way for the Development of China's Aquaculture Industry. Journal of Fisheries of China, 2011, (10): 1595-1600). The deterioration of aquaculture water bodies has caused serious losses to the aquaculture industry. The microbial method in the bioremediation method has become the most studied bioremediation technology due to its low cost, low pollution, good remediation effect and wide application range. However, free microorganisms are easily affected by water environmental factors, the effect is unstable, and even more toxic products are produced. Embedded immobilization technology is an emerging microbial immobilization technology in the field of modern bioengineering. Studies have shown that using embedded immobilization technology to immobilize ammonia oxidizing sludge can effectively prevent the loss of anaerobic ammonia oxidizing bacteria and maintain the biomass in the bioreactor (Zhang, D., Y. Zhang, F. Shen, et al., Removal of cadmium and lead from heavy metals loaded PVA–SA immobilized Lentinus edodes. Desalination and Water Treatment, 2014. 52 (25-27): 4792-4801). How to apply this technology to the restoration of aquaculture water is of great significance.

[0003] Oysters are widely cultivated in coastal areas of China, but a large number of oyster shells formed after eating oysters are randomly piled up, and a large amount of organic matter will produce a large amount of unpleasant gases during the stacking process, which has become a serious environmental problem. In 2020, the output of marine aquaculture oysters in my country reached 5.4246 million tons (Ministry of Agriculture and Rural Affairs, Fisheries Administration, 2021 Fisheries Statistical Yearbook. 2021: China Agricultural Press). Oyster shells account for 70% of the mass of oysters. Every year, the oyster shells in coastal areas reach millions of tons, most of which are directly discarded as waste, causing serious environmental pollution and waste of resources. Oyster shells are a kind of porous material that can be modified to increase the specific surface area several times. The main component of natural material oyster shells is CaCO3, which contains a variety of trace elements and a large number of micropores of 2 to 10 μm. After modification, it can become a porous material with a specific surface area increased several times, giving it a strong adsorption capacity.

[0004] Therefore, the discarded oyster shells are modified and used to fix denitrifying probiotics to prepare biological fillers for aquaculture water restoration, which can not only realize the resource reuse of oyster shells, but also improve the water treatment performance of the biofilter. Summary of the invention

[0005] In order to overcome the defects of the prior art, the present invention provides a method for preparing an oyster shell-immobilized probiotic biofiller. The technical solution of the present invention is:

[0006] A method for preparing an oyster shell-immobilized probiotic biofiller comprises the following steps:

[0007] S1, optimizing the treatment of oyster shells to prepare modified oyster shell powder;

[0008] S2, testing the ability of the modified oyster shell powder prepared in step S1 to immobilize microorganisms and preparing filler raw materials;

[0009] S3. The preparation of the biofiller is completed by modifying the oyster shell powder.

[0010] The step S1 is specifically as follows:

[0011] 1-1. Use clean water to clean the mud and sand attached to the surface of the oyster shell and the oyster meat residue, then place it in an oven for drying at 105°C for 2 hours, and pass it through a 50-100 mesh grinder to form oyster shell powder, which is stored in a dryer for later use;

[0012] 1-2. The oyster shell powder was placed in a muffle furnace and calcined at 400°C for 2 hours to prepare modified oyster shell powder, which was marked as OS. 400 Another oyster shell powder was placed in a muffle furnace and calcined at 600 ° C for 2 h to prepare a modified oyster shell powder marked as OS 600 Modified oyster shell powder; uncalcined oyster shell powder is marked as OS.

[0013] The step S2 is specifically as follows:

[0014] 2-1. Cultivate the Chengdu Pseudomonas BF6 culture solution at 30°C, 200 rpm / min for 16 h, centrifuge at 5000 rpm for 10 min, rinse 2-3 times with sterile saline, resuspend with sterile saline and prepare 100-120 mL OD 600 0.64 suspension; take 1g sterilized OS 400 Oyster shell powder and 1g sterilized OS 600 Oyster shell powder was placed in 15 ml sterilized centrifuge tubes, and then 10 ml of bacterial suspension was added to each tube. A 10 ml bacterial suspension group without oyster shell powder was set up as the control group C bacterial suspension. 400The mixture of oyster shell powder and bacterial suspension formed the mixed solution of experimental group A, OS 600 The mixture of oyster shell powder and bacterial suspension formed the mixed solution of experimental group B, and each group was set up with 3 replicates;

[0015] 2-2. Take 1 mL of the mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C at 1 h, 6 h, 24 h and 72 h respectively, and then centrifuge at 300 rpm for 5 min. Use a full-wavelength microplate reader to measure the concentration of the supernatant at OD600, make an adsorption kinetic curve, and calculate the adsorption capacity of different oyster shell powders on probiotics. The fixed number calculation method for Chengdu Pseudomonas BF6 bacterial solution is as follows:

[0016] OD = CT, where C is the OD value of the blank control group and T is the OD value of the experimental group;

[0017] 2-3. After 72 hours, the mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C were centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded; the obtained mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C were rinsed with sterile phosphate buffer, PBS was discarded, 2.5% glutaraldehyde was added, and they were fixed at 4°C for 2 hours to form group A samples, group B samples and group C samples; the fixed samples of group A, group B and group C were rinsed with 0.1M phosphate buffer of pH 7.4 for 3 times, each time for 15 minutes; then they were dehydrated with 30%, 50%, 70%, 85%, 90%, and 95% ethanol once each, and dehydrated with 100% ethanol for 15 minutes each time to form group A sample raw materials, group B sample raw materials and group C sample raw materials, respectively, and their microscopic morphology was observed and analyzed using a scanning electron microscope.

[0018] The step S3 is specifically as follows:

[0019] 3-1. Use an electronic multipurpose electric furnace to melt polybutylene succinate, and mix the polybutylene succinate with the sample raw materials of group A, group B and group C in a ratio of 1:1 to make OS biofiller, OS biofiller and other flakes. 400 Biofillers and OS 600 Biological filler, sterilized and ready for use;

[0020] 3-2. Pseudomonas chengduensis BF6 and Pseudomonas putida DS2 cultured at 30°C, 200 rpm / min for 16 h were centrifuged at 5000 rpm for 10 min, rinsed 2-3 times with sterile saline, and resuspended with sterile saline to prepare 3000-3500 mL OD 600 The BF6 bacterial solution and OD of 0.5 600 DS2 bacterial solution of 0.5;

[0021] 3-3. Take 500g sterilized OS biofiller and add 300ml OD 600 0.5 BF6 bacterial solution and 300mL OD 600 DS2 bacterial solution of 0.5; in 500g OS 400 Add 300ml OD 600 0.5 BF6 bacterial solution and 300mL OD 600 DS2 bacterial solution of 0.5; in OS 600 Add 300mlOD to the biofiller 600 0.5 BF6 bacterial solution and 300mL OD 600 The DS2 bacterial solution was 0.5 and incubated in a shaker at 30°C and 150 rpm for 24 hours to prepare OS-P biofiller, OS 400 -P Biofiller and OS 600 -P biological filler.

[0022] An oyster shell-immobilized probiotic biological filler is prepared by a preparation method of an oyster shell-immobilized probiotic biological filler.

[0023] Application of an oyster shell-immobilized probiotic biofiller in aquaculture water treatment. The invention has the advantages that, compared with the PE filler used in production, after the oyster shell-immobilized probiotic biofiller has been treated at high temperatures of 400°C and 600°C and has been running in a biofilter for 18 days, the community diversity of microorganisms on its surface increases, and the relative abundance of the immobilized microorganisms in the biofilm increases. This indicates that oysters treated at high temperatures of 400°C and 600°C are suitable for use as microbial immobilization materials, and the oyster shell-immobilized probiotic biofiller can increase the relative abundance of the immobilized microorganisms at the genus level, has an efficient purification effect on aquaculture water, and can be used to improve the aquaculture water environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The microstructure of the oyster shell before and after the heat treatment of the present invention;

[0025] Figure 2 The immobilization capacity of oyster shells treated with different methods according to the present invention on microorganisms;

[0026] Figure 3 The microstructure of microorganisms immobilized in oyster shells treated differently according to the present invention;

[0027] Figure 4 The NH4-N treatment effects of different biological fillers in the biofilter of the present invention;

[0028] Figure 5 The NO2-N treatment effects of different biological fillers in the biofilter of the present invention;

[0029] Figure 6 The NO3-N treatment effects of different biological fillers in the biofilter of the present invention;

[0030] Figure 7 The TN treatment effects of different biological fillers in the biofilter of the present invention;

[0031] Figure 8 The diversity of microbial communities of the surface biofilm of different biological fillers in the biofilter after running for 18 days according to the present invention;

[0032] Fig. 9 The relative abundance of microorganisms in the surface biofilm of different biological fillers in the biofilter after running for 18 days according to the present invention. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0034] Embodiment 1: The invention relates to a method for preparing an oyster shell-immobilized probiotic biofiller, comprising the following steps:

[0035] S1, optimizing the treatment of oyster shells to prepare modified oyster shell powder;

[0036] S2, testing the ability of the modified oyster shell powder prepared in step S1 to immobilize microorganisms and preparing filler raw materials;

[0037] S3. The preparation of the biofiller is completed by modifying the oyster shell powder.

[0038] The step S1 is specifically as follows:

[0039] 1-1. Use clean water to clean the mud and sand attached to the surface of the oyster shell and the oyster meat residue, then place it in an oven for drying at 105°C for 2 hours, pass it through a 50-mesh grinder to form oyster shell powder, and store it in a dryer for later use;

[0040] 1-2. The oyster shell powder was placed in a muffle furnace and calcined at 400°C for 2 hours to prepare modified oyster shell powder, which was marked as OS. 400 Another oyster shell powder was placed in a muffle furnace and calcined at 600 ° C for 2 h to prepare a modified oyster shell powder marked as OS 600 Modified oyster shell powder; uncalcined oyster shell powder is marked as OS.

[0041] The step S2 is specifically as follows:

[0042] 2-1. The Chengdu Pseudomonas BF6 bacterial suspension cultured at 30°C and 200 rpm / min for 16 h was centrifuged at 5000 rpm for 10 min, rinsed twice with sterile saline, and resuspended with sterile saline to prepare 100 mL OD 600 0.64 suspension; take 1g sterilized OS 400 Oyster shell powder and 1g sterilized OS 600 Oyster shell powder was placed in 15 ml sterilized centrifuge tubes, and then 10 ml of bacterial suspension was added to each tube. A 10 ml bacterial suspension group without oyster shell powder was set up as the control group C bacterial suspension. 400 The mixture of oyster shell powder and bacterial suspension formed the mixed solution of experimental group A, OS 600 The mixture of oyster shell powder and bacterial suspension formed the mixed solution of experimental group B, and each group was set up with 3 replicates;

[0043] 2-2. Take 1 mL of the mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C at 1 h, 6 h, 24 h and 72 h respectively, and then centrifuge at 300 rpm for 5 min. Use a full-wavelength microplate reader to measure the concentration of the supernatant at OD600, make an adsorption kinetic curve, and calculate the adsorption capacity of different oyster shell powders on probiotics. The fixed number calculation method for Chengdu Pseudomonas BF6 bacterial solution is as follows:

[0044] OD = CT, where C is the OD value of the blank control group and T is the OD value of the experimental group;

[0045] 2-3. After 72 hours, the mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C were centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded; the obtained mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C were rinsed with sterile phosphate buffer, PBS was discarded, 2.5% glutaraldehyde was added, and they were fixed at 4°C for 2 hours to form group A samples, group B samples and group C samples; the fixed samples of group A, group B and group C were rinsed with 0.1M phosphate buffer of pH 7.4 for 3 times, each time for 15 minutes; then they were dehydrated with 30%, 50%, 70%, 85%, 90%, and 95% ethanol once each, and dehydrated with 100% ethanol for 15 minutes each time to form group A sample raw materials, group B sample raw materials and group C sample raw materials, respectively, and their microscopic morphology was observed and analyzed using a scanning electron microscope.

[0046] The step S3 is specifically as follows:

[0047] 3-1. Use an electronic multipurpose electric furnace to melt polybutylene succinate, and mix the polybutylene succinate with the sample raw materials of group A, group B and group C in a ratio of 1:1 to make OS biofiller, OS biofiller and other flakes. 400 Biofillers and OS 600 Biological filler, sterilized and ready for use;

[0048] 3-2. Pseudomonas chengduensis BF6 and Pseudomonas putida DS2 cultured at 30°C, 200 rpm / min for 16 h were centrifuged at 5000 rpm for 10 min, rinsed 2-3 times with sterile saline, and resuspended with sterile saline to prepare 3000 mL OD 600 The BF6 bacterial solution and OD of 0.5 600 DS2 bacterial solution of 0.5;

[0049] 3-3. Take 500g sterilized OS biofiller and add 300ml OD 600 0.5 BF6 bacterial solution and 300mL OD 600 DS2 bacterial solution of 0.5; in 500g OS 400 Add 300ml OD 600 0.5 BF6 bacterial solution and 300mL OD 600 DS2 bacterial solution of 0.5; in OS 600 Add 300mlOD to the biofiller 600 0.5 BF6 bacterial solution and 300mL OD 600 The DS2 bacterial solution was 0.5 and incubated in a shaker at 30°C and 150 rpm for 24 hours to prepare OS-P biofiller, OS 400 -P Biofiller and OS 600 -P biological filler.

[0050] The invention also relates to an oyster shell-immobilized probiotic biological filler, which is prepared by a preparation method of the oyster shell-immobilized probiotic biological filler.

[0051] The invention also relates to the application of an oyster shell-immobilized probiotic biological filler in aquaculture water treatment.

[0052] Embodiment 2: A method for preparing an oyster shell immobilized probiotic biofiller, comprising the following steps:

[0053] S1, optimizing the treatment of oyster shells to prepare modified oyster shell powder;

[0054] S2, testing the ability of the modified oyster shell powder prepared in step S1 to immobilize microorganisms and preparing filler raw materials;

[0055] S3. The preparation of the biofiller is completed by modifying the oyster shell powder.

[0056] The step S1 is specifically as follows:

[0057] 1-1. Use clean water to clean the mud and sand attached to the surface of the oyster shell and the oyster meat residue, then place it in an oven for drying at 105°C for 2 hours, and pass it through a 80-mesh grinder to form oyster shell powder, which is stored in a dryer for later use;

[0058] 1-2. The oyster shell powder was placed in a muffle furnace and calcined at 400°C for 2 hours to prepare modified oyster shell powder, which was marked as OS. 400 Another oyster shell powder was placed in a muffle furnace and calcined at 600 ° C for 2 h to prepare a modified oyster shell powder marked as OS 600 Modified oyster shell powder; uncalcined oyster shell powder is marked as OS.

[0059] The step S2 is specifically as follows:

[0060] 2-1. The Chengdu Pseudomonas BF6 bacterial suspension cultured at 30°C and 200 rpm / min for 16 h was centrifuged at 5000 rpm for 10 min, rinsed three times with sterile saline, and resuspended with sterile saline to prepare 110 mL OD 600 0.64 suspension; take 1g sterilized OS 400 Oyster shell powder and 1g sterilized OS 600 Oyster shell powder was placed in 15 ml sterilized centrifuge tubes, and then 10 ml of bacterial suspension was added to each tube. A 10 ml bacterial suspension group without oyster shell powder was set up as the control group C bacterial suspension. 400 The mixture of oyster shell powder and bacterial suspension formed the mixed solution of experimental group A, OS 600 The mixture of oyster shell powder and bacterial suspension formed the mixed solution of experimental group B, and each group was set up with 3 replicates;

[0061] 2-2. Take 1 mL of the mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C at 1 h, 6 h, 24 h and 72 h respectively, and then centrifuge at 300 rpm for 5 min. Use a full-wavelength microplate reader to measure the concentration of the supernatant at OD600, make an adsorption kinetic curve, and calculate the adsorption capacity of different oyster shell powders on probiotics. The fixed number calculation method for Chengdu Pseudomonas BF6 bacterial solution is as follows:

[0062] OD = CT, where C is the OD value of the blank control group and T is the OD value of the experimental group;

[0063] 2-3. After 72 hours, the mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C were centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded; the obtained mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C were rinsed with sterile phosphate buffer, PBS was discarded, 2.5% glutaraldehyde was added, and they were fixed at 4°C for 2 hours to form group A samples, group B samples and group C samples; the fixed samples of group A, group B and group C were rinsed with 0.1M phosphate buffer of pH 7.4 for 3 times, each time for 15 minutes; then they were dehydrated with 30%, 50%, 70%, 85%, 90%, and 95% ethanol once each, and dehydrated with 100% ethanol for 15 minutes each time to form group A sample raw materials, group B sample raw materials and group C sample raw materials, respectively, and their microscopic morphology was observed and analyzed using a scanning electron microscope.

[0064] The step S3 is specifically as follows:

[0065] 3-1. Use an electronic multipurpose electric furnace to melt polybutylene succinate, and mix the polybutylene succinate with the sample raw materials of group A, group B and group C in a ratio of 1:1 to make OS biofiller, OS biofiller and other flakes. 400 Biofillers and OS 600 Biological filler, sterilized and ready for use;

[0066] 3-2. Pseudomonas chengduensis BF6 and Pseudomonas putida DS2 cultured at 30°C, 200 rpm / min for 16 h were centrifuged at 5000 rpm for 10 min, rinsed 2-3 times with sterile saline, and resuspended with sterile saline to prepare 3200 mL OD 600 The BF6 bacterial solution and OD of 0.5 600 DS2 bacterial solution of 0.5;

[0067] 3-3. Take 500g sterilized OS biofiller and add 300ml OD 600 0.5 BF6 bacterial solution and 300mL OD 600 DS2 bacterial solution of 0.5; in 500g OS 400 Add 300ml OD 600 0.5 BF6 bacterial solution and 300mL OD 600 DS2 bacterial solution of 0.5; in 500gOS 600 Add 300mlOD to the biofiller 600 0.5 BF6 bacterial solution and 300mL OD 600 The DS2 bacterial solution was 0.5 and incubated in a shaker at 30°C and 150 rpm for 24 hours to prepare OS-P biofiller, OS 400 -P Biofiller and OS600 -P Biofiller.

[0068] Embodiment 3: A method for preparing an oyster shell immobilized probiotic biofiller, comprising the following steps:

[0069] S1, optimizing the treatment of oyster shells to prepare modified oyster shell powder;

[0070] S2, testing the ability of the modified oyster shell powder prepared in step S1 to immobilize microorganisms and preparing filler raw materials;

[0071] S3. The preparation of the biofiller is completed by modifying the oyster shell powder.

[0072] The step S1 is specifically as follows:

[0073] 1-1. Use clean water to clean the mud and sand attached to the surface of the oyster shell and the oyster meat residue, then place it in an oven for drying at 105°C for 2 hours, and pass it through a 100-mesh grinder to form oyster shell powder, which is stored in a dryer for later use;

[0074] 1-2. The oyster shell powder was placed in a muffle furnace and calcined at 400°C for 2 hours to prepare modified oyster shell powder, which was marked as OS. 400 Another oyster shell powder was placed in a muffle furnace and calcined at 600 ° C for 2 h to prepare a modified oyster shell powder marked as OS 600 Modified oyster shell powder; uncalcined oyster shell powder is marked as OS.

[0075] The step S2 is specifically as follows:

[0076] 2-1. The Chengdu Pseudomonas BF6 bacterial suspension cultured at 30°C and 200 rpm / min for 16 h was centrifuged at 5000 rpm for 10 min, rinsed twice with sterile saline, and resuspended with sterile saline to prepare 120 mL OD 600 0.64 suspension; take 1g sterilized OS 400 Oyster shell powder and 1g sterilized OS 600 Oyster shell powder was placed in 15 ml sterilized centrifuge tubes, and then 10 ml of bacterial suspension was added to each tube. A 10 ml bacterial suspension group without oyster shell powder was set up as the control group C bacterial suspension. 400 The mixture of oyster shell powder and bacterial suspension formed the mixed solution of experimental group A, OS 600 The mixture of oyster shell powder and bacterial suspension formed the mixed solution of experimental group B, and each group was set up with 3 replicates;

[0077] 2-2. Take 1 mL of the mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C at 1 h, 6 h, 24 h and 72 h respectively, and then centrifuge at 300 rpm for 5 min. Use a full-wavelength microplate reader to measure the concentration of the supernatant at OD600, make an adsorption kinetic curve, and calculate the adsorption capacity of different oyster shell powders on probiotics. The fixed number calculation method for Chengdu Pseudomonas BF6 bacterial solution is as follows:

[0078] OD = CT, where C is the OD value of the blank control group and T is the OD value of the experimental group;

[0079] 2-3. After 72 hours, the mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C were centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded; the obtained mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C were rinsed with sterile phosphate buffer, PBS was discarded, 2.5% glutaraldehyde was added, and they were fixed at 4°C for 2 hours to form group A samples, group B samples and group C samples; the fixed samples of group A, group B and group C were rinsed with 0.1M phosphate buffer of pH 7.4 for 3 times, each time for 15 minutes; then they were dehydrated with 30%, 50%, 70%, 85%, 90%, and 95% ethanol once each, and dehydrated with 100% ethanol for 15 minutes each time to form group A sample raw materials, group B sample raw materials and group C sample raw materials, respectively, and their microscopic morphology was observed and analyzed using a scanning electron microscope.

[0080] The step S3 is specifically as follows:

[0081] 3-1. Use an electronic multipurpose electric furnace to melt polybutylene succinate, and mix the polybutylene succinate with the sample raw materials of group A, group B and group C in a ratio of 1:1 to make OS biofiller, OS biofiller and other flakes. 400 Biofillers and OS 600 Biological filler, sterilized and ready for use;

[0082] 3-2. Pseudomonas chengduensis BF6 and Pseudomonas putida DS2 cultured at 30°C, 200 rpm / min for 16 h were centrifuged at 5000 rpm for 10 min, rinsed 2-3 times with sterile saline, and resuspended with sterile saline to prepare 3500 mL OD 600 The BF6 bacterial solution and OD of 0.5 600 DS2 bacterial solution of 0.5;

[0083] 3-3. Take 500g sterilized OS biofiller and add 300ml OD 600 0.5 BF6 bacterial solution and 300mL OD 600 DS2 bacterial solution of 0.5; in 500g OS 400Add 300ml OD 600 0.5 BF6 bacterial solution and 300mL OD 600 DS2 bacterial solution of 0.5; in OS 600 Add 300mlOD to the biofiller 600 0.5 BF6 bacterial solution and 300mL OD 600 The DS2 bacterial solution was 0.5 and incubated in a shaker at 30°C and 150 rpm for 24 hours to prepare OS-P biofiller, OS 400 -P Biofiller and OS 600 -P biological filler. Combining the test data in Examples 1 to 3, the test results are shown in Table 1:

[0084] Table 1 Structural parameters of oyster shells treated with different

[0085]

[0086] It can be seen from Table 1 that with the increase of heat treatment temperature, the specific surface area of ​​oyster shell increases and the microporosity increases.

[0087] refer to Figures 1 to 9 ,from Figure 1 (A) Scanning electron microscopy shows that the microstructure of oyster shells is lamellar, with the lamellar layers arranged in parallel and relatively regular, but with fewer pores. After calcination at 400°C, the lamellar structure of the oyster shells is further reduced, replaced by irregular fragments and pores, with diversified pore sizes, significantly increased numbers, and good connectivity [ Figure 1 (B)]. The oyster shells calcined at 600℃ showed a completely different surface microstructure from the other groups. The lamellar structure observed in the oyster shells of the other groups was almost not observed in the 600℃ calcined group. The oyster shells of this group mainly showed granular crystals with smooth grain surfaces and many circular pores between the crystals. The pores were interconnected. Figure 1 (C)].

[0088] like Figure 2 As shown in the figure, after 1h and 6h of adsorption, the immobilization efficiency of OS on Chengdu Pseudomonas BF6 was the highest, reaching 0.15 and 0.19 OD respectively; after 24h of adsorption, OS 400 The immobilization efficiency of NY5 was the highest, reaching 0.20OD; after 72h of adsorption and fixation, the immobilization efficiency of OS on NY5 was the highest, reaching 0.29OD.

[0089] from Figure 3It can be seen that after 72 hours, Chengdu Pseudomonas BF6 was embedded or attached to the surface of the oyster shell. In addition, since the bacterial solution contained water, after high-temperature calcination, the structure of the oyster shell changed after it came into contact with water, especially the oyster shell treated at 400°C, whose structure changed from lamellar to petal-like and arranged in clusters; the surface of the oyster shell grains treated at 600°C became rough.

[0090] The operating effect of the oyster shell immobilized probiotic filler in the biofilter of the present invention:

[0091] 1. Experimental Methods

[0092] (1) Commercially available annular fillers PE, OS-P, and OS 400 -P, OS 600 -P biofiller was added to a 2.5L organic glass biofilter (H = 40cm, ), the filler addition amount of each biofilter was 480 mL, and 3 replicates were set for each group;

[0093] (2) Artificial wastewater (KNO2 0.3 g / L, NaNO3 0.3 g / L, (NH4)2SO4 0.3 g / L, NaCl 0.01 g / L, K2HPO4 0.2 g / L, MgSO4〃7H2O 0.05 g / L, FeSO4〃7H2O 0.01 g / L, MnSO4 0.01 g / L) was used as the influent of the biofilter. The hydraulic retention time (HRT) of each system was 6 h. The environmental conditions for the system operation were 29.77±0.21℃, pH 7.30±0.34, and DO 7.35±0.45.

[0094] (3) Collect 50 ml of water sample from each system every 3 days, and use the multi-parameter water quality meter [5B-3B (V8)] and total nitrogen meter (LH-3BN) of Beijing Lianhua Yongxing Technology Development Co., Ltd. to measure the content of ammonia nitrogen (NH4-N), nitrate nitrogen (NO3-N), nitrite nitrogen (NO2-N) and total nitrogen (TN) in the water. Among them, the NH4-N content is determined by Nessler's reagent method; the NO3-N content is determined by phenol disulfonic acid method; the NO2-N content is determined by naphthylethylenediamine hydrochloride spectrophotometry; the TN content is determined by alkaline potassium persulfate digestion ultraviolet spectrophotometry. The removal rate of each water quality index is calculated using the following formula:

[0095] Removal rate (%) = (C in -C ef ) / C in ×100%

[0096] Among them, C in is the concentration of each index at the water inlet, Cef is the concentration of each indicator at the outlet.

[0097] (4) At the end of the experiment, a piece of biological filler was taken from each biofilter and placed in a sterile centrifuge tube. The total DNA of the sample was extracted using the Environmental Sample DNA Purification Kit (ZYMO Research) and the TIANamp Soil DNA Kit (Tiangen Biochemical Technology Co., Ltd.). The library was constructed using the TruSeq DNA PCR-Free Sample Prep Kit, and sequencing was performed using the PE250 mode of the HiSeq2500 platform of Ronin Biotechnology.

[0098] like Figure 4 As shown, Figure 4 The NH4-N treatment effect of commercially available PE ring fillers and oyster shell immobilized probiotic biofillers treated at different temperatures in the biofilter for 18 days. As can be seen from the figure, on the third day of the biofilter operation, there was no significant difference in the removal rate of NH4-N among the groups (P>0.05). 400 -P group had the highest NH4-N removal rate, reaching 97.43%. On the 6th day of the experiment, the removal rate of NH4-N by OS-P was significantly lower than that of the other three groups (P<0.05), and the removal rate of NH4-N by PE group was the highest, reaching 96.73%. 400 -P and OS 600 The removal efficiency of NH4-N in the -P group was significantly higher than that in the PE and OS-P groups (P<0.05). 400 -P group had the highest NH4-N removal rate, reaching 84.59%; on the 12th day of the experiment, OS 400 -P and OS 600 The removal rate of NH4-N in the -P group was significantly higher than that in the PE and OS-P groups (P<0.05). 400 -P group had the highest NH4-N removal rate, reaching 97.94%; on the 15th and 18th days of the experiment, OS 400 The -P group had the highest NH4-N removal rate, reaching 99.25% and 97.36% respectively.

[0099] like Figure 5 As shown, Figure 5 The NO2-N treatment effect of commercially available PE ring fillers and oyster shell immobilized probiotic biofillers treated at different temperatures in the biofilter for 18 days. As can be seen from the figure, on the 3rd and 6th days of the biofilter operation, there was no significant difference in the removal rate of NO2-N among the groups (P>0.05), but OS 600 -P group had the highest NO2-N removal rate, reaching 42.08% and 6.42% respectively. 600-P had a significantly higher removal rate of NO2-N than the other three-year groups (P<0.05), reaching 71.59%. 600 -P achieved a NO2-N removal rate of 82.85%, significantly higher than the other three groups. The NO2-N accumulation in the PE group was 72.03%. On the 18th day of the experiment, OS 600 -P had a NO2-N removal rate of 87.14%, and the NO2-N accumulation of the PE group was 71.91%.

[0100] Figure 6 The NO3-N treatment effect of commercially available PE ring fillers and oyster shell immobilized probiotic biofillers treated at different temperatures in the biofilter for 18 days. As can be seen from the figure, on the third day of the biofilter operation, there was no significant difference in the removal rate of NO3-N among the groups (P>0.05), but OS 400 -P group NO3-N removal rate reached the highest 42.71%. 600 -P had a significantly higher removal rate of NO3-N than the other three groups (P<0.05), reaching 46.06%. 400 -P and OS 600 -P had a significantly higher removal rate of NO3-N than PE and OS-P groups (P<0.05), reaching 85.73% and 86.55%, and 85.84% and 90.90%, respectively. 400 -P group had a NO3-N removal rate of 96.90% and 96.20% respectively; on the 18th day of the experiment, there was no significant difference in the NO3-N removal rate among the groups (P>0.05), but OS 400 The removal rate of NO3-N by -P group reached the highest level of 94.11%.

[0101] Figure 7 The TN treatment effect of commercially available PE ring fillers and oyster shell immobilized probiotic biofillers treated at different temperatures in the biofilter for 18 days. As can be seen from the figure, on the third day of the biofilter operation, there was no significant difference in the removal rate of TN among the groups (P>0.05), but OS 600 -P group TN removal rate reached the highest 46.37%. 600 -P had a significantly higher removal rate of TN than other groups (P<0.05), reaching 47.53%. 600 -P group had a TN removal rate of 78.55%, which was significantly higher than that of PE, OS-P and OS 400 -P group, and the PE group had the lowest TN removal rate (P<0.05), only 1.21%; on the 12th day of the experiment, OS600 -P group had a significantly higher removal rate of TN than other groups (P<0.05), reaching 73.90%. 400 -P group had a significantly higher removal rate of TN than PE (P<0.05), reaching 80.78%. 400 The -P group had the highest TN removal rate, reaching 63.60%.

[0102] The above results show that OS 400 -P and OS 600 -P biological filler has a high treatment performance for NH4-N, NO2-N, NO3-N and TN in aquaculture water.

[0103] Figure 8 Diversity of microbial communities on the surface biofilm of different biofilter media after 18 days of operation.

[0104] from Figure 8 It can be seen that the diversity of the microbial community of the biofilm on the annular PE surface was the lowest. There was no significant difference in the diversity of the microbial community of the surface biofilm between the different treatment oyster shell biofiller groups (P>0.05), but the diversity of the microbial community of the biofilm in the OS-P group was the highest, followed by the OS 600 -P Group and OS 400 -P Group.

[0105] Fig. 9 Relative abundance of microorganisms in the surface biofilm of different biological media after running in the biofilter for 18 days.

[0106] The distribution patterns of microorganisms on the surface of different biological fillers at the genus level are as follows: Fig. 9 As shown in the figure, it can be seen that the microbial richness of the surface of the annular PE filler and the oyster shell fillers with different treatments is quite different. Among them, the top five microorganisms in the PE group according to relative richness are Ralstonia, Bradyrhizobium, Fluvicola, Flavobacterium and Pseudomonas; the top five microorganisms in the OS-P group according to relative richness are Pseudomonas, Ralstonia, Pedobacter, Flavobacterium and Fluvicola; OS 400-The top five microorganisms in relative abundance in group P were Ralstonia, Flavobacterium, Pseudomonas, Arthrobacter, and Pedobacter; OS 600 The top five microorganisms in relative abundance in OS-P group were Pseudomonas, Ralstonia, Comamonas, Fluvicola and Pedobacter. Among them, Pseudomonas to which Chengdu Pseudomonas BF6 and Pseudomonas putida DS2 belonged were more abundant in OS-P, OS 400 -P, and OS 600 -P group was significantly enriched, which once again proved that OS-P, OS 400 -P, and OS 600 -P biofiller successfully immobilized the added exogenous microorganisms.

[0107] There was a correlation between the microbial community of the surface biofilm of different biofillers and water quality indicators at the genus level after running for 18 days in the biofilter. The removal rate of NO2-N was significantly positively correlated with Rhabdobacter and Paracoccus (P<0.05), extremely significantly positively correlated with Pedobacter, Taibaiela, Halomonas, Geosporobacter and Devosia (P<0.01), and extremely significantly positively correlated with Dyadobacter and Pseudomonas (P<0.001). The removal rate of NO3-N was extremely significantly positively correlated with Pseudopedobacter (P<0.01). The removal rate of TN was significantly negatively correlated with Sphingomonas (P<0.05). The NH4-N removal rate was significantly positively correlated with the genus Aquimonas (P<0.05).

[0108] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing an oyster shell immobilized probiotic biofiller, characterized in that: The following steps are involved: S1, optimizing the treatment of oyster shells to prepare modified oyster shell powder; S2, testing the ability of the modified oyster shell powder prepared in step S1 to immobilize microorganisms and preparing filler raw materials; S3, completing the preparation of the biofiller by modifying the oyster shell powder; The step S1 is: 1-1. Use clean water to clean the mud and sand attached to the surface of the oyster shell and the oyster meat residue, then place it in an oven for drying at 105°C for 2 hours, and pass it through a 50-100 mesh grinder to form oyster shell powder, which is stored in a dryer for later use; 1-2. The oyster shell powder was placed in a muffle furnace and calcined at 400°C for 2 hours to prepare modified oyster shell powder, which was marked as OS. 400 Another oyster shell powder was placed in a muffle furnace and calcined at 600 ° C for 2 h to prepare a modified oyster shell powder marked as OS 600 Modified oyster shell powder; uncalcined oyster shell powder is marked as OS; The step S2 is: 2-1. Cultivate the Chengdu Pseudomonas BF6 culture solution at 30°C and 200 rpm / min for 16 hours, centrifuge at 5000 rpm for 10 minutes, rinse 2-3 times with sterile saline, resuspend with sterile saline and prepare 100-120 mL OD 600 0.64 suspension; take 1g sterilized OS 400 Oyster shell powder and 1g sterilized OS 600 Oyster shell powder was placed in 15 ml sterile centrifuge tubes, and then 10 ml of bacterial suspension was added to each tube. A 10 ml bacterial suspension group without oyster shell powder was set up as the control group C bacterial suspension. 400 The mixture of oyster shell powder and bacterial suspension formed the mixed solution of experimental group A, OS 600 The mixture of oyster shell powder and bacterial suspension formed the mixed solution of experimental group B, and each group was set up with 3 replicates; 2-2. Take 1 mL of the mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C at 1 h, 6 h, 24 h and 72 h respectively, centrifuge at 300 rpm for 5 min, and read the mixture at OD using a full-wavelength microplate reader. 600 The concentration of the supernatant solution was determined, the adsorption kinetic curve was made, and the adsorption capacity of different oyster shell powders on probiotics was calculated. The fixed number calculation method for Chengdu Pseudomonas BF6 solution was as follows: OD = CT, where C is the OD value of the blank control group and T is the OD value of the experimental group; 2-3. After 72 hours, the mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C were centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded; the obtained mixed solution of group A, the mixed solution of group B and the bacterial suspension of group C were rinsed with sterile phosphate buffer, PBS was discarded, 2.5% glutaraldehyde was added, and they were fixed at 4°C for 2 hours to form the samples of group A, group B and group C; the fixed samples of group A, group B and group C were rinsed with 0.1M phosphate buffer of pH 7.4 for 3 times, each time for 15 minutes; then they were dehydrated with 30%, 50%, 70%, 85%, 90%, and 95% ethanol for 1 time, and dehydrated with 100% ethanol for 15 minutes each time to form the raw materials of group A, group B and group C, and their microscopic morphology was observed and analyzed using a scanning electron microscope; The step S3 is: 3-1. Use an electronic multipurpose electric furnace to melt polybutylene succinate, and mix the polybutylene succinate with the sample raw materials of group A, group B and group C in a ratio of 1:1 to make OS biofiller, OS biofiller and other flakes. 400 Biofillers and OS 600 Biological filler, sterilized and ready for use; 3-2. Pseudomonas chengduensis BF6 and Pseudomonas putida DS2 cultured at 30°C, 200 rpm / min for 16 h were centrifuged at 5000 rpm for 10 min, rinsed 2-3 times with sterile saline, and resuspended with sterile saline to prepare 3000-3500 mL OD 600 The BF6 bacterial solution and OD of 0.5 600 DS2 bacterial solution of 0.5; 3-3. Take 500g sterilized OS biofiller and add 300ml OD 600 0.5 BF6 bacterial solution and 300mL OD 600 DS2 bacterial solution of 0.5; in 500g OS 400 Add 300ml OD 600 0.5 BF6 bacterial solution and 300mL OD 600 DS2 bacterial solution of 0.5; in OS 600 Add 300mlOD to the biofiller 600 0.5 BF6 bacterial solution and 300mL OD 600 The DS2 bacterial solution was 0.5 and incubated in a shaker at 30°C and 150 rpm for 24 hours to prepare OS-P biofiller, OS 400 -P Biofiller and OS 600 -P Biofiller.

2. An oyster shell immobilized probiotic biofiller, characterized in that: The biofiller is prepared by the method for preparing the oyster shell-immobilized probiotic biofiller according to claim 1.

3. Use of the oyster shell-immobilized probiotic biofiller according to claim 2 in aquaculture water treatment.

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