Lake sediment reduction bacterium, screening method, immobilized microbial agent and application

Abstract

The invention relates to a lake sediment reduction bacterium which is classified and named as bacillus amyloliquefaciens WP02, the preservation number is CCTCC (China Center for Type Culture Collection) NO: M2020696, and the lake sediment reduction bacterium is preserved in the China Center for Type Culture Collection. The invention relates to an application of a lake sediment reduction bacterium in sediment denitrification, phosphorus uptake and quality degradation. The method has the beneficial effects that the immobilized microbial agent obtained by respectively embedding and adsorbing lake sediment reduction bacteria can effectively reduce sediment organic matters, reduce sediment total nitrogen and absorb phosphorus in overlying water, namely effectively perform denitrification, phosphorus absorption and quality degradation on the sediment, and the microbial agent can improve the oxidation reduction potential of the sediment and reduce the pollution of the sediment. And the method has important significance and application value in the aspect of maintaining the ecological balance of the water body, and has the characteristics of high in-situ remediation efficiency, small environmental disturbance and no secondary pollution compared with a physical and chemical remediation method.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to lake sediment reducing bacteria, a screening method, an immobilized bacterial agent and applications. Background Art

[0002] The long-term siltation of lake sediments causes systemic harm to the ecosystem and functions of water bodies. As a sediment carrier of pollutants, the sediments continuously release nitrogen, phosphorus, heavy metals and persistent organic matter in an anaerobic environment, forming a "time bomb" of endogenous pollution. The released pollutants will trigger a chain reaction. The increase in nitrogen and phosphorus concentrations will lead to explosive proliferation of algae. The decomposed algae residues that sink to the bottom further consume dissolved oxygen, causing the oxygen content in the bottom water to be lower than 2 mg / L, forming a seasonal hypoxic zone. At the same time, the continuous siltation of the sediments causes the lake volume to show irreversible decline. The volume shrinkage not only weakens the flood control and drought resistance capabilities, but also causes the ecological space to compress sharply. Therefore, the control of lake sediment pollution has become a core link in water ecological restoration. Scientific disposal can effectively reduce the total amount of pollutants in sediments and is a key technical means to control secondary pollution of water bodies and achieve endogenous control.

[0003] In order to alleviate the endogenous pollution load of lakes, most eutrophic waters need to rely on physical dredging, chemical passivation or single-function bacterial agents for sediment remediation. Traditional treatment methods are prone to secondary pollution, short remediation cycles, and inability to simultaneously reduce nitrogen, phosphorus and organic matter. These problems lead to the degradation of the self-purification function of the sediment, algae outbreaks in the water body, the extinction of submerged plants, and the collapse of the aquatic ecosystem. Therefore, the development of multi-effect bacterial agents with efficient denitrification, phosphorus absorption and decomposition functions to improve the synergistic degradation efficiency of sediment pollutants is the core research direction in the field of lake ecological restoration.

[0004] Based on this, the targeted breeding of wild bacterial strains with efficient denitrification, phosphorus absorption and decomposition functions is of great strategic significance for building a "one bacteria, multiple effects, long-term and stable" sediment bioremediation technology system. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide lake sediment reducing bacteria, screening methods, immobilized bacterial agents and applications to overcome the deficiencies in the above-mentioned prior art.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] Lake sediment-reducing bacteria, the bacteria are classified and named: Bacillus amyloliquefaciens WP02, and were deposited on November 6, 2020 with the preservation number: CCTCC NO: M2020696, and preserved in: China Center for Type Culture Collection.

[0008] Based on the above technical solution, the present invention also provides an application of lake sediment reducing bacteria in sediment denitrification, phosphorus absorption and decomposition.

[0009] Based on the above technical solution, the present invention also provides a method for screening lake sediment-reducing bacteria, comprising the following steps:

[0010] S1. Take an appropriate amount of Yezhi Lake sediment sample and place it in an enrichment acclimation medium. Acclimation is carried out in a shake flask at 25°C to 30°C and 100 rpm to 150 rpm for 2 to 3 days. Then, a certain amount of the enrichment solution is added to a new enrichment acclimation medium. The sample is enriched and cultured under the same conditions for another 2 to 3 days to obtain the final enrichment solution.

[0011] S2. Take the corresponding amount of final enrichment solution and add it to sterile saline and mix well to obtain 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 For each dilution, take a certain amount of bacterial solution and spread it on solid culture medium, and culture it in a constant temperature incubator at 25℃~30℃ for 2d~3d to obtain colonies visible to the naked eye;

[0012] S3. Spread the enriched colonies on a solid culture medium, select plates with 30 to 300 colonies, and separate and purify colonies with different morphological characteristics by streaking to obtain plates with single colonies;

[0013] S4. Inoculate the single colony grown after the application into the BTB medium and observe the BTB medium until the colony in the BTB medium turns blue.

[0014] S5. Add Giltay medium to the test tube, place a Dulbecco's tube, inoculate the bacteria that turned blue in the BTB medium into the test tube, place it in an incubator at 25℃~30℃ for 1d~2d, and observe whether bubbles are generated. If so, select strains with the ability to remove nitrogen and phosphorus, which are lake sediment reducing bacteria.

[0015] Further, 10 -1 The dilution was to take 1 mL of the final enrichment solution and mix it in 9 mL of sterile saline. Other dilutions were obtained in the same way. For each dilution in S2, 100 μL of the bacterial solution was spread on the solid culture medium.

[0016] Furthermore, the solid culture medium is prepared by adding 20 g of agar to the enriched acclimation medium components; the enriched acclimation medium comprises: 4.7 g of sodium succinate, 0.45 g of ammonium chloride, 0.72 g of potassium nitrate, 1.5 g of potassium dihydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, 0.03 g of ferrous sulfate heptahydrate, 2 mL of trace elements, and 1000 mL of distilled water, and the pH is adjusted to 7.

[0017] Furthermore, the BTB culture medium is composed of: 4.7 g sodium succinate, 0.72 g potassium nitrate, 1.5 g potassium dihydrogen phosphate, 7.9 g sodium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 2 mL trace elements, 1 mL 1% BTB ethanol solution, 1000 mL distilled water, and the pH is adjusted to 7.

[0018] Furthermore, Giltay medium is composed of: 1.0 g potassium nitrate, 1.0 g glycine, 1 mL 1% BTB ethanol solution, 8.5 g anhydrous sodium citrate, 1.0 g magnesium sulfate heptahydrate, 1.0 g potassium dihydrogen phosphate, 0.05 g ferric chloride hexahydrate, 0.15 g calcium chloride, and 1000 mL distilled water, and the pH is adjusted to 7.

[0019] Based on the above technical solution, the present invention also provides an immobilized bacterial agent, the material composition of which is: an adhesive, an auxiliary material, a cross-linking agent, and a bacterial liquid obtained by activating and culturing lake sediment-reducing bacteria; 10mL to 50mL of bacterial liquid; an adhesive: 5% to 15% polyvinyl alcohol, 1% to 6% sodium alginate, 0.1% to 0.3% sodium carboxymethyl cellulose; an auxiliary material: 1% to 3% zeolite, 1% to 3% bamboo charcoal; a cross-linking agent: 2% to 4% calcium chloride saturated boric acid solution.

[0020] Based on the above technical solution, the present invention also provides a method for preparing an immobilized bacterial agent, comprising the following steps:

[0021] S1. Activate lake sediment-reducing bacteria stored on a slant, inoculate them onto LB solid medium, place them in a constant temperature incubator, and incubate them at 25°C to 30°C for 24 to 48 hours. Then, pick the bacteria from the LB solid medium and inoculate them into LB liquid medium. Shake and incubate them at 100 rpm to 150 rpm at 25°C to 30°C for 24 to 48 hours to obtain a bacterial solution. The LB solid medium is composed of LB liquid medium components plus 20 g of agar.

[0022] S2, mixing and stirring the bacterial liquid, the binder, and the auxiliary materials to obtain a mixture;

[0023] S3. The mixture is aspirated and slowly injected into a cross-linking agent, and cross-linked in the cross-linking agent for 24 to 48 hours to obtain immobilized bacterial pellets and a finished immobilized bacterial agent.

[0024] Furthermore, in S3, the mixture is drawn up using a needleless syringe.

[0025] Furthermore, the LB liquid culture medium is composed of: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and 1000 mL distilled water, adjusted to pH 7, and divided into 250 mL Erlenmeyer flasks, and each bottle of 100 mL is sterilized in a sterilizer at 121°C for 20 min.

[0026] Furthermore, the activity of the immobilized bacterial agent was determined by randomly selecting multiple immobilized bacterial pellets and adding them to 10-fold diluted LB liquid culture medium. The culture was placed in a full-temperature culture shaker at 30°C and 150 rpm for 48 hours, and then the number of viable bacteria was counted by dilution coating.

[0027] Based on the above technical solution, the present invention also provides an immobilized bacterial agent, the material composition of which is: adsorption material and bacterial liquid obtained by activating and culturing lake sediment reduction bacteria, the bacterial liquid is 10mL to 50mL; adsorption material: polyurethane sponge or fiber ball, the volume of polyurethane sponge is 16cm 3 ~48cm 3 , the volume of the fiber ball is 32cm 3 ~96cm 3 .

[0028] Based on the above technical solution, the present invention also provides a method for preparing an immobilized bacterial agent, comprising the following steps:

[0029] S1. Activate lake sediment-reducing bacteria stored on a slant, inoculate them onto LB solid medium, place them in a constant temperature incubator, and incubate them at 25°C to 30°C for 24 to 48 hours. Then, pick the bacteria from the LB solid medium and inoculate them into LB liquid medium. Shake and incubate them at 100 rpm to 150 rpm at 25°C to 30°C for 24 to 48 hours to obtain a bacterial solution. The LB solid medium is composed of LB liquid medium components plus 20 g of agar.

[0030] S2, mixing the bacterial solution and the adsorption material to obtain a mixture;

[0031] S3. Place the mixture in a culture shaker at 25°C to 30°C and 100 rpm to 150 rpm for 24 to 48 hours, wash the free bacteria with sterile water, and obtain a finished immobilized bacterial agent.

[0032] Furthermore, the LB liquid culture medium is: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 1000 mL distilled water, adjusted to pH 7, and divided into 250 mL Erlenmeyer flasks, 100 mL per bottle is sterilized in a sterilizer at 121°C for 20 min; the LB solid culture medium is LB liquid culture medium components plus 20 g agar.

[0033] Furthermore, the activity of the immobilized bacteria was determined by randomly selecting a plurality of the immobilized bacteria, and the volume of the immobilized bacteria randomly selected was 16 cm 3 Polyurethane sponge or a volume of 32cm 3 The fiber balls were added to LB liquid culture medium diluted 10 times, and cultured in a full-temperature culture shaker at 25℃~30℃ and 100rpm~150r / min for 24h~48h, and then the number of viable bacteria was obtained by dilution coating and counting.

[0034] Compared with the prior art, the present invention has the following beneficial technical effects:

[0035] The immobilized bacterial agents obtained by encapsulating and adsorbing lake sediment-reducing bacteria can not only effectively reduce sediment organic matter, reduce sediment total nitrogen and absorb phosphorus in overlying water, that is, effectively denitrify, absorb phosphorus and decompose the sediment, but also reduce the volume of the sediment by decomposing as much organic matter in the sediment as possible, convert the organic nitrogen in the sediment into nitrogen gas and release it into the air, reduce the nitrogen content in the sediment and overlying water as much as possible, absorb phosphorus in the water, reduce the phosphorus concentration in the overlying water, and improve the water quality of the overlying water. In addition, the application of this bacterial agent can enhance the redox potential of the sediment, promote the mineralization and transformation of endogenous pollutants, and reduce the nutrient concentration of the water body. It has important significance and application value in maintaining the ecological balance of the water body. Compared with physical and chemical remediation methods, the in-situ remediation efficiency is high, the environmental disturbance is small and there is no secondary pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 A diagram showing the colony morphology of lake sediment-reducing bacteria during identification using streaking on a plate, as provided in Example 4 of the present invention;

[0037] Figure 2 The results of the determination of the denitrification, phosphorus removal and decomposition capabilities of the lake sediment reduction bacteria provided in Example 5 of the present invention;

[0038] Figure 3 Showing the bacterial morphology of WP02 and the actual pictures of the prepared bacterial agent products in Examples 6 and 7;

[0039] Figure 4 The immobilized bacterial agent for embedding and adsorbing lake sediment-reducing bacteria provided in Application Example 1 of the present invention has an effect on reducing sediment without overlying water;

[0040] Figure 5 The immobilized bacterial agent for encapsulating and adsorbing lake sediment-reducing bacteria provided in Application Example 2 of the present invention has an effect on reducing overlying water sediment. DETAILED DESCRIPTION

[0041] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0042] Example 1

[0043] Lake sediment reducing bacteria, the bacteria are classified and named: Bacillus amyloliquefaciens WP02, and were deposited on November 6, 2020 with the deposit number: CCTCC NO: M2020696, at: China Center for Type Culture Collection, Address: Wuhan University, Wuhan, China. The colony morphology of Bacillus amyloliquefaciens is shown in the attached Figure 1 As shown, the biochemical identification results are shown in Example 4.

[0044] Example 2

[0045] An application of lake sediment reducing bacteria as described in Example 1 in sediment denitrification, phosphorus absorption and decomposition.

[0046] Example 3

[0047] A method for screening lake sediment-reducing bacteria as described in Example 1 comprises the following steps:

[0048] S1. Take an appropriate amount of Yezhi Lake sediment sample and place it in an enrichment acclimation medium. Acclimation is carried out in a shake flask at 25°C to 30°C and 100 rpm to 150 rpm for 2 to 3 days. Then, a certain amount of the enrichment solution is added to a new enrichment acclimation medium. The sample is enriched and cultured under the same conditions for another 2 to 3 days to obtain the final enrichment solution.

[0049] S2. Take the corresponding amount of final enrichment solution and add it to sterile saline and mix well to obtain 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Diluted bacterial solution, for example, 10 -1 Taking the dilution as an example, 1 mL of the final enrichment solution was mixed with 9 mL of sterile physiological saline. This is only an example and can be adjusted accordingly during actual application. Other dilutions were obtained in the same way. A certain amount of bacterial solution was applied to the solid culture medium at each dilution. For example, 100 μL of bacterial solution was applied to the solid culture medium at each dilution. This is only an example and can be adjusted accordingly during actual application. The culture was then placed in a constant temperature incubator at 25°C to 30°C for 2 to 3 days to obtain visible colonies.

[0050] S3. Spread the enriched colonies on a solid culture medium, select plates with 30 to 300 colonies, and separate and purify colonies with different morphological characteristics by streaking to obtain plates with single colonies;

[0051] S4. Inoculate the single colony grown after the application into the BTB medium and observe the BTB medium until the colony in the BTB medium turns blue.

[0052] S5. Add Giltay medium to the test tube, place a Dulbecco's tube, inoculate the bacteria that turned blue in the BTB medium into the test tube, place it in an incubator at 25℃~30℃ for 1d~2d, and observe whether bubbles are generated. If so, select strains with the ability to remove nitrogen and phosphorus, which are lake sediment reducing bacteria.

[0053] The results showed that Bacillus amyloliquefaciens WP02 turned the culture medium blue, indicating that it could carry out denitrification and consume nitrate to make the culture medium alkaline, thereby turning the indicator blue. At the same time, bubbles were generated in the test tube of the WP02 bacterial solution, indicating that thorough denitrification had occurred in the tube and the product escaped in a gaseous form. The strain with the ability to denitrify and remove phosphorus, namely Bacillus amyloliquefaciens WP02 obtained by screening according to the present invention, was inoculated into a glycerol tube and stored in a -80°C refrigerator. A strain numbered WP02 was obtained through the above isolation route, demonstrating that the strain has the ability to denitrify and remove phosphorus.

[0054] In this embodiment, the solid culture medium is prepared by adding 20 g of agar to the components of the enriched acclimation medium. The enriched acclimation medium contains: 4.7 g of sodium succinate, 0.45 g of ammonium chloride, 0.72 g of potassium nitrate, 1.5 g of potassium dihydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, 0.03 g of ferrous sulfate heptahydrate, 2 mL of trace elements, and 1000 mL of distilled water. The pH is adjusted to 7 and the medium is dispensed into 250 mL Erlenmeyer flasks. 100 mL of each bottle is sterilized in an autoclave at 121° C. for 20 min.

[0055] BTB culture medium is: 4.7 g sodium succinate, 0.72 g potassium nitrate, 1.5 g potassium dihydrogen phosphate, 7.9 g sodium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 2 mL trace elements, 1 mL 1% BTB ethanol solution, 1000 mL distilled water, adjust the pH to 7, and dispense into 250 mL Erlenmeyer flasks. Each bottle of 100 mL is sterilized in a sterilizer at 121°C for 20 min.

[0056] Giltay medium consists of: 1.0 g potassium nitrate, 1.0 g glycine, 1 mL 1% BTB ethanol solution, 8.5 g anhydrous sodium citrate, 1.0 g magnesium sulfate heptahydrate, 1.0 g potassium dihydrogen phosphate, 0.05 g ferric chloride hexahydrate, 0.15 g calcium chloride, and 1000 mL distilled water. The pH is adjusted to 7 and the medium is dispensed into 250 mL Erlenmeyer flasks. 100 mL of each bottle is sterilized in an autoclave at 121°C for 20 min.

[0057] Example 4

[0058] Identification of lake sediment-reducing bacteria

[0059] The strain obtained above was identified as Bacillus amyloliquefaciens WP02 through morphological identification, biochemical identification and 16S rRNA sequencing analysis;

[0060] 1. The results of colony morphology are as shown in the attached Figure 1 The growth physiological morphology of the bacteria is: forming biofilm in liquid culture medium, and the colonies on solid culture medium are light yellow, opaque, with rough surface and irregular edges;

[0061] 2. Biochemical identification results showed that the organism required oxygen, was short rod-shaped (0.7 μm to 3.0 μm), motile, and had round spores; it was positive for nitrate reduction, catalase, hydrolyzed starch, liquefied gelatin, and indole, but negative for methyl red.

[0062] 3. Bacterial genomic DNA extraction and 16S rDNA sequence analysis:

[0063] 1) Bacterial genomic DNA was extracted using the SDS alkaline lysis-phenol:chloroform:isoamyl alcohol (25:24:1, v / v / v) extraction-ethanol precipitation method (Maloy, 1990);

[0064] 2) PCR amplification of strain ZM-1 was performed using universal bacterial 16S rDNA primers (27F: AGAGTTTGATCMTGGCTCAG and 1492R: TACGGYTACCTTGTTACGACTT) to obtain a 1422 bp PCR product. The PCR product was purified and sent to Shanghai Sunny Co., Ltd. for sequencing. The specific sequence is shown in the sequence listing in the instruction manual. Part of the nucleotide sequence is shown in SEQ ID NO: 1.

[0065] 3) The determined sequences were assembled and manually proofread using the DNAStar software package, and then compared for nucleotide homology in GenBank using the NCBI (National Center for Biotechnology Information) BLAST program. The results showed that the WP02 strain had 100% similarity with the accession number KM091682.1 in GenBank, and was identified as Bacillus amyloliquefaciens, a member of the genus Bacillus. It was named Bacillus amyloliquefaciens because it can produce liquefying amylase to decompose starch.

[0066] Example 5

[0067] Determination of Nitrogen Removal, Phosphorus Uptake and Degradation Capacity of Bacillus amyloliquefaciens WP02

[0068] 1) LB liquid medium was divided into 250 mL Erlenmeyer flasks, with 100 mL per flask. Under sterile operation, 1% inoculum of WP02 bacteria in the logarithmic growth phase was inoculated (activate lake sediment-reducing bacteria stored on a slant, inoculate on LB solid medium, place in a constant temperature incubator at 25°C to 30°C, and incubate at this temperature for 24 to 48 hours. Then, the bacteria on the LB solid medium were picked and inoculated into LB liquid medium, and shaken at 100 rpm to 150 rpm at 25°C to 30°C for 24 to 48 hours to obtain a bacterial solution). The flasks were placed in a constant temperature shaker at 25°C to 30°C and 100 rpm to 150 rpm for 24 to 48 hours;

[0069] 2) Culture the bacterial solution to OD 600 =3, inoculation amount 1%, denitrification experiment was carried out on wastewater, the total nitrogen of wastewater inlet was 10mg / L, COD was 150mg / L, and total phosphorus was 2mg / L;

[0070] 3) The nitrogen and phosphorus removal effects of Bacillus amyloliquefaciens WP02 are shown in the attached figure. Figure 2 As shown in the figure, it can be seen that the nitrogen removal capacity of Bacillus amyloliquefaciens WP02 is 8.47 mg / L, the removal rate is 84.73%, the phosphorus removal capacity is 1.33 mg / L, the removal rate is 66.51%, and it has strong denitrification and phosphorus removal capabilities, and strong ability to convert organic matter. The COD removal rate is as high as 59.91%, and the organic matter reduction is 89.87 mg / L.

[0071] LB liquid medium is composed of: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and 1000 mL distilled water. The pH is adjusted to 7 and divided into 250 mL Erlenmeyer flasks. 100 mL of each bottle is sterilized in an autoclave at 121°C for 20 min. LB solid medium is composed of 20 g agar added to the components of LB liquid medium.

[0072] Example 6

[0073] Based on Example 1, the present invention also provides an immobilized bacterial agent, the material composition of which is: a binder, an auxiliary material, a cross-linking agent, and a bacterial solution obtained by activating and culturing the lake sediment-reducing bacteria described in Example 1, wherein the bacterial solution is 10 mL to 50 mL; the binder comprises 5% to 15% polyvinyl alcohol, 1% to 6% sodium alginate, and 0.1% to 0.3% sodium carboxymethyl cellulose; the auxiliary material comprises 1% to 3% zeolite and 1% to 3% bamboo charcoal; and the cross-linking agent comprises 2% to 4% calcium chloride saturated boric acid solution. The percentages of the binder, auxiliary material, and cross-linking agent are all ratios of their added amounts relative to the volume of the bacterial solution, and the unit is: g (binder, auxiliary material, or cross-linking agent) / mL (bacterial solution).

[0074] The immobilized bacterial agent is prepared by the embedding method, comprising the following steps:

[0075] S1. Activate lake sediment-reducing bacteria stored on a slant, inoculate them onto LB solid medium, place them in a constant temperature incubator, and incubate them at 25°C to 30°C for 24 to 48 hours. Then, pick the bacteria from the LB solid medium and inoculate them into LB liquid medium. Shake and incubate them at 100 rpm to 150 rpm at 25°C to 30°C for 24 to 48 hours to obtain a bacterial solution. The LB solid medium is composed of LB liquid medium components plus 20 g of agar.

[0076] S2, mixing and stirring the bacterial liquid, the binder, and the auxiliary materials to obtain a mixture;

[0077] S3. Absorb the mixture, for example, by using a needleless syringe, for example, a 10 mL needleless syringe, and slowly inject the mixture into the cross-linking agent, for example, 100 mL of the cross-linking agent, and cross-link in the cross-linking agent for 24 to 48 hours to obtain an immobilized bacterial ball, thereby obtaining a finished immobilized bacterial agent, such as Figure 3 shown.

[0078] Multiple immobilized bacterial agents were randomly selected and added to 10-fold diluted LB liquid culture medium. Multiple particles can refer to three, four, five, etc. For example, three particles were selected, which is just an example. After incubation in a full-temperature incubation shaker at 30°C and 150 rpm for 48 hours, the number of viable bacteria was obtained by dilution coating and counting.

[0079] LB liquid culture medium is composed of: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and 1000 mL distilled water. The pH is adjusted to 7 and divided into 250 mL Erlenmeyer flasks. 100 mL of each bottle is sterilized in a sterilizer at 121°C for 20 min.

[0080] Example 7

[0081] Based on Example 1, the present invention also provides an immobilized bacterial agent, the material composition of which is: adsorption material and bacterial liquid obtained by activating and culturing the lake sediment reduction bacteria described in Example 1, the bacterial liquid being 10mL to 50mL; adsorption material: polyurethane sponge or fiber ball, the volume of the polyurethane sponge being 16cm 3 ~48cm 3 , the volume of the fiber ball is 32cm 3 ~96cm 3 .

[0082] The preparation of the above-mentioned immobilized bacterial agent by adsorption method includes the following steps:

[0083] S1. Activate lake sediment-reducing bacteria stored on a slant, inoculate them onto LB solid medium, place them in a constant temperature incubator, and incubate them at 25°C to 30°C for 24 to 48 hours. Then, pick the bacteria from the LB solid medium and inoculate them into LB liquid medium. Shake and incubate them at 100 rpm to 150 rpm at 25°C to 30°C for 24 to 48 hours to obtain a bacterial solution. The LB solid medium is composed of LB liquid medium components plus 20 g of agar.

[0084] S2. Mix the bacterial solution and the adsorption material to obtain a mixture, wherein the composition of each material is as follows: 10mL to 50mL of bacterial solution, 16cm of adsorption material 3 ~96cm 3 ;

[0085] S3. Place the mixture in a culture shaker at 25°C to 30°C and 100 rpm to 150 rpm for 24 to 48 hours, wash the free bacteria with sterile water, and obtain a finished immobilized bacterial agent, such as Figure 3 shown.

[0086] Randomly select multiple particles of the above immobilized bacterial agent. Multiple particles can refer to three, four, five, etc. For example, selecting three particles is just an example. The volume of the randomly selected immobilized bacterial agent is 16 cm 3 Polyurethane sponge or a volume of 32cm 3 The fiber balls were added to LB liquid culture medium diluted 10 times, and cultured in a shaker at 25℃~30℃ and 100rpm~150r / min for 24h~48h, and then the number of viable bacteria was counted by dilution coating.

[0087] LB liquid culture medium is composed of: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and 1000 mL distilled water. The pH is adjusted to 7 and divided into 250 mL Erlenmeyer flasks. 100 mL of each bottle is sterilized in a sterilizer at 121°C for 20 min.

[0088] The immobilized bacteria were added to the sediment without overlying water and the sediment with overlying water to verify its effect on sediment denitrification, phosphorus absorption and decomposition.

[0089] Application Example 1: Effect of Embedded and Adsorbed Immobilized Bacteria on Reduction of Uncovered Bottom Mud

[0090] The embedded and adsorbed immobilized bacterial agents in Examples 6 and 7 were respectively added to the Yezhi Lake sediment of Huazhong Agricultural University to verify their degradation effects on endogenous pollutants in the sediment. The initial volume of the sediment was 325 mL, and the initial total nitrogen, total phosphorus and organic matter contents were measured. The diameter of each sediment simulator was 5 cm, the depth of the added sediment was 16.5 cm, and the depth of the sediment covering water (ultrapure water) was 5 cm. The bacterial agent was added once every 7 days for a total of 4 times, with each addition amount of 2 g of immobilized bacterial balls and 32 cm of adsorbed bacterial agent fiber balls. 3 After adding, stir evenly and take samples to determine the total nitrogen, total phosphorus and organic matter content after 42 days.

[0091] The effects of embedded and adsorbed immobilized bacteria on nitrogen removal, phosphorus absorption and decomposition of sediment without overlying water are shown in the attached figure. Figure 4 As shown, the initial organic matter, total nitrogen and total phosphorus contents of the sediment were 11.3%, 137.86 mg / kg and 1.93 mg / kg respectively. After addition, the organic matter content decreased to 10.25% and 10.24% respectively, and the relative content changes reached 90.71% and 90.62%, respectively. The immobilized bacterial agent showed a relatively strong decomposition ability; after addition, the total nitrogen content decreased to 93.14 mg / kg and 97.89 mg / kg respectively, and the relative content changes reached 67.56% and 71.01%; the total phosphorus content increased to 2.12 mg / kg and 2.09 mg / kg respectively, and the relative content changes reached 1.09% and 1.08%, respectively, indicating that the immobilized bacterial agent showed a strong denitrification and phosphorus absorption ability.

[0092] Application Example 2: Effect of Embedded and Adsorbed Immobilized Bacteria on Reduction of Overlying Water Sediment

[0093] The embedded and adsorbed immobilized bacterial agents in Examples 6 and 7 were directly added to the Yezhi Lake sediment of Huazhong Agricultural University, and the upper layer of the sediment was injected with Yezhi Lake water to verify its degradation effect on endogenous pollutants in the sediment. The initial volume of the sediment was 325 mL, and its initial total nitrogen, total phosphorus and organic matter contents were measured respectively. The effective water depth of each sediment simulator was 85 cm and the diameter was 5 cm. The depth of the added sediment was 16.5 cm, and the dosage was 2 g of immobilized bacterial balls and 32 cm of adsorbed bacterial fiber balls. 3 After adding, stir evenly and take samples to determine the total nitrogen, total phosphorus and organic matter content after 75 days.

[0094] The effects of embedded and adsorbed immobilized bacteria on nitrogen removal, phosphorus absorption and decomposition of sediment with overlying water are shown in the attached figure. Figure 5 As shown in the figure, the initial organic matter, total nitrogen and total phosphorus contents of the sediment were 6.09%, 116.28mg / kg and 0.78mg / kg respectively. After addition, the organic matter contents decreased to 5.01% and 5.29% respectively, and the relative content changes were 82.27% and 86.86% respectively. The immobilized bacterial agent showed a strong decomposition ability. After addition, the total nitrogen content decreased to 42.51mg / kg and 47.21mg / kg respectively, and the relative content changes were 36.56% and 40.60% respectively; the total phosphorus content increased to 0.91mg / kg and 0. .94mg / kg (absorbs phosphorus in the overlying water into the body, reduces the phosphorus concentration in the water body, and is beneficial to the overlying water treatment), the relative content changes are 1.17% and 1.21%, and the immobilized bacterial agent shows a strong ability to remove nitrogen and absorb phosphorus. Through continuous monitoring of the overlying water body, it is found that the total phosphorus content has a slight fluctuation in the early stage, and slowly decreases and tends to be stable in the later stage, which once again verifies the phosphorus absorption capacity of the immobilized bacterial agent applied to the bottom mud. At the same time, the final stabilization of the total nitrogen, ammonia nitrogen, phosphate and chemical oxygen demand of the overlying water body shows that the bacterial agent has the potential for synergistic purification of the bottom mud-water system.

[0095] The above two application examples show that after the embedded and adsorbed immobilized bacterial agents are added to the sediment, the redox potential of the sediment can be enhanced, the mineralization and transformation of endogenous pollutants can be promoted, and the nutrient concentration of the water body can be reduced. They have important significance and application value in maintaining the ecological balance of the water body. Compared with the physical and chemical remediation methods, the in-situ remediation efficiency is high, the environmental disturbance is small, and there is no secondary pollution.

[0096] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. Lake sediment reducing bacteria, characterized in that The bacteria was classified and named: Bacillus amyloliquefaciens WP02, and was deposited in: China Center for Type Culture Collection on November 6, 2020 with the preservation number: CCTCC NO: M2020696.

2. Use of the lake sediment reduction bacteria as claimed in claim 1 in denitrification, phosphorus absorption and decomposition of sediment.

3. A method for screening lake sediment-reducing bacteria according to claim 1, characterized in that: The steps include: S1. Take an appropriate amount of Yezhi Lake sediment sample and place it in an enrichment acclimation medium. Acclimation is carried out in a shake flask at 25°C to 30°C and 100 rpm to 150 rpm for 2 to 3 days. Then, a certain amount of the enrichment solution is added to a new enrichment acclimation medium. The sample is enriched and cultured under the same conditions for another 2 to 3 days to obtain the final enrichment solution. S2. Take the corresponding amount of final enrichment solution and add it to sterile saline and mix well to obtain 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 For each dilution, take a certain amount of bacterial solution and spread it on solid culture medium, and culture it in a constant temperature incubator at 25℃~30℃ for 2d~3d to obtain colonies visible to the naked eye; S3. Spread the enriched colonies on a solid culture medium, select plates with 30 to 300 colonies, and separate and purify colonies with different morphological characteristics by streaking to obtain plates with single colonies; S4. Inoculate the single colony grown after the application into the BTB medium and observe the BTB medium until the colony in the BTB medium turns blue. S5. Add Giltay medium to the test tube, place a Dulbecco's tube, inoculate the bacteria that turned blue in the BTB medium into the test tube, place it in an incubator at 25℃~30℃ for 1d~2d, and observe whether bubbles are generated. If so, select strains with the ability to remove nitrogen and phosphorus, which are lake sediment reducing bacteria.

4. The screening method according to claim 3, wherein The solid culture medium is prepared by adding 20 g of agar to the enriched acclimation culture medium components; the enriched acclimation culture medium comprises: 4.7 g of sodium succinate, 0.45 g of ammonium chloride, 0.72 g of potassium nitrate, 1.5 g of potassium dihydrogen phosphate, 0.1 g of magnesium sulfate heptahydrate, 0.03 g of ferrous sulfate heptahydrate, 2 mL of trace elements, and 1000 mL of distilled water, and the pH is adjusted to 7.

5. The screening method according to claim 3 or 4, characterized in that The BTB culture medium comprises: 4.7 g sodium succinate, 0.72 g potassium nitrate, 1.5 g potassium dihydrogen phosphate, 7.9 g sodium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 2 mL trace elements, 1 mL 1% BTB ethanol solution, 1000 mL distilled water, and the pH is adjusted to 7.

6. The screening method according to claim 3, 4 or 5, characterized in that The Giltay medium comprises: 1.0 g potassium nitrate, 1.0 g glycine, 1 mL 1% BTB ethanol solution, 8.5 g anhydrous sodium citrate, 1.0 g magnesium sulfate heptahydrate, 1.0 g potassium dihydrogen phosphate, 0.05 g ferric chloride hexahydrate, 0.15 g calcium chloride, and 1000 mL distilled water, and the pH is adjusted to 7.

7. An immobilized bacterial agent, characterized in that: The materials are composed of: adhesive, auxiliary materials, cross-linking agent and bacterial liquid obtained by activating and culturing the lake sediment reducing bacteria described in claim 1; 10mL to 50mL of bacterial liquid; Adhesive: polyvinyl alcohol 5% to 15%, sodium alginate 1% to 6%, sodium carboxymethyl cellulose 0.1% to 0.3%; auxiliary materials: zeolite 1% to 3%, bamboo charcoal 1% to 3%; cross-linking agent: 2% to 4% calcium chloride saturated boric acid solution.

8. A method for preparing the immobilized bacterial agent according to claim 7, characterized in that: The steps include: S1. Activate lake sediment-reducing bacteria stored on a slant, inoculate them onto LB solid medium, place them in a constant temperature incubator, and incubate them at 25°C to 30°C for 24 to 48 hours. Then, pick the bacteria from the LB solid medium and inoculate them into LB liquid medium. Shake and incubate them at 100 rpm to 150 rpm at 25°C to 30°C for 24 to 48 hours to obtain a bacterial solution. The LB solid medium is composed of LB liquid medium components plus 20 g of agar. S2, mixing and stirring the bacterial liquid, the binder, and the auxiliary materials to obtain a mixture; S3. The mixture is aspirated and slowly injected into a cross-linking agent, and cross-linked in the cross-linking agent for 24 to 48 hours to obtain immobilized bacterial pellets and a finished immobilized bacterial agent.

9. An immobilized bacterial agent, characterized in that: The materials are composed of: adsorption material and bacterial liquid obtained by activating and culturing the lake sediment reduction bacteria described in claim 1, 10mL to 50mL of bacterial liquid; adsorption material: polyurethane sponge or fiber ball, the volume of the polyurethane sponge is 16cm 3 ~48cm 3 , the volume of the fiber ball is 32cm 3 ~96cm 3 .

10. A method for preparing the immobilized bacterial agent according to claim 9, characterized in that: The steps include: S1. Activate lake sediment-reducing bacteria stored on a slant, inoculate them onto LB solid medium, place them in a constant temperature incubator, and incubate them at 25°C to 30°C for 24 to 48 hours. Then, pick the bacteria from the LB solid medium and inoculate them into LB liquid medium. Shake and incubate them at 100 rpm to 150 rpm at 25°C to 30°C for 24 to 48 hours to obtain a bacterial solution. The LB solid medium is composed of LB liquid medium components plus 20 g of agar. S2, mixing the bacterial solution and the adsorption material to obtain a mixture; S3. Place the mixture in a culture shaker at 25°C to 30°C and 100 rpm to 150 rpm for 24 to 48 hours, wash the free bacteria with sterile water, and obtain a finished immobilized bacterial agent.

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