Gordonia terra C5 and application thereof in sewage treatment

Abstract

The invention belongs to the field of environmental microorganisms, and discloses Gordonia sp. C5 and application thereof in sewage treatment. According to the invention, a Gordonia sp. C5 is screened, the preservation number is CCTCC NO: M 2024622, and after the Gordonia sp. C5 is fermented in a denitrifying phosphorus-deficient culture medium for 28-36 hours, denitrifying phosphorus-accumulating bacteria capable of effectively treating pig farm wastewater can be obtained. The denitrifying phosphorus-accumulating bacteria are easy to produce and good in treatment effect, and a new source is provided for phosphorus removal in the pig farm wastewater treatment process.

Description

Technical Field

[0001] The invention belongs to the field of environmental microorganisms, and in particular relates to Gordonia spp. C5 and its application in sewage treatment. Background Art

[0002] As the aquaculture industry becomes increasingly large-scale, the treatment of aquaculture wastewater has gradually become an important part of environmental control. Livestock and poultry aquaculture wastewater contains a large amount of organic matter, ammonia nitrogen, phosphorus and other components that are easy to cause eutrophication of water bodies. Direct discharge into the water body can easily lead to water pollution, which may affect human health. Phosphorus is the main cause of eutrophication of water bodies. Effective control of the phosphorus content in aquaculture wastewater is of great significance for the treatment of eutrophication of water bodies. At present, the main methods of phosphorus removal are physical, chemical and biological methods. Physical and chemical methods have good treatment effects, but the high cost is not suitable for large-scale promotion, especially in the aquaculture industry. It is difficult to promote, and chemical methods are also prone to secondary pollution. Biological methods are superior to their low cost and no secondary pollution and have attracted much attention.

[0003] Biological phosphorus removal from sewage mainly relies on polyphosphate-accumulating bacteria (PAOs) to release phosphates in their bodies under anaerobic conditions, and to utilize organic matter in the water and absorb a large amount of phosphates under aerobic conditions, so that phosphorus in sewage is enriched in the bacteria, and phosphorus can be further removed through sludge discharge. Most sewage treatment facilities use microorganisms to remove N and P simultaneously. However, due to the different ages of nitrifying bacteria and traditional polyphosphate-accumulating bacteria and the competition between denitrifying bacteria and PAOs in carbon source demand, there are major contradictions between them in the process of nitrogen and phosphorus removal, and it is difficult for the system to reach the optimal operating conditions. Current research has found a type of microorganism that has the function of nitrogen removal and phosphorus removal at the same time, called denitrifying polyphosphate-accumulating bacteria (DAPB). This type of microorganism can utilize organic matter in sewage to synthesize poly-β-hydroxybutyric acid (PHB) under anaerobic conditions and store it in the cell, releasing orthophosphate. Under anaerobic conditions, they use PHB as a carbon source and NO3 - The discovery of these two methods provides a possible solution to the contradiction between carbon source utilization and sludge age differences, allowing nitrogen and phosphorus removal to be carried out simultaneously. Compared with traditional biological phosphorus removal, denitrification and phosphorus removal can reduce COD consumption by 50% and oxygen consumption by about 30%, thus saving costs. Summary of the invention

[0004] The object of the present invention is to provide an isolated Gordonia terrae strain, the deposit number of which is CCTCC NO: M2021438.

[0005] Another object of the present invention is to provide an application of Gordonia spp. in sewage treatment. The strain of the present invention has the characteristics of denitrification and phosphorus absorption, which can effectively reduce the concentration of pollutants such as nitrate nitrogen and phosphorus in pig farm wastewater, reduce the subsequent process load and ensure the stability of effluent water quality.

[0006] In order to achieve the above object, the present invention adopts the following technical measures:

[0007] Acquisition of Gordonia terrae C5:

[0008] The applicant obtained soil from Huazhong Agricultural University, measured the denitrification phosphorus removal rate, and comprehensively compared the colony morphology, growth status, and denitrification denitrification and phosphorus removal effects of denitrifying polyphosphate bacteria. Finally, a denitrifying polyphosphate bacteria was screened out from 8 samples, which was identified as Gordonia terrae by 16srDNA. The bacterium was sent to the China Center for Type Culture Collection for preservation on April 3, 2024, and the classification name is Gordonia terrae C5, the preservation number is CCTCC NO: M 2024622, and the address is Wuhan University, Wuhan, China.

[0009] Gordonia terrestris C5 is a Gram-negative bacterium with red, opaque colonies.

[0010] The protection content of the present invention also includes:

[0011] The fermentation liquid of Gordonia spp. C5 contains live bacteria of Gordonia spp. C5.

[0012] A composition containing Gordonia terrestris C5 or the fermentation liquid thereof.

[0013] Application of soil Gordonia C5, fermentation liquid or composition of soil Gordonia C5 in sewage treatment;

[0014] In the above application, preferably, the ambient temperature of the application is 16-37°C, more preferably 23-30°C;

[0015] In the above application, preferably, the pH of the sewage is 7-11;

[0016] In the above application, preferably, the inoculation amount of the strain is 2-25%;

[0017] In the above application, preferably, the initial phosphorus concentration is 9 to 45 mg / L, more preferably 9 to 27 mg / L;

[0018] In the above application, specifically, the sewage treatment includes treating total phosphorus, nitrate nitrogen, COD and / or ammonia nitrogen in the sewage.

[0019] The strain of the present invention is also particularly suitable for sewage treatment under non-room temperature conditions.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The present invention provides for the first time a denitrifying and phosphorus-accumulating strain with good denitrification and phosphorus removal efficiency: Gordonia terrestris. The strain provides a high-quality seed strain for biological denitrification and phosphorus removal. A certain optimization screening is carried out for pig farm wastewater, providing an effective strain source for denitrification and phosphorus removal treatment of pig farm wastewater.

[0022] At the same time, it was found through tests that the strain can still exert its biological functions under non-room temperature conditions, has environmental tolerance, and is suitable for industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The removal efficiency of nitrate nitrogen and phosphorus by Gordonia ciliata C5 at different temperatures.

[0024] Figure 2 The removal efficiency of nitrate nitrogen and phosphorus by Gordonia species C5 in different pH soils.

[0025] Figure 3 The removal efficiency of nitrate nitrogen and phosphorus by Gordonia species C5 under different rotation speeds.

[0026] Figure 4 is the removal efficiency of nitrate nitrogen and phosphorus by Gordonia ciliata C5 at different initial phosphorus concentrations.

[0027] Figure 5 The removal efficiency of nitrate nitrogen and phosphorus by Gordonia species C5 at different initial inoculation doses. DETAILED DESCRIPTION

[0028] In the technical solutions of the present invention, the reagents, unless otherwise specified, are purchased from biochemical stores, and the technical solutions, unless otherwise specified, are conventional techniques in the art.

[0029] LB medium: yeast extract 5g / L, peptone 10g / L, NaCl 5g / L, pH 7.0-7.2 (add 20g agar if solid).

[0030] Enrichment medium: CH3COONa·3H2O 5g, KNO3 2.0g, MgSO4.7H2O 0.2g, K2HPO4 2.0g, trace elements 2mL, deionized water: 1L, pH=7.2.

[0031] BTB-denitrification medium: CH3COONa·3H2O 5.0 g, KNO 32.0 g, MgSO4.7 H2O 0.2 g, agar 20 g, 1%-BTB 4 mL, H2O 1000 mL, pH 7.6.

[0032] Denitrification phosphorus-deficient culture medium: KNO3 2g / L, CH3COONa·3H2O 5g / L, K2HPO4 0.05g / L, MgSO4·7H2O 0.2g / L, CaCl2 0.5g / L, trace elements 2mL / L.

[0033] Denitrification phosphorus-rich medium: KNO3 2g / L, CH3COONa·3H2O 5g / L, K2HPO4 0.05g / L, KH2PO40.2g / L, MgSO4·7H2O 0.2g / L, CaCl2 0.5g / L, trace elements 2mL / L.

[0034] The formula of nitrogen-rich and phosphorus-rich culture medium is: CH3COONa·3H2O 3.32mg, K2HPO4 25mg, NH4CI305.52mg, MgSO4·7H2O 91.26mg, CaCI2·2H2O 25.68mg, KNO3 300mg, Na2EDTA 1g, NaCI20mg, trace elements 2ml, deionized water: 1L, pH=7.2.

[0035] Trace element solution: FeCl3·6H2O 1.5g / L, H3BO3 0.15g / L, CuSO4·5H2O 0.03g / L, KI0.03g / L, Na2MoO4·2H2O 0.06g / L, MnCl2·4H2O 0.12g / L, ZnSO4·7H2O 0.12g / L, CoCl2·2H2O 0.12g / L.

[0036] All of the above culture media need to be sterilized by high pressure for 30 minutes before use.

[0037] The formula for the removal efficiency of nitrate nitrogen and phosphorus is:

[0038] Phosphorus removal rate: η1=(CC t ) / C×100%, denitrification rate: η2=(AA t ) / A×100%.

[0039] Where: η1—phosphorus removal rate (%), C—PO4 3- -The initial concentration of P, C t —PO4 in solution after 36h treatment 3—P concentration, η2—reverse digestibility (%), A—NO3 - -The initial concentration of N, A t —NO3 in solution after 36h treatment - -N concentration.

[0040] Embodiment 1:

[0041] Isolation, purification and identification of Gordonia spp. C5

[0042] 1. Sample Collection and Pretreatment

[0043] Take 20g each of the facultative pond mud, experimental fish pond mud and experimental field soil from the boutique pig farm of Huazhong Agricultural University, add them into a 250ml conical flask, add 100ml of physiological saline, seal the bottle and shake on a shaker for 30min, let it stand and stratify, and collect the supernatant.

[0044] 2. Enrichment of bacterial strains

[0045] Add 5 ml of supernatant to 100 ml of enrichment medium, seal and shake culture (30°C, 160 r / min) for more than 24 hours. Take 5 ml of culture solution as seed solution, mix with 100 ml of enrichment medium, seal and shake culture again (30°C, 160 r / min) for more than 24 hours, take 5 ml of cultured bacterial solution and mix with 100 ml of enrichment medium and shake culture again. Repeat this process 2-3 times. After the culture is completed, take 0.5 mL of enrichment solution and shake culture for 10 -1 ~10 -9 Dilution was performed in a concentration gradient. 0.1 mL of each dilution was taken and spread on LB solid medium. Each dilution was repeated 3 times and cultured in a biochemical incubator at 26°C for 2-3 days. Single colonies with clear morphology were selected for streaking and isolation culture. The grown single colonies were transferred and streaking culture was repeated 5-7 times to obtain pure single colonies.

[0046] 3. Initial screening of blue-white spots

[0047] The pure single colonies obtained above were inoculated into 1% BTB-denitrification solid medium and cultured at 25°C for 3 days. The strains whose medium turned blue were selected as the primary screening strains.

[0048] 4. Poly-P particle dyeing and rescreening

[0049] After the strains screened above were cultured anaerobically for 12 hours, Poly-P particles were respectively produced, and the strains with Poly-P particles were selected as denitrifying polyphosphate bacteria. LB slant medium was inoculated and stored at 4°C for future use.

[0050] 5. Preparation of Seed Liquid Culture

[0051] The denitrifying polyphosphate-accumulating bacteria screened above were separated and purified by multiple streaking and then inoculated into 100 ml LB liquid culture medium respectively, and cultured in a shaking incubator at 25° C. and 160 r / min for 24 h. This bacterial liquid was used as the seed liquid.

[0052] 6. Denitrification phosphorus removal rate determination

[0053] Take 5 ml of seed liquid and inoculate it into 100 ml of denitrification phosphorus-deficient medium. Seal the culture bottle for anaerobic phosphorus release for 12 hours. Centrifuge at 8000 r / min for 10 minutes to obtain the bacteria. Wash twice with sterile saline and resuspend in 100 ml of denitrification phosphorus-rich medium. Culture on a shaking table at 25°C and 160 r / min for anoxic phosphorus absorption. After 36 hours, take an appropriate amount of culture solution and centrifuge it to determine the PO4 in the supernatant. 3- -P, NO3 - -N, NO2 - -N absorbance, and according to the change of absorbance, the denitrification denitrification rate and phosphorus removal rate of the strain were calculated respectively. The strains with denitrification denitrification rate and phosphorus removal rate greater than 70% and 50% respectively were selected as the test strains.

[0054] 7. Purification and identification of denitrifying phosphorus accumulation strains

[0055] After the isolated strain was streaked multiple times to obtain a pure single colony, the colony morphology was observed for biochemical identification, and the genomic DNA was extracted for sequencing and comparison analysis of its 16SrDNA. Comprehensive identification and analysis showed that the bacteria were changed to the genus Gordonia terrae. The bacteria were sent to the China Center for Type Culture Collection for preservation on April 3, 2024, with classification name: Gordonia terrae C5, preservation number: CCTCC NO: M 2024622, address: Wuhan University, Wuhan, China.

[0056] The culture medium was denitrification phosphorus-rich medium: KNO3 2g / L, CH3COONa·3H2O 5g / L, K2HPO4 0.05g / L, KH2PO4 0.2g / L, MgSO4·7H2O 0.2g / L, CaCl2 0.5g / L, trace elements 2mL / L. The culture temperature was 30℃, the fermentation time was 48h, and the inoculation size was 7.7×10 7 cfu / ml, stirring speed 180r / min, the effective concentration after fermentation is 3.7×10 8 cfu / ml.

[0057] Embodiment 2:

[0058] Optimal treatment conditions for Gordonia terrae C5

[0059] 1. Experiment on the optimal temperature selection of Gordonia terrae C5

[0060] The optimal temperature for the bacteria to treat wastewater was selected through selection tests. 10 ml of bacterial solution was inoculated (inoculation volume was 10%) in 100 ml of nitrogen-rich and phosphorus-rich culture medium, and placed in a constant temperature shaker at 16°C, 23°C, 30°C, and 37°C for 36 hours at 160 r / min, centrifuged at 8000 r / min for 10 minutes, and the supernatant was taken to detect the nitrate nitrogen concentration and phosphorus concentration, and the removal efficiency of nitrate nitrogen and phosphorus was calculated. The results are shown in Table 1.

[0061] Table 1. Effect of temperature on soil P and N removal by Gordonia

[0062]

[0063] 2. Optimal pH selection test for Gordonia terrae C5

[0064] The optimal pH for the bacteria to treat wastewater was selected through selection tests. Take 10 ml of bacterial solution and inoculate (inoculation volume is 10%) in 100 ml of nitrogen-rich and phosphorus-rich culture medium, and place it in a constant temperature shaker (25°C, 160r / min) with pH values ​​of 6, 7, 8, 9, 10, 11, 12, and 13 for 36 hours of shaking culture, centrifuge at 8000r / min for 10 minutes, take the supernatant to detect nitrate nitrogen concentration and phosphorus concentration, and calculate the removal efficiency of nitrate nitrogen and phosphorus. The specific experimental results are shown in Table 2.

[0065] Table 2. Effect of pH on P and N removal by Gordonia gordonii

[0066]

[0067] 3. Experiment on the optimal rotation speed selection of Gordonia terrae C5

[0068] The optimal speed for the bacteria to treat wastewater was selected through selection tests. Take 10 ml of bacterial solution and inoculate (inoculation volume is 10%) in 100 ml of nitrogen-rich and phosphorus-rich culture medium, and place it in a constant temperature shaker at a speed of 120, 140, 160, 180, 200 r / min and a temperature of 23°C for 36 hours of shaking, centrifuge at 8000 r / min for 10 minutes, take the supernatant to detect the nitrate nitrogen concentration and phosphorus concentration, and calculate the removal efficiency of nitrate nitrogen and phosphorus. The specific experimental results are shown in Table 3.

[0069] Table 3. Effect of rotation speed on removal of P and N by Gordonia spp.

[0070]

[0071] 4. Experiment on the selection of the optimal initial phosphorus concentration of Gordonia terrae C5

[0072] The optimal initial phosphorus concentration for treating wastewater with this bacteria was selected through selection test. Take 10ml of bacterial solution and inoculate (the inoculation amount is 10%) in 100ml with initial phosphorus concentrations of 9, 18, 27, 36, and 45mg / L, and transfer the phosphorus concentration by adding different weights of phosphate. In nitrogen-rich and phosphorus-rich culture medium, place them in a constant temperature shaker (23℃, 160r / min) for shaking culture for 36h, centrifuge at 8000r / min for 10min, take the supernatant to detect nitrate nitrogen concentration and phosphorus concentration, and calculate the removal efficiency of nitrate nitrogen and phosphorus. The specific experimental results are shown in Table 4.

[0073] Table 4. Effect of initial concentration on the removal of P and N by Gordonia gordonii

[0074]

[0075] 5. Experiment on the selection of the optimal initial inoculum amount of Gordonia terrae C5

[0076] The optimal initial inoculation amount of the bacteria for treating wastewater was selected through selection tests. 5ml, 10ml, 15ml, 20ml, and 25ml of bacterial solution were inoculated into 100ml of nitrogen-rich and phosphorus-rich culture medium, respectively, and placed in a constant temperature shaker (23℃, 160r / min) for shaking culture for 36h, centrifuged at 8000r / min for 10min, and the supernatant was taken to detect the nitrate nitrogen concentration and phosphorus concentration, and the nitrate nitrogen and phosphorus removal efficiency were calculated. The specific experimental results are shown in Table 5.

[0077] Table 5. Effect of initial inoculation amount on the removal of P and N by Gordonia spp.

[0078]

[0079] 6 Orthogonal test

[0080] Select the optimal experimental conditions to design orthogonal experimental groups, combine according to different conditions, design four-factor three-level orthogonal experiments, the culture medium is phosphorus-rich culture medium: KNO3 2g / L, CH3COONa·3H2O 5g / L, K2HPO4 0.05g / L, KH2PO4 0.2g / L, MgSO4·7H2O 0.2g / L, CaCl2 0.5g / L, trace elements 2mL / L. The speed is 180r / min, the culture time is 48h, and then the optimal conditions of C5 strain under different conditions are determined. The group design is as shown in the figure below:

[0081] Number of groups Temperature ℃ pH Initial inoculum volume (cfu / ml) Initial p concentration (mg / L) L1 23 7 <![CDATA[2.5×10 7 ]]> 9 L2 23 9 <![CDATA[7.7×10 7 ]]> 18 L3 23 11 <![CDATA[1.26×10 8 ]]> 27 L4 30 7 <![CDATA[7.7×10 7 ]]> 27 L5 30 9 <![CDATA[1.26×10 8 ]]> 9 L6 30 11 <![CDATA[2.5×10 7 ]]> 18 L7 37 7 <![CDATA[1.26×10 8 ]]> 18 L8 37 9 <![CDATA[2.5×10 7 ]]> 27 L9 37 11 <![CDATA[7.7×10 7 ]]> 9

[0082] The test results are shown in the figure below:

[0083]

[0084] Embodiment 2:

[0085] Effective application of Gordonia terrae C5 in piggery wastewater

[0086] This embodiment mainly tests the denitrification, denitrification and phosphorus removal of the bacteria under actual application conditions. Untreated feces and urine flushing water was selected for direct testing. 100 ml of the collected pig farm flushing water was taken, and its original COD, ammonia nitrogen and total phosphorus concentrations are shown in the table below. The bacterial solution (10 ml) was added at a 10% addition amount, and placed in a constant temperature shaker (23°C, 160r / min) for shaking culture for 36h, and centrifuged at 8000r / min for 10min. The supernatant was taken to detect the concentrations of four indicators of COD, ammonia nitrogen, total phosphorus and nitrate nitrogen in the pig farm wastewater, and the corresponding removal rate of each sample was calculated. The specific experimental results are shown in the table below.

[0087]

Claims

1. An isolated strain of Gordonia spp. Gordonia terrae )C5, the preservation number of the land Gordonia bacteria is CCTCC NO: M 2024622.

2. The fermentation liquid of Gordonia australis C5 according to claim 1, wherein the fermentation liquid contains live bacteria of Gordonia australis C5.

3. A composition comprising the Gordonia australis C5 according to claim 1 or the fermentation liquid according to claim 2.

4. Use of the Gordonia spp. C5 according to claim 1, the fermentation liquid according to claim 2 or the composition according to claim 3 in sewage treatment.

5. The use according to claim 4, wherein the ambient temperature of the application is 16-37°C.

6. The use according to claim 4, wherein the pH of the sewage is 7-11.

7. The use according to claim 4, wherein the inoculation amount of the strain is 5-25%.

8. The use according to claim 4, wherein the initial phosphorus concentration of the sewage is 9-45 mg / L.

9. The use according to claim 4, wherein the sewage treatment comprises treating total phosphorus, nitrate nitrogen, COD and / or ammonia nitrogen in the sewage.

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