Flavobacterium S3 and its application
By screening and identifying the S3 strain of Flavobacterium, the problem of poor degradation of polycyclic aromatic hydrocarbons in the aquatic environment was solved, and the efficient degradation effect was achieved in the adapted bottom sludge environment, especially the degradation of pyrene in lakes and rivers. Good pollution recovery potential.
Patent Information
- Application Number
- CN202211022601.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-08-24
- Publication Date
- 2024-12-13
- Estimated Expiration
- 2042-08-24
AI Technical Summary
The prior art has poor degradation effect on polycyclic aromatic hydrocarbons in actual environments, especially in sediments in aquatic environments such as lakes and rivers, and lacks effective microbial repair measures.
Flavobacterium S3 strain was screened and identified. This strain came from the rhizosphere environment of bitter grass, adapted to the bottom sediment environment, and efficiently degrades polycyclic aromatic hydrocarbons, especially pyrene under specific culture methods and conditions, forming an effective degradation mechanism.
Flavobacterium S3 can degrade 1 mg/L of pyrene 90.26% in one week under specific conditions, showing efficient degradation ability in aquatic environments and has good application prospects for pollution repair.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioengineering and environmental restoration, and specifically relates to Flavobacterium S3 and application thereof. Background Art
[0002] PAHs are volatile hydrocarbons produced by incomplete combustion of coal, oil, wood, tobacco, etc. With the rapid development of industry, the concentration of PAHs in the environment has continued to increase, posing a major threat to the environment and food safety. PAHs in land and air enter rivers and lakes through various means such as surface runoff and atmospheric deposition, and are eventually deposited in the sediments. However, lake and river sediments, as the largest reservoir of PAHs, have received little attention, and most research is still focused on the surface.
[0003] There are various ways to treat PAHs, including physical methods such as heating, flocculation precipitation, adsorption, and chemical methods such as photooxidation and reagent oxidation. Considering economic cost and environmental friendliness, microbial degradation is still a very promising way to remove PAHs. At present, more than 100 genera of bacteria have been found to have the ability to degrade PAHs, including. However, most microbial sampling habitats are limited to farmland, factory surroundings, sewage treatment plants and other relatively special places with high concentrations of PAHs. Most of the screened bacteria are only effective in degrading PAHs in the laboratory, and the effect in the actual environment is not satisfactory. Aquatic environments such as lakes and rivers are closely related to human life, and few studies have focused on the degradation and bioremediation of PAHs in sediments in freshwater aquatic environments.
[0004] Studies have shown that Vallisneria, a common plant in southern lakes, has a good degradation effect on PAHs. The more similar the root community of submerged plants is to Vallisneria, the stronger the degradation effect on PAHs. In view of the increasing concentration of PAHs in lake sediments, finding microorganisms that are adapted to the environment of common lake sediments and have the ability to degrade PAHs has become an important part of bioremediation of sediments. Summary of the invention
[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a strain Xanthobacteraceae bacterium S3 that is adapted to the rhizosphere environment of the submerged plant Vallisneria and can degrade polycyclic aromatic hydrocarbons, and its application.
[0006] Technical solution: In order to solve the above technical problems, the present invention provides Xanthobacteraceae bacterium S3, which has been deposited in the China Center for Type Culture Collection (CCTCC) on May 27, 2022, with a deposit number of CCTCC NO: M 2022743, and a deposit address of: Wuhan University, Wuhan, China, Postal Code: 430072. The Xanthobacteraceae bacterium of the present invention is screened from the rhizosphere sediment of Vallisneria, and is a functional degrading bacteria that can adapt to the sediment environment and degrade polycyclic aromatic hydrocarbons.
[0007] The present invention also includes a method for culturing Xanthobacteraceae bacterium S3, wherein Xanthobacteraceae bacterium S3 is cultured in LB medium in a light-proof and shaking manner, with a pH value of 5 to 9, a culture temperature of 25 to 40° C., and a salinity of 0.1 to 5%.
[0008] Wherein, the pH is 7, the temperature is 37°C, and the salinity is 0.1-1%.
[0009] Wherein, the culture medium also includes pyrene solution.
[0010] The present invention also includes the use of the Xanthobacteraceae bacterium S3 in the degradation of polycyclic aromatic hydrocarbons.
[0011] Wherein, the polycyclic aromatic hydrocarbon is pyrene.
[0012] Wherein, the pyrene concentration is 1-200 mg / L.
[0013] The application conditions are: culture temperature of 25-40° C., pH of 5-7, and salinity of 0-1.5%.
[0014] The present invention also includes a method for degrading the pollutant pyrene, comprising the following steps:
[0015] 1) Cultivating Xanthobacteraceae bacterium S3 in LB medium until turbid, centrifuging in a sterile centrifuge tube, washing with basal salt medium, and diluting and mixing the prepared bacterial solution with basal salt medium;
[0016] 2) Take the pyrene solution in a sterilized test tube, after the acetone evaporates, add the basal salt culture medium, adjust the pH to 5-7 and the NaCl% to 0-1.5%, sterilize at high temperature, add it to the bacterial solution prepared in step 1), and culture it in the dark at 25-40° C. for 1-28 days.
[0017] Wherein, the basic salt culture medium includes disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium chloride, calcium tetrahydrate and trace elements.
[0018] The method determines the optimal pyrene degradation conditions of Xanthobacteraceae bacterium S3 by establishing a regression model, and the regression model is:
[0019] Y(%)=85.59-5.47A-6.47B-21.80C-4.60AB-4.85AC+7.52BC+0.2662A 2 -5.19B 2 -32.09C 2 .
[0020] Beneficial effects: Compared with existing strains, the advantages of the strains screened out this time are: Xanthobacteraceae bacterium S3 is screened from the roots of underwater Vallisneria, and can adapt to the underwater sediment environment while being able to efficiently degrade polycyclic aromatic hydrocarbons. Under the conditions of pH 7.0, salinity of 0, and temperature of 28.5°C, 1 mg / L of pyrene can be degraded by 90.26% within one week, and it has good application prospects in the remediation of polycyclic aromatic hydrocarbons pollution in sediments. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The growth curves of S3 strain under different salinity (a), pH (b) and temperature (c) conditions;
[0022] Figure 2 It is the response surface curve reflecting the interaction effect of various factors on the degradation rate of S3 strain. DETAILED DESCRIPTION
[0023] The present study is further described below through specific embodiments. It should be noted that a person skilled in the art can make several modifications and improvements without departing from the experimental principles.
[0024] Example 1 Screening process of Flavobacterium S3
[0025] 1. Culture medium:
[0026] A. LB medium (g / L): yeast extract 5, tryptone 10, NaCl 10, agar 15, pH = 7.0
[0027] B. Basic salt medium (g / L): disodium hydrogen phosphate 2.8, potassium dihydrogen phosphate 1.0, ammonium sulfate 0.5, magnesium chloride 0.053, calcium tetrahydrate 0.05, trace elements 1mL
[0028] C trace elements (g / L): EDTA 0.5, ferrous sulfate 0.2, zinc sulfate heptahydrate 0.01, manganese chloride tetrahydrate 0.003, boric acid 0.03, cobalt chloride hexahydrate 0.02, cupric chloride dihydrate 0.001, nickel chloride hexahydrate 0.002, sodium borate dihydrate 0.003
[0029] D. Screening medium (g / L): Add 20 g·L -1 Agar powder was used to make a plate, and then 8 mg / mL pyrene solution (dissolved in acetone) was evenly applied.
[0030] All culture media were prepared with deionized water, sterilized at 0.1 MPa, 121°C for 15 minutes, and cooled for later use.
[0031] 2. The initial screening method is as follows: take 10g of rhizosphere sediment collected from Vallisneria, put it into 100mL of basal salt culture medium containing 400mg / L pyrene, and culture it on a shaker for 20 days; after standing for 20 minutes, take 1ml of supernatant, add it to 9ml sterilized water, mix it evenly to make sediment bacterial suspension, then take 1ml of dilution and add it to different volumes of sterile water to make sediment solutions of different dilutions of 0.01, 0.001, 0.0001, 0.00001, 0.000001, take 100μL of each for coating or plate streaking, and cultivate it in the dark at 30℃ for 2-3 weeks. This method obtained 10 strains that can survive high concentrations of pyrene, of which 6 strains can form obvious plaques in the screening culture medium.
[0032] In order to further examine the ability of the strains to degrade the polycyclic aromatic hydrocarbon pyrene, the 6 strains that can degrade polycyclic aromatic hydrocarbons selected in the preliminary screening were inoculated into a basal salt culture medium containing pyrene as the only carbon source (5 mg / L pyrene), 6 bottles of each strain (10 mL bottle), 35°C, 180 rpm, and cultured for 14 days, with an interval of 7 days. Three bottles of each strain were taken to determine the residual concentration of pyrene in the culture medium. Finally, a strain of Xanthobacteraceae S3 with a strong ability to degrade pyrene was screened. The Xanthobacteraceae bacterium S3 was deposited in the China Center for Type Culture Collection (CCTCC) on May 27, 2022, with a preservation number of CCTCC NO: M2022743.
[0033] 3. Detection method of residual pyrene concentration: The experiment adopted a destructive collection method. An equal volume of dichloromethane was added to the culture medium, vortexed for 5 minutes, and allowed to stand for 20 minutes. The lower organic phase was taken, dried over anhydrous sodium sulfate, and then the pyrene concentration was determined by GC-MS. The GC-MS analysis parameters were: DB-5MS (30nm×0.25mm×0.25um), split ratio: 20:1, injection volume: 1.0μL, injection port temperature: 280℃, column flow rate: 10mL min-1, heating program: 90℃ for 2min, 20℃ min-1) to 180℃, maintained for 5min, 10℃ min-1 to 300℃ and maintained for 5min, EI ion source, ionization energy 70ev, ion source temperature: 230℃, quadrupole temperature: 150℃, solvent delay: 5.5min, mass number scanning range: 45~450u, SIM mode.
[0034] Example 2: Identification of biological properties of strain S3
[0035] Biological properties of strain S3: After 96 hours of growth on a solid culture medium plate, the colonies are round, about 1 mm in diameter, with a smooth, light yellow, opaque surface, flat edges, and a protrusion in the middle; when cultured on a slant surface, the colonies grow along the streaks with flat edges; when cultured in a shake flask, the culture fluid is yellow and opaque.
[0036] The NCBI comparison and identification result of the S3 strain is: Xanthobacteraceae bacterium S3, Proability: 100%. That is, the PCR product of the 16s rDNA sequence (shown in SEQ ID NO: 1) is 1332bp, and the accession number of the 16s rDNA sequence in GenBank is ON527741. The sequencing results were compared with the known sequences (homology>99%) through the NCBI BLAST database, and the results showed that the homology of the S3 strain and the Xanthobacteraceae can reach 100%. Combining the two identification results, the bacteria can be identified as a species and named as Xanthobacteraceae bacterium S3.
[0037] Example 3: Growth characteristics of S3 strain
[0038] In this experiment, the bacterial culture was at OD 600 The absorbance at the 40°C was used as the assessment standard to examine the optimal growth temperature, pH and salinity of the S3 strain. The Flavobacterium L1I3 strain was cultured in LB medium until turbid, and then centrifuged in a sterile centrifuge tube at 4°C, 10,000 rpm, for 5 minutes. It was then washed and centrifuged three times with basal salt medium, and diluted and mixed with basal salt medium to prepare OD 600The bacterial suspension was inoculated into LB medium containing 1% NaCl at a volume ratio of 10%, and a control group was set up, with 5 parallels in each group. The initial pH of the LB medium was set to 5, 6, 7, 8, and 9, respectively, and the culture medium was stirred at 175 rpm·min -1 , shake and culture in a 35℃ shaker in the dark, and continuously measure the OD of the bacterial solution 600 The value changes to explore the optimal growth pH of S3. According to the above steps, the bacterial suspension was inoculated in LB medium with a pH of 7, and the NaCl content in the LB medium was set to 0.1%, 0.5%, 1%, 2%, and 5% to explore the optimal growth salinity. The bacterial suspension was inoculated in LB medium with a pH of 7 and a NaCl content of 1%, and the shaking temperature was set to 25, 30, 35, and 40°C to explore the optimal growth temperature of the S3 strain.
[0039] The OD values of the bacterial solution at different temperatures, pH values and salinities were measured, and the growth curves of the S3 strain under different conditions were drawn. Figure 1 The growth curves of S3 strain under different salinity (a), pH (b) and temperature (c) conditions. Figure 1 The results showed that the optimal growth conditions for the S3 strain were pH 7, temperature 37°C, and salinity range 0.1-1%.
[0040] Example 4: This experiment illustrates the pyrene degradation characteristics of the S3 strain
[0041] This experiment mainly investigates the effects of degradation temperature, medium pH and medium salinity on the degradation efficiency of pyrene by S3 strain. The temperature range is set to 25-40℃, pH5-7, and salinity is expressed as NaCl percentage, with the NaCl% range set to 0-1.5. The Box-Behnken experimental design was performed using Design Expert 12 software, and the specific experiments are shown in Table 1.
[0042] The S3 strain was cultured in LB medium until turbid, centrifuged in a sterile centrifuge tube at 4°C, 10,000 rpm for 5 min, washed and centrifuged three times with basal salt medium, and diluted and mixed with basal salt medium to prepare OD 600 The bacterial solution has a value of 1.000.
[0043] Take 100 μL of 100 mg / L pyrene solution (prepared with acetone) in a sterilized test tube, and after the acetone evaporates, add 9 mL of basal salt medium (pH and NaCl% adjusted to the target values set in Table 1 and sterilized at 121°C for 15 min), and add the S3 experimental bacterial solution prepared previously. Cultivate in the dark for 4 days at each set temperature, the specific temperature is shown in Table 1. Each group of experiments is repeated three times.
[0044] Establish a regression model based on experimental results:
[0045] Y(%)=85.59-5.47A-6.47B-21.80C-4.60AB-4.85AC+7.52BC+0.2662A 2 -5.19B 2 -32.09C 2
[0046] From the results of the regression model variance analysis, we can see that the P value of the model is less than 0.0001, the regression equation is extremely significant, and the P value of the lack of fit term is 0.3566, which is not significant, indicating that the model has a good degree of fit. 2 The value is 0.993, which is a good correlation, indicating that the model fully optimizes the degradation parameters. According to the F value in Table 2, the strength of each influencing factor can be judged, which is C>B>A.
[0047] The response surface diagram drawn according to the regression equation is shown in Figure 2 ,The relationship between the influencing factors and the response surface can be intuitively seen in the figure, that the steeper the curve, the greater impact of the factor on the degradation efficiency of pyrene. Figure 2 The interaction of various factors on the degradation rate of the S3 strain shows that factor C (temperature) has the greatest impact on the response value, which is shown by a steep curve, followed by factor B (NaCl%) and factor A (pH). The curve of factor A is the smoothest, which is consistent with the results of variance analysis. Through this regression model, the optimal degradation conditions of Flavobacterium S3 are pH 7, NaCl% 0, and temperature 28.50. It is predicted that the theoretical degradation rate of pyrene by the S3 strain under this experimental condition can reach 95.37%. In order to verify the accuracy of the model prediction, the above optimal degradation conditions were used, and the actual degradation rate of pyrene obtained by three parallel experiments was 90.26%, which is relatively close, indicating that the model is a good optimization of the conditions for S3 to degrade pyrene.
[0048] Table 1 Box-Behnken experimental design and degradation rate results
[0049]
[0050]
[0051] Table 2
[0052]
[0053]
[0054] Example 5: Application of S3 strain to degrade different pyrene concentrations
[0055] This experiment uses the pyrene degradation rate of the S3 strain as the assessment standard to investigate the degradation efficiency of the S3 strain on different concentrations of pyrene. The experimental degradation conditions are set at pH 7, NaCl% 0, temperature 28.50, pyrene concentration gradient is set at 1, 5, 100, 200 mg / L, the detection interval is 7 days, and the specific experimental operation is the same as Example 4. The experimental results are shown in Table 3.
[0056] Table 3
[0057]
[0058] The results show that when the pyrene concentration is 1 mg / L, the pyrene degradation rate can reach 98.29% on the 7th day of culture. When the pyrene concentration is 5 mg / L, the degradation rate can reach 91.42% after 14 days of culture. When the pyrene concentration in the culture system is increased to 100 mg / L, the degradation rate reaches 92.04% after 21 days of culture. When the pyrene concentration is increased to 200 mg / L, the pyrene degradation rate reaches 99.54% after 28 days.
[0059] The results showed that the S3 strain could effectively degrade pyrene in the system at both low concentrations of 1 mg / L and high concentrations of 200 mg / L.
Claims
1. Flavobacterium Xanthobacteraceae bacteria ) S3, characterized in that, The Flavobacterium Xanthobacteraceae bacteria )S3 was deposited in the China Center for Type Culture Collection (CCTCC) on May 27, 2022, with the deposit number CCTCC NO: M 2022743.
2. The Flavobacterium according to claim 1 ( Xanthobacteraceae bacteria ) The culture method of S3, characterized in that, The following steps are involved: Flavobacterium ( Xanthobacteraceae bacteria ) S3 was cultured in LB medium with shaking in the dark, pH 5-9, culture temperature 25-40℃, and salinity 0.1-5%.
3. The culture method according to claim 2, characterized in that The culture medium also includes a pyrene solution.
4. The Flavobacterium according to claim 1 ( Xanthobacteraceae bacteria ) Application of S3 in the degradation of polycyclic aromatic hydrocarbons, wherein the polycyclic aromatic hydrocarbons are pyrene.
5. The use according to claim 4, characterized in that: The pyrene concentration is 1-200 mg / L.
6. The use according to claim 4, characterized in that: The application conditions are: culture temperature of 25-40°C, pH of 5-7, and salinity of 0-1.5%.
7. A method for degrading the pollutant pyrene, characterized in that: The following steps are involved: 1) The Flavobacterium ( Xanthobacteraceae bacteria ) S3 was cultured in LB medium until turbid, centrifuged in a sterile centrifuge tube, washed with basal salt medium, and diluted and mixed with basal salt medium to prepare the bacterial solution; 2) Place the pyrene solution in a sterilized test tube. After the acetone evaporates, add the basal salt culture medium, adjust the pH to 5-7 and the NaCl to 0-1.5%. After high-temperature sterilization, add it to the bacterial solution prepared in step 1) and culture it in the dark at 25-40°C for 1-28 days.
8. The method for degrading the pollutant pyrene according to claim 7, characterized in that: The basal salt culture medium includes disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium chloride, calcium chloride and trace elements, and the trace elements include EDTA, ferrous sulfate, manganese chloride, zinc sulfate, manganese chloride, boric acid, cobalt chloride, cupric chloride, nickel chloride or sodium borate.
9. The method for degrading the pollutant pyrene according to claim 7, characterized in that: The method determines the presence of Flavobacterium by establishing a regression model. Xanthobacteraceae bacteria ) The optimal pyrene degradation condition of S3, the regression model is: 。