Application of Siamese Bacillus L5 in Phenol Degradation

Abstract

The present invention discloses the application of Siamese Bacillus L5 in phenol degradation; the bacterium has been deposited in China Center for Type Culture Collection on November 1, 2019, with the deposit number of CCTCC M 2019885. Strain L5 can efficiently degrade phenolic compounds such as phenol, catechol, resorcinol, 3,5-dimethylphenol, 2-methoxyphenol and 2,6-dimethoxyphenol. The degradation bacterial agent prepared by mixing the fermentation liquid of strain L5 with a porous glass carrier can further improve the degradation rate, and the strain can be applied to the degradation of phenolic compounds in the environment by direct addition. The preparation method of the bacterial agent is simple, convenient and efficient to use, and can safely and quickly degrade the phenol remaining in water and soil, and has a good application prospect in the pollution control of phenolic compounds such as phenol.

Description

Technical Field

[0001] The invention belongs to the field of environmental microorganisms, and particularly relates to the application of siam bacillus in phenol degradation. Background Art

[0002] Pyrethroid pesticides are a class of broad-spectrum insecticides containing multiple benzene ring structures that are synthesized by simulating natural pyrethrins. Due to their high efficiency, low toxicity, broad spectrum and safety, they have been widely used as substitutes for organochlorine and organophosphorus pesticides to control agricultural pests and sanitary pests. According to literature reports, some pyrethroid pesticides will be accompanied by the production of phenol as an intermediate product during the microbial degradation process. Phenol is a major pollutant in industrial wastewater such as oil refining, coking, and papermaking. It has been listed in the list of 65 priority pollutants by the US Environmental Protection Agency, and its potential harm to the ecological environment is greater than that of pyrethroid pesticides. 1 g of phenol is enough to be fatal. Short-term exposure to phenolic compounds usually causes immediate skin blisters, respiratory problems and eye burns. Long-term exposure may cause lung problems, immune system damage and cancer. Many international environmental regulatory agencies have set strict emission limits for phenol and its derivatives. The US Agency for Toxic Substances and Disease Registry stipulates that the detection limit of phenol in surface water is 1 ppb.

[0003] Patent document ZL201911175618.0 discloses a strain of Bacillus siamensis that can efficiently degrade high-efficiency cypermethrin ( Bacillus siamensis )L5, which was deposited in the China Center for Type Culture Collection (CCTCC M 2019885) on November 1, 2019, but it was not involved in the degradation of phenolic compounds such as phenol. Summary of the invention

[0004] The purpose of the present invention is to provide application of siam bacillus L5 in degradation of phenolic compounds.

[0005] The technical solution adopted by the present invention is:

[0006] In a first aspect of the present invention, a method for degrading phenolic compounds using Siamese Bacillus subtilis L5 is provided. Bacillus siamensis ) was deposited in the China Center for Type Culture Collection on November 1, 2019, with the accession number CCTCC M 2019885.

[0007] In some embodiments of the present invention, the phenolic compound comprises at least one of phenol, catechol, resorcinol, 3,5-dimethylphenol, 2-methoxyphenol and 2,6-dimethoxyphenol.

[0008] In some preferred embodiments of the present invention, the phenolic compound is phenol.

[0009] In a second aspect of the present invention, there is provided a use of Siamese Bacillus L5 in preparing a product for degrading phenolic compounds, wherein the Siamese Bacillus L5 ( Bacillus siamensis ) was deposited in the China Center for Type Culture Collection on November 1, 2019, with the accession number CCTCC M 2019885.

[0010] In some embodiments of the present invention, the phenolic compound comprises at least one of phenol, catechol, resorcinol, 3,5-dimethylphenol, 2-methoxyphenol and 2,6-dimethoxyphenol.

[0011] In some preferred embodiments of the present invention, the phenolic compound is phenol.

[0012] The third aspect of the present invention provides a bacterial agent, comprising the siam Bacillus L5 described in the first aspect of the present invention and a carrier and / or an auxiliary material.

[0013] In some embodiments of the present invention, the carrier is porous glass.

[0014] In some embodiments of the present invention, the mass concentration of the carrier is 5-15%.

[0015] The fourth aspect of the present invention provides a method for preparing the bacterial agent described in the third aspect of the present invention, comprising the following steps: inoculating the Siamese Bacillus L5 described in the first aspect of the present invention into a culture medium with phenolic compounds as the sole carbon source for activation, expanding the culture, and uniformly mixing the obtained fermentation solution with a carrier to obtain a phenol-degrading bacterial agent.

[0016] In some embodiments of the present invention, the activation conditions are: shaking culture at 29-33° C. and 100-200 rpm for 20-28 h for activation.

[0017] In some embodiments of the present invention, the carrier is porous glass.

[0018] In some embodiments of the present invention, the mass concentration of the carrier is 5-15%.

[0019] In some embodiments of the present invention, the phenolic compound comprises at least one of phenol, catechol, resorcinol, 3,5-dimethylphenol, 2-methoxyphenol and 2,6-dimethoxyphenol.

[0020] In some preferred embodiments of the present invention, the phenolic compound is phenol.

[0021] A fifth aspect of the present invention provides a method for degrading phenolic compounds, using the siam Bacillus L5 described in the first aspect of the present invention or the bacterial agent described in the third aspect of the present invention to degrade phenolic compounds.

[0022] In some embodiments of the present invention, it can be used to degrade phenolic compounds in water and soil environments.

[0023] In some embodiments of the present invention, the degradation conditions are: 15-45°C, pH 4.0-10.0.

[0024] In some embodiments of the present invention, the degradation conditions are: 20-45°C, pH 5-9.

[0025] In some embodiments of the present invention, the degradation conditions are: 25-40°C, pH 6-8.

[0026] In some embodiments of the present invention, the inoculation amount of the siam Bacillus L5 is 0.5-10%.

[0027] In some preferred embodiments of the present invention, the inoculation amount of the siam Bacillus L5 is 1-8%.

[0028] In some preferred embodiments of the present invention, the inoculation amount of Bacillus siamese L5 is 2-6%.

[0029] In some embodiments of the present invention, the phenolic compound comprises at least one of phenol, catechol, resorcinol, 3,5-dimethylphenol, 2-methoxyphenol and 2,6-dimethoxyphenol.

[0030] The beneficial effects of the present invention are:

[0031] The invention finds that Siamese Bacillus L5 has a good degradation effect on phenolic compounds, and can be used for the degradation of phenolic compounds such as phenol, catechol, resorcinol, 3,5-dimethylphenol, 2-methoxyphenol and 2,6-dimethoxyphenol, or the preparation of a degradation bacterial agent; and the strain can be applied to the degradation of phenolic compounds in the environment by direct addition, and the preparation method of the bacterial agent is simple, convenient and efficient to use, and the residual phenol in water bodies and soil can be safely and quickly degraded, and has a good application prospect in the pollution control of phenolic compounds such as phenol. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The relationship between the growth and phenol degradation of strain L5.

[0033] Figure 2 Effect of inoculum size on phenol degradation by strain L5.

[0034] Figure 3 Effect of temperature on phenol degradation by strain L5.

[0035] Figure 4 Effect of pH on phenol degradation by strain L5.

[0036] Figure 5 Degradation of phenolic compounds by strain L5.

[0037] Figure 6 Degradation kinetics of phenol by degrading bacteria. DETAILED DESCRIPTION

[0038] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0039] Preparation of culture medium

[0040] Basal salt culture medium: (NH4)2SO42.0 g, MgSO4•7H2O 0.2 g, CaCl2•2H2O 0.01 g, FeSO4•7H2O 0.001 g, Na2HPO4•12H2O 1.5 g, KH2PO41.5 g, add distilled water to make up to 1 L, pH 7.0, sterilize with high pressure steam and set aside.

[0041] LB medium: 10 g peptone, 5 g yeast extract, 10 g sodium chloride, add distilled water to 1 L, pH 7.0, add 15 g / L agar powder when preparing solid culture medium, sterilize with high pressure steam and set aside.

[0042] Example 1 Relationship between growth and phenol degradation of strain L5

[0043] A single colony of strain L5 was picked and pre-cultured in LB liquid medium for 12 h. The resulting bacterial solution was centrifuged at 4000 r / min for 2 min, the supernatant was discarded, and the bacteria were rinsed with 50 mL of 0.9% sterile saline and resuspended as seed suspension. During degradation determination, 2 mL of the above bacterial solution was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of sterile MSM medium, and phenol mother solution was added to make the final mass concentration 100 mg / L. It was cultured at 30 °C, pH 7.0, and 150 r / min for 7 days. Samples were taken every day, and the phenol residue was determined by gas chromatography. The phenol degradation curve and bacterial growth curve were drawn. The bacterial growth was measured by the absorbance value OD at 600 nm. 600The control was non-inoculated, and each treatment was repeated 3 times.

[0044] Pour the above 50 mL culture medium into a 250 mL separatory funnel, add 3 g of sodium chloride, shake to dissolve, add 30 mL of a 1:1 volume ratio of dichloromethane / ethyl acetate mixed solvent, shake, release gas, shake and extract for 5-10 min, let stand for more than 10 min until the organic phase and the aqueous phase are fully separated, and collect the organic phase. Repeat the extraction 1-2 times and combine the organic phases. Dehydrate the organic phase with anhydrous sodium sulfate, and wash the anhydrous sodium sulfate with an appropriate amount of dichloromethane / ethyl acetate mixed solvent, collect the organic phase extract, condense to dryness by rotary evaporation at 45 ° C, add 2.0 mL of dichloromethane / ethyl acetate mixed solvent, and then concentrate to 1.0 mL for testing. Gas chromatography program temperature: 100 ℃ for 3 min, then increase to 250 ℃ at 15 ℃ / min and maintain for 1 min; injection port temperature: 260 ℃; FID detector temperature: 280 ℃; carrier gas flow rate: 1.5 mL / min; hydrogen flow rate: 30.0 mL / min; air flow rate: 300.0 mL / min; tail gas flow rate: 35.0 mL / min; injection mode: splitless injection; injection volume: 1.0 μL.

[0045] The results are as follows Figure 1 As shown in the figure, strain L5 can use phenol as a growth substance and can quickly degrade phenol, and the degradation of phenol is positively correlated with the growth of the strain. In the basic salt culture medium containing phenol, strain L5 quickly entered the logarithmic growth phase, and 2-3 days was the logarithmic growth phase of the strain, during which the degradation rate of phenol by strain L5 was the fastest; when the growth of strain L5 reached the stable period on the 4th to 5th day, the degradation curve of phenol also tended to be flat; after 6 days of culture, the strain entered the decline phase, and by the 7th day, the degradation rate of phenol reached 91.2%, while in the control group without inoculation, the degradation rate of phenol was 8.5% on the 7th day.

[0046] Example 2 Effect of inoculum size of strain L5 on phenol degradation

[0047] To the basal salt medium with a phenol concentration of 100 mg / L, the bacterial suspension was added with a volume ratio of 0.5%, 1%, 2%, 4%, 6%, 8%, and 10%, respectively. The culture was shaken at 30 ℃ and pH 7.0 at 150 r / min, and the phenol concentration was detected by gas phase on the 7th day.

[0048] The results are as follows Figure 2As shown in the figure, strain L5 showed good phenol degradation effect when the inoculation amount was in the range of 0.5-10%, among which 2% was the optimal inoculation amount, under which the phenol degradation rate was 90.5%. When the inoculation amount was 0.5-2%, the phenol degradation rate increased significantly with the increase of inoculation amount, reaching 58.3%, 75.7% and 90.5% respectively; in the range of 2-6%, the phenol degradation rate decreased slightly with the increase of inoculation amount; when the inoculation amount continued to increase to 10%, the phenol degradation rate decreased significantly to 61.6%.

[0049] Example 3 Effect of temperature on phenol degradation

[0050] After selecting the optimal inoculum size of the strain, the strain was added to a basal salt medium with a phenol concentration of 100 mg / L and cultured at 15, 20, 25, 30, 35, 40 and 45°C, pH 7.0, and 150 r / min shaking. The phenol concentration on the 7th day was detected by gas phase.

[0051] The degradation effect of strain L5 on phenol in the range of 15-45℃ is shown in Figure 3 , the optimum temperature is 30℃. When the culture temperature is 15 and 20℃, the phenol degradation rate is 37.4% and 49.7%; when the temperature rises to 25-35℃, the phenol degradation rate increases significantly, all above 85%, and the highest degradation rate reaches 92.5% at 30℃; when the temperature continues to rise to 40 and 45℃, the phenol degradation rate decreases significantly, to 68.2% and 53.8%.

[0052] Example 4 Effect of pH on Phenol Degradation

[0053] After selecting the optimal inoculum size and culture temperature of the strain, the pH of the basal salt culture medium was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, and the strains were added to the above basal salt culture medium with a phenol concentration of 100 mg / L, respectively, and cultured at 30 ℃ and 150r / min with shaking, and the phenol concentration on the 7th day was detected by gas phase.

[0054] pH can affect the dissociation of related groups and substrates on the active site of microbial degradation enzyme molecules. Both excessive acidity and alkali can denature and inactivate the enzyme. The degradation effect of strain L5 on phenol under different pH conditions is shown in Figure 4 , the optimum pH was 7.0, and the phenol degradation rate was 91.2%. When the pH was 4.0-6.0, the phenol degradation rate increased with the increase of pH value, reaching 47.4%, 59.5% and 88.7% respectively; at pH 6.0-8.0, the phenol degradation rate was above 88%; when the pH continued to rise to 9.0 and 10.0, the phenol degradation rate decreased significantly, reaching 62.1% and 44.6%. The strain L5 tolerated a wide pH range, and the phenol degradation rate was above 50% in the range of 5.0-9.0.

[0055] Example 5 Degradation of phenolic compounds by strain L5

[0056] The strain L5 was inoculated at a 2% inoculum size into a basal salt medium with a pH of 7.0 and a final concentration of 100 mg / L of catechol, resorcinol, 3,5-dimethylphenol, 2-methoxyphenol and 2,6-dimethoxyphenol. The culture was shaken at 30 ℃ and 150 rpm for 7 days, and the degradation rate of phenolic compounds by strain L5 was detected by gas phase.

[0057] like Figure 5 As shown in the figure, strain L5 has a wide degradation spectrum. After 7 days of cultivation, it showed good degradation effects on catechol, resorcinol, 3,5-dimethylphenol, 2-methoxyphenol and 2,6-dimethoxyphenol, with degradation rates of 88.2%, 84.9%, 73.4%, 69.5% and 50.3%, respectively.

[0058] Example 6 Preparation of the degradation bacterial agent and its degradation kinetics for phenol

[0059] (1) Preparation of carrier porous glass: Shell powder and glass powder (the chemical compositions of shell powder and glass powder obtained by XRF test are shown in Table 1) were sieved through a 200-mesh sieve and dried in a vacuum drying oven at 80 °C for 5 hours for use. Weigh an appropriate amount of shell powder and glass powder (mass percentage is 15:85), add an appropriate amount of water, mix well, press into shape manually with a metal film mold, and calcine in a muffle furnace. The heating program is: hold at 600 °C for 5 min, then increase to 850 °C at 25 °C / min, hold for 5 min, and finally increase to 950 °C at 20 °C / min and hold for 10 min. The prepared sample is placed in 10 °C cold water to obtain porous glass.

[0060] Table 1 Chemical composition of shell powder and glass powder

[0061]

[0062] (2) Preparation of degradation agent: Take an appropriate amount of strain L5 and inoculate it into a liquid basal salt medium containing 100 mg / L phenol as the sole carbon source. According to the optimized conditions, shake and culture at 150 rpm for 24 h. Inoculate 10% by volume into LB liquid medium with pH 7.0 and shake and culture at 30°C and 150 rpm for 24 h to activate it. Mix the obtained 50 mL bacterial solution with 5 g sterilized porous glass and shake in a shaker for 2 h to obtain the bacterial agent.

[0063] (3) Degradation kinetics of phenol by degradation bacteria

[0064] The initial concentration of phenol in the MSM liquid medium was set to 100 mg / L, 30 ℃, 150 r / min for 7 days, and samples were taken every day. A total of 4 treatments were set, including no inoculation, bacterial solution, foam glass, and bacterial agent, with 3 replicates for each treatment. The first-order degradation kinetic model was used to calculate the phenol residues ( C ) and time( t ) to perform the fitting.

[0065] The first-order degradation kinetic model is:

[0066]

[0067] In the formula, C t for t Residual amount of phenol at the moment (mg / L); C 0 is the initial concentration of phenol (mg / L); k is the degradation rate constant (d -1 ).

[0068] Degradation kinetics of phenol by degradation bacteria Figure 6 As shown in the figure, with the extension of degradation time, the degradation rate of phenol by bacterial solution and bacterial agent increases, but the degradation effect of bacterial agent is better than that of bacterial solution, indicating that the prepared foamed glass is conducive to the fixation and growth of microorganisms and can improve the degradation effect of strain L5 on phenol; the degradation rate in the treatment without bacteria and foamed glass is only about 10%. The degradation process conforms to the first-order kinetic equation, and the correlation coefficient R of the fitting equation of the four treatments without bacteria, foamed glass, bacterial solution and bacterial agent is 2 They were 0.9368, 0.9923, 0.9786 and 0.9824 respectively, and the degradation half-lives were 53.32, 49.51, 1.76 and 1.39 days respectively.

[0069] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. The application of Siamese Bacillus L5 in the degradation of phenolic compounds is characterized in that: The siam bacillus ( Bacillus siamensis )L5 was deposited in China Center for Type Culture Collection on November 1, 2019, with the accession number CCTCC M 2019885; The phenolic compound is at least one of phenol, catechol, resorcinol, 3,5-dimethylphenol, 2-methoxyphenol and 2,6-dimethoxyphenol.

2. The use of Siamese Bacillus L5 in preparing a product for degrading phenolic compounds, characterized in that: The siam bacillus ( Bacillus siamensis )L5 was deposited in China Center for Type Culture Collection on November 1, 2019, with the accession number CCTCC M 2019885; The phenolic compound is at least one of phenol, catechol, resorcinol, 3,5-dimethylphenol, 2-methoxyphenol and 2,6-dimethoxyphenol.

3. A method for degrading phenolic compounds, using the siam bacillus L5 described in claim 1 to degrade phenolic compounds; The phenolic compound is at least one of phenol, catechol, resorcinol, 3,5-dimethylphenol, 2-methoxyphenol and 2,6-dimethoxyphenol.

4. The method according to claim 3, characterized in that The degradation temperature is 15-45°C.

5. The method according to claim 3, characterized in that: The pH of the degradation is 4.0-10.

0.

6. The method according to claim 3, characterized in that The inoculation amount of the Siamese Bacillus L5 is 0.5-10%.

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