High-efficiency phenol degrading bacterium with aerobic denitrification characteristic and application of high-efficiency phenol degrading bacterium

Abstract

The invention relates to a high-efficiency phenol degrading bacterium with an aerobic denitrification characteristic and application thereof, and belongs to the technical field of coking wastewater treatment and microorganisms. The strain is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M20242868, and the preservation time is December 20, 2024. The strain can grow and breed by taking phenol as a unique carbon source, complete degradation of 1250mg / L phenol is realized, and phenol with the concentration range of 1000mg / L can be rapidly degraded within 36 hours. The strain PD-1 can also realize efficient and synchronous removal of phenol and a nitrogen source under the condition that NH4 < + >-N, NO2 <->-N or NO3 <->-N is used as the only nitrogen source. The strain PD-1 provided by the invention can quickly degrade high-concentration phenol in a water body, has the advantages of high efficiency, low energy consumption and no secondary pollution, and has important theoretical and application values.

Description

Technical Field

[0001] The present invention relates to the fields of coking wastewater treatment and microbial technology, and more specifically, to a phenol-degrading bacterium with aerobic denitrification characteristics and its application. Background Art

[0002] As an important chemical raw material and product, phenol is commonly found in the wastewater generated by various industrial processes such as coal chemical industry, petrochemical industry, and pharmaceutical production. Due to the strong carcinogenicity and bioaccumulation of phenol, discharging it without treatment will pose a serious threat to the water ecosystem and human health. The US Environmental Protection Agency (EPA) and the Ministry of Ecology and Environment of China have both included phenol in the list of priority pollutants. Relevant standards in China stipulate that the limits of phenol in surface water and domestic drinking water are 0.1 and 0.002 mg / L respectively.

[0003] Currently, the methods for removing phenol mainly include physical methods, chemical methods, and biological methods. Due to the problems of high instrument costs, high treatment costs, and secondary pollution in traditional physical and chemical methods, the biological method with low cost, high efficiency, and no secondary pollution has become the best choice for removing phenol. More than 200 phenol-degrading strains have been reported so far, among which bacteria account for about 75%, mainly including Pseudomonas, Bacillus, Alcaligenes, and Rhodococcus. Due to the toxic effect of high-concentration phenol on bacteria, most of the degrading bacteria have problems such as a long adaptation time and a low degradation rate. At the same time, industrial wastewater is often accompanied by compound nitrogen pollution such as ammonia nitrogen, nitrite, and nitrate, and most bacteria are difficult to adapt to complex water quality conditions. Therefore, screening out efficient degrading multi-functional strains that can simultaneously remove nitrogen sources and phenol will greatly improve the biological treatment efficiency of phenol wastewater and is of great significance for enriching the microbial resource library and improving the synchronous removal efficiency of pollutants. Summary of the Invention

[0004] The primary object of the present invention is to provide a phenol-degrading bacterium aiming at the problems of a long adaptation time and a low degradation rate in the existing phenol degradation technology, and to achieve the synchronous removal of phenol and nitrogen source, providing a resource of highly efficient degrading strains for the biodegradation of phenol, which has important economic value and practical significance for environmental pollution control.

[0005] According to the first aspect of the present invention, there is provided a phenol-degrading bacterium with aerobic denitrification characteristics, and the phenol-degrading bacterium is Pseudomonas monteilii with the preservation number of CCTCC NO: M20242868.

[0006] According to another aspect of the present invention, there is provided the application of the phenol-degrading bacterium with aerobic denitrification in degrading phenol.

[0007] Preferably, the application is specifically as follows: inoculating the phenol-degrading bacterium into a liquid containing phenol, and then performing shaking culture.

[0008] Preferably, the concentration of phenol in the liquid is less than or equal to 1250 mg / L.

[0009] Preferably, the pH of the liquid is 7 - 9.

[0010] Preferably, the rotation speed of the shaking culture is 100 rpm - 180 rpm.

[0011] Preferably, the liquid contains a nitrogen source, and the degrading bacterium can achieve synchronous removal of phenol and the nitrogen source.

[0012] Preferably, the nitrogen source is ammonia nitrogen NH4 + , nitrite nitrogen NO2 - and nitrate nitrogen NO3 - and at least one of them.

[0013] A phenol-degrading bacterium of the present invention is strain PD-1, which is identified as Pseudomonas monteilii. The strain was deposited at the China Center for Type Culture Collection on December 20, 2024. The address is: Wuhan University, Wuhan, China, and the deposit number is: CCTCC NO: M20242868, and the classification name is: Pseudomonas monteilii PD-1. Strain PD-1 can grow and reproduce with phenol as the sole carbon source within the range where the phenol concentration is less than or equal to 1250 mg / L, achieving complete degradation of phenol, and can rapidly and completely degrade 1000 mg / L of phenol within 36 hours. In addition, strain PD-1 can also efficiently synchronously remove phenol and the nitrogen source when using NH4 + -N, NO2 - -N or NO3 - -N as the sole nitrogen source.

[0014] Generally speaking, compared with the prior art through the above technical solutions conceived by the present invention, the following technical advantages are mainly possessed:

[0015] (1) The phenol-degrading bacterium Pseudomonas monteilii PD-1 provided by the present invention can grow and reproduce with phenol as the sole carbon source within the range where the phenol concentration is within 1250 mg / L, and the removal rate of phenol reaches more than 99%.

[0016] (2) The phenol-degrading bacterium Pseudomonas monteilii PD-1 provided by the present invention can use NH4 + -N, NO2 - -N or NO3- Synchronous removal of phenol and nitrogen source is achieved under the condition that -N is the sole nitrogen source.

[0017] (3) The phenol-degrading bacterium Pseudomonas monteilii PD-1 provided by the present invention has the advantages of high efficiency, no need for additional carbon source, no secondary pollution and low cost when applied to biodegradation of phenol-containing wastewater, and has good application prospects. Description of the Drawings

[0018] Figure 1 It is a colony morphology diagram of the strain Pseudomonas monteilii PD-1 of the present invention on an LB solid medium.

[0019] Figure 2 It is a scanning electron micrograph of the cell morphology of the strain Pseudomonas monteilii PD-1 of the present invention in an LB medium (a) and an inorganic salt medium containing phenol (b).

[0020] Figure 3 It is a schematic phylogenetic tree constructed after 16S rRNA sequence analysis and comparison of the strain Pseudomonas monteilii PD-1 of the present invention.

[0021] Figure 4 It is a growth curve diagram of the strain Pseudomonas monteilii PD-1 of the present invention in an LB liquid medium.

[0022] Figure 5 It is the OD 600 value and the change curve diagram of phenol concentration over time of the strain Pseudomonas monteilii PD-1 of the present invention in an inorganic salt medium containing 1000 mg / L phenol.

[0023] Figure 6 It is a data diagram of the phenol removal rate (a) and degradation rate (b) of the strain Pseudomonas monteilii PD-1 of the present invention in inorganic salt media containing different phenol concentrations.

[0024] Figure 7 It is a data diagram of the change in phenol concentration (a) and degradation rate (b) of the strain Pseudomonas monteilii PD-1 of the present invention at different pH values.

[0025] Figure 8 It is a data diagram of the change in phenol concentration (a) and degradation rate (b) of the strain Pseudomonas monteilii PD-1 of the present invention at different rotation speeds.

[0026] Figure 9 It is a data graph showing the changes in phenol and nitrogen source concentrations of the strain Pseudomonas monteilii PD-1 of the present invention under different nitrogen sources. Detailed implementation manners

[0027] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0028] The screening method of the phenol-degrading bacteria of the present invention includes the following steps:

[0029] (1) Pretreat the activated sludge taken from the first-stage aerobic tank of a coking wastewater treatment plant in Wuhan, and then inoculate it into a laboratory-scale sequencing batch reactor (SBR). Gradually increase the influent concentration of the next day according to the effluent concentration of the reactor every day, so as to domesticate and enrich the phenol-degrading functional bacteria in the activated sludge;

[0030] (2) After the effluent is stable, take the sludge in the reactor, and use the dilution plate method to spread the sludge on the LB solid medium, and culture it at 30 °C until single colonies grow. Pick single colonies into fresh LB solid medium and repeat streaking multiple times until purified single bacteria are obtained;

[0031] (3) Inoculate the screened strain into an inorganic salt liquid medium containing phenol, measure the phenol content after shaking culture for a certain period of time, evaluate its phenol degradation ability, and finally screen out the strain PD-1 with the strongest degradation performance.

[0032] The following are specific embodiments

[0033] Example 1 Isolation and identification of strains

[0034] (1) Sample collection and pretreatment

[0035] Collect activated sludge from the first-stage aerobic tank of a coking wastewater treatment plant in Wuhan, and perform anaerobic aeration treatment for 2 days to allow the activated sludge to fully absorb oxygen and wake up the microorganisms therein, and deplete the remaining available nutrient sources in the sludge.

[0036] (2) Domestication and isolation of strains

[0037] A laboratory-scale sequencing batch reactor (SBR) with an effective volume of 1 L was set up using synthetic wastewater at room temperature. The automatic influent / effluent system was controlled by a peristaltic pump and a flow valve, aeration was provided by an air pump, and a mechanical stirrer was used to mix the liquid in the SBR reactor. The initial mixed liquor suspended solids concentration (MLSS) was approximately 4000 mg / L. The working process was as follows: influent for 5 min, continuous stirring and aeration for 23 h, sedimentation for 50 min, and drainage for 5 min. The daily liquid exchange volume of the SBR reactor was 500 mL / d. The phenol concentration in the influent was determined by the remaining phenol concentration in the effluent of the previous day. The initial phenol concentration was 50 mg / L. The method of precise aeration combined with a gradually increasing phenol concentration was used to gradually domesticate and enrich the phenol-degrading bacteria in the activated sludge.

[0038] The domesticated sludge was taken from the SBR reactor, and the sludge was spread on LB solid medium by the dilution plate method and cultured at 30 °C until single colonies grew. Single colonies were picked and streaked repeatedly on fresh LB solid medium until purified single bacteria were obtained.

[0039] Furthermore, the selected single strain was inoculated into an inorganic salt medium containing phenol for cultivation, and its phenol degradation rate was measured to determine its phenol degradation ability. Finally, the strain with the strongest phenol degradation ability was screened out and named PD-1. The single strain with confirmed phenol degradation characteristics was stored in a cell cryopreservation solution and reserved at -80 °C.

[0040] Composition of synthetic wastewater: MgSO4 0.12 g / L, KH2PO4 0.4 g / L, NaCl 0.5 g / L, NaHCO3 0.2 g / L, FeSO4·7H2O 0.02 g / L, NH4Cl 0.5 g / L, phenol 500 mg / L, trace element solution (1.5 g / L FeCl2·4H2O, 190 mg / L CoCl2·6H2O, 100 mg / L MnCl2·6H2O, 70 mg / L ZnCl2, 62 mg / L H3BO3, 36 mg / L Na2MoO4·2H2O, 24 mg / L NiCl2·6H2O, 17 mg / L CuCl2·2H2O) 1 mL / 1000 mL. The final pH was controlled at 7.

[0041] Composition of LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L, and the pH was adjusted to 7 with 1 mol / L NaOH solution.

[0042] (3) Morphological characteristics of the strain

[0043] Single colony morphology of the strain obtained by separation and purification in the logarithmic growth phase with stable colony size was described. The results showed that the degrading bacteria isolated and purified in the above steps were Gram-negative, grew well on LB medium, with yellow opaque, round colonies, no serrated edges, uneven surfaces, and were easily picked up( Figure 1 ). The results of scanning electron microscopy are shown in Figure 2 . The strain grew well in LB medium and inorganic salt medium (phenol concentration was 1000 mg / L), and the cells were rod-shaped.

[0044] (4) Physiological and biochemical characteristics

[0045] The physiological and biochemical characteristics of the degrading bacteria were determined with reference to "Manual for Systematic Identification of Common Bacteria" (Dong Xiuzhu, Cai Miaoying. Manual for Systematic Identification of Common Bacteria. Beijing: Science Press, 2011). The specific results of physiological and biochemical determination are shown in the following table.

[0046] Table 1 Physiological and biochemical characteristics of phenol-degrading bacterium Pseudomonas monteilii PD-1

[0047] Test Items Results Oxidase + Glucose - Maltose - Sucrose - Xylose - Hydrogen Sulfide - Nitrate Reduction + Methyl Red (M.R.) - V.P. -

[0048] +: Positive result; -: Negative result

[0049] (5) 16S rRNA identification

[0050] Universal primers for 16S rRNA bacteria (27F 5’-AGAGTTTGATCCTGGCTCA-3’ and 1492R 5’AGAGTTTGATCCTGGCTCA-3’) were used for amplification, and the amplification results were sent to a sequencing company for sequencing. After sequencing, a gene sequence with a length of about 1400 bp was obtained. This sequence was submitted to NCBI (National Center for Biotechnology Information (nih.gov)) for BLAST alignment to obtain related sequences with high homology, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA11 software( Figure 3 ).

[0051] Based on the comprehensive results of cell morphology, physiological and biochemical characteristics, and 16S rRNA gene sequence, the strain PD-1 was identified as Pseudomonas monteilii.

[0052] Example 2 Growth and degradation curves of the strain

[0053] The optical density method was used to measure the absorbance value of the bacterial solution at a wavelength of 600 nm to determine the cell concentration. The selected phenol-degrading bacterium PD-1 was inoculated into 100 mL of LB medium and inorganic salt medium (containing 1000 mg / L phenol) at an inoculation amount of 5%, and then cultured in a shaker at 30 °C and 150 rpm. During this period, the phenol concentration and the OD 600 value of the bacterial solution were measured regularly to study the growth of the strain in LB and inorganic salt media.

[0054] The growth curve results of the strain are as Figure 4 、 Figure 5 shown. In the LB medium, the strain quickly entered the logarithmic phase, and then the growth rate gradually slowed down, and entered the stationary phase after 18 h. In the inorganic salt medium, in the stage of 0-10 h, the strain was in the adaptation period. Due to the certain biological toxicity of phenol, the OD 600 value decreased, and some strains died, and the phenol degradation was relatively slow; after culturing for 10 h, the strain adapted to the environment, the OD 600 value increased, the strain entered the logarithmic growth phase, and the phenol degradation rate accelerated; after 36 h, the phenol removal rate reached 96%, and after continuing to culture for a period of time, 1000 mg / L phenol was completely degraded. As the phenol concentration decreased, the OD 600 value gradually increased, indicating that the degradation of phenol by the strain and its own growth trend were basically consistent, and the strain could use phenol as the sole carbon source and energy source to synthesize its own nutrients for growth and reproduction.

[0055] Experiment on the phenol degradation performance of the strain in Example 3

[0056] The growth and phenol removal characteristics of the phenol-degrading bacterium PD-1 were studied through experiments at different phenol concentrations, pH values and rotation speeds.

[0057] (1) Degradation characteristics of the phenol-degrading bacterium PD-1 at different phenol concentrations

[0058] The strain PD-1 was inoculated into 100 mL of inorganic salt liquid medium with a pH of 7 at an inoculation amount of 5%, and the phenol concentrations were 1000, 1200, 1250, and 1300 mg / L respectively, and cultured in a shaker at 30 °C and 150 rpm. An appropriate amount of the culture solution was taken regularly, and the residual phenol concentration was measured by the 4-aminoantipyrine spectrophotometric method to evaluate the degradation effect of the strain PD-1. The results are as Figure 6As shown by a and b in the figure, when the initial concentration of phenol was 0 - 1250 mg / L, the degradation rate of phenol by strain PD-1 could reach 100% respectively. And as the concentration increased, the degradation rate decreased, being 20.75, 16.67, 12.68 mg / (L·h) respectively. However, when the phenol concentration rose to 1300 mg / L, perhaps due to the enhanced toxicity of high-concentration phenol to the strain, strain PD-1 could not grow normally and stably, and the degradation rate of phenol decreased significantly. Subsequently, the effects of other conditions on the degradation performance of strain PD-1 were studied under the condition of a phenol concentration of 1000 mg / L (with a fast degradation rate).

[0059] (2) Degradation characteristics of phenol-degrading bacterium PD-1 at different pH values

[0060] Strain PD-1 was inoculated into 100 mL of inorganic salt liquid medium with a phenol concentration of 1000 mg / L at an inoculation amount of 5%, where the pH values were 5, 7, 9, and 11 respectively, and cultured with shaking in a shaker at 30 °C and 150 rpm. An appropriate amount of the culture solution was taken regularly, and the residual concentration of phenol was measured by 4-aminoantipyrine spectrophotometry to evaluate the degradation effect of strain PD-1, and the OD value of the bacterial solution was measured by sampling at 24 h of culture. 600 value. The results are as Figure 7 shown by a and b in the figure. In the range of pH 7 - 9, strain PD-1 could achieve complete degradation of 1000 mg / L phenol within 48 hours, and when pH = 7, the phenol degradation rate was the highest.

[0061] (3) Degradation characteristics of phenol-degrading bacterium PD-1 at different rotation speeds

[0062] Strain PD-1 was inoculated into 100 mL of inorganic salt liquid medium with a pH of 7 at an inoculation amount of 5%, where the phenol concentration was 1000 mg / L, and cultured with shaking at rotation speeds of 100, 150, and 180 rpm respectively, and the culture temperature was 30 °C. An appropriate amount of the culture solution was taken regularly, and the residual concentration of phenol was measured by 4-aminoantipyrine spectrophotometry to evaluate the degradation effect of strain PD-1. The results are as Figure 8 shown by a and b in the figure. At the three rotation speeds, strain PD-1 could achieve complete degradation of 1000 mg / L, and as the rotation speed increased, the degradation rate increased, being 16.26, 20.75, 29.76 mg / (L·h) respectively.

[0063] Example 4 Aerobic denitrification characteristics experiment of the strain

[0064] Inoculate the strain PD-1 bacterial suspension into 100 mL of inorganic salt liquid medium with a phenol concentration of 500 mg / L at an inoculation amount of 5%, using ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen as the sole nitrogen sources respectively, with the other components remaining unchanged, and culture it in a shaker at 30 °C and 150 rpm. Regularly take an appropriate amount of the culture solution and measure the concentrations of phenol, ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in the liquid respectively. As Figure 9 shown, when using ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen as the sole nitrogen sources respectively (corresponding to Figure 9 a, b, and c in it), the nitrogen source and phenol are removed synchronously. Specifically, the removal rates of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen are 95.04%, 99.97%, and 99.99% respectively, and the phenol removal rate reaches 99.9%. The results show that the strain PD-1 can grow using ammonia nitrogen (NH4 + ), nitrite nitrogen (NO2 - ), and nitrate nitrogen (NO3 - ) as the sole nitrogen sources and has good removal effects. No obvious ammonia nitrogen accumulation was observed during the whole culture process, and it is speculated that aerobic denitrification may be the dominant pathway for nitrate nitrogen removal.

[0065] It is easy for those skilled in the art to understand that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A phenol-degrading bacterium with aerobic denitrification characteristics, characterized in that, The phenol-degrading bacterium is Pseudomonas monteilii with the preservation number of CCTCC NO: M20242868.

2. Use of the phenol-degrading bacterium with aerobic denitrification as described in claim 1 for degrading phenol.

3. The application according to claim 2, wherein The specific use is as follows: inoculate the phenol-degrading bacterium into a liquid containing phenol, and then perform shaking culture.

4. The application according to claim 3, wherein The concentration of phenol in the liquid is less than or equal to 1250 mg / L.

5. The application according to claim 3, wherein The pH of the liquid is 7 - 9.

6. The application according to claim 3, wherein The rotation speed of the shaking culture is 100 rpm - 180 rpm.

7. The application according to claim 3, wherein The liquid contains a nitrogen source, and the degrading bacterium can achieve synchronous removal of phenol and the nitrogen source.

8. The application according to claim 7, characterized in that, The nitrogen source is ammonia nitrogen NH4 + , nitrite nitrogen NO2 - and nitrate nitrogen NO3 - or at least one of them.

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