CN114940961B

CN114940961B - Salt-tolerant pyridine degradation strain and application thereof in high-salt pyridine wastewater

Info

Publication number: CN114940961B
Application number: CN202210705506.7A
Authority: CN
Inventors: 王莹; 陈虎; 徐梦迪; 吕永康
Original assignee: Taiyuan University of Technology
Current assignee: Taiyuan University of Technology
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2023-09-15
Anticipated expiration: 2042-06-21
Also published as: CN114940961A

Abstract

The invention discloses a salt-tolerant pyridine degradation strain and application thereof in high-salt pyridine wastewater; the strain is rhodococcus genus [ (]Rhodococcussp.) bacterial LV4 with a preservation number of CGMCC No.25045, which can grow and completely degrade pyridine under the conditions of no salt and high salt with a salinity range of 10g/L-60g/L by taking pyridine as a sole carbon nitrogen source, and can completely degrade up to 900mg/L of pyridine under the conditions of high salt with a salinity of 40g NaCl/L, the tolerance concentration of the strain to pyridine is improved to 2100mg/L after domestication, and the pyridine removal efficiency can reach 95.54% within 72 hours; under the high-salt condition, the strain can not only grow by taking quinoline as the sole carbon and nitrogen source and completely degrade the quinoline, but also degrade pyridine and the quinoline simultaneously, and the coexistence of the pyridine and the quinoline accelerates the degradation of the quinoline, so that the strain has great significance for the treatment of high-salt wastewater containing the pyridine and the quinoline.

Description

Salt-tolerant pyridine degradation strain and application thereof in high-salt pyridine wastewater

Technical Field

The invention belongs to the technical field of water pollutant control, and particularly relates to a salt-tolerant pyridine degradation strain and application thereof in high-salt pyridine wastewater.

Background

Pyridine is a typical organic pollutant in wastewater discharged by coal chemical enterprises such as coking and coal gasification, and is also an important basic raw material for the production of pesticides, medicines, dyes, disinfectants and other daily chemicals. Pyridine has a simple chemical structure but has remarkable teratogenic, oncogenic and mutagenic properties, and if not properly treated, emissions can be detrimental to human health and the ecosystem. Therefore, it is necessary to find an effective removal method.

The method for removing pyridine is mainly divided into a physical method, a chemical method and a biological method. The physical method is mainly divided into an extraction method, an adsorption method, a coagulation method and the like, and the chemical method is mainly an oxidation method. Compared with the physical method and the chemical method, the biological method has the advantages of less investment, high efficiency, mild operation environment, no secondary pollution and the like, but the biological degradability of the pyridine molecule is greatly reduced due to the 'pi-electron lack structure' in the pyridine molecule, and the pyridine molecule is difficult to be utilized by common microorganisms, so that the conventional biological method for treating the wastewater has poor treatment effect. Therefore, the exploration and development of efficient treatment technology for pyridine is a key for realizing green sustainable development of related industries.

The biological strengthening treatment technology based on efficient pyridine degrading bacteria screening and separation is an economic and effective way for solving the environmental pollution caused by pyridine. However, industrial waste water such as coking waste water, printing waste water, pharmaceutical waste water and the like contains not only pyridine substances but also high-concentration salts, resulting in high-salt pyridine waste water. The waste water has higher salinity, can destroy cell membranes and biological enzymes of microorganisms, and inhibit the growth and propagation of the microorganisms, thereby restricting the biological treatment effect of the waste water.

It is found that screening halophilic bacteria can be used for improving the treatment effect of high-salt organic wastewater. Yellow and the like prepares 9 salt-tolerant bacteria obtained by screening from different bacteria sources into composite salt-tolerant bacteria agent which continuously operates to treat the actual coal chemical reverse osmosis strong brine, and the organic matter removal rate can reach about 30 percent (chemical engineering journal, 2021, 72 (9): 4881-4891.). The Mehdi et al uses 3 salt-tolerant bacteria obtained by screening petrochemical wastewater to construct salt-tolerant bacteria groups, and adds the salt-tolerant bacteria into an SBR reactor to treat the actual salt-containing wastewater, wherein the organic matter removal rate of the SBR reinforced reactor can reach 78.7% at most (Journal of Environmental Management, 2017, 191:198-208.). Chen et al constructed a complex flora with salt tolerant petroleum degrading bacteria and surfactant producing bacteria, which could reach 95.8% crude oil degradation rate within 10 d, and could degrade crude oil efficiently within the pH (4-10) and salinity (0-120 g/L) (International Biodeterioration & biodegration, 2020, 154: 105047.). Therefore, the screening and obtaining of salt-tolerant pyridine degrading bacteria aiming at high-salt pyridine waste water is probably a key point for the biologically enhanced treatment of the waste water, but most of pyridine degrading microorganisms reported in the literature are carried out in a salt-free or low-salt environment, and the pyridine degradation in the high-salt environment is still freshly reported. Therefore, development of microorganisms which have pyridine degradation performance and can tolerate high salinity is important for realizing efficient treatment of high-salinity pyridine wastewater.

Disclosure of Invention

Aiming at the defects existing in the actual high-salt pyridine wastewater treatment, the invention provides a salt-resistant pyridine degradation strain which can grow and completely degrade pyridine under the high-salt condition with the salt-free and salinity range of 10g/L-60g/L, and the strain can not only grow and completely degrade quinoline by taking quinoline as the sole carbon-nitrogen source under the high-salt condition, but also degrade pyridine and quinoline simultaneously, and the coexistence of pyridine and quinoline accelerates the degradation of quinoline, so that the strain has great significance for the treatment of high-salt pyridine and quinoline-containing wastewater.

In order to solve the technical problems, the invention adopts the following technical scheme: salt-tolerant pyridine degradation strain named rhodococcus @, and its preparation methodRhodococcus sp.) bacteria LV4, deposited at the China general microbiological culture Collection center, address: the institute of microbiology, national academy of sciences, north chen xi lu 1, 3, the region of the morning sun in beijing; the preservation number is CGMCC NO. 25045.

The salt-resistant pyridine degradation strainRhodococcus sp.) LV4 screening method as follows:

(1) And taking a water sample from a biochemical aerobic section of the coking wastewater treatment plant, and performing enrichment culture at 30 ℃ and 120rpm for 2d.

(2) Then, a part of the enriched culture solution was inoculated into a pyridine removal medium having a pyridine concentration of 500mg/L, and pyridine degrading bacteria were selectively cultured at 30℃and 120rpm for 2d.

(3) Subjecting the mixed bacterial liquid in the step (2) to 10 ⁻¹ ~10 ⁻⁹ Gradient diluting, and coating the diluted solutionAnd removing the culture medium from the solid pyridine with the pyridine concentration of 500mg/L, and then placing the culture medium in an incubator for 3-5 d, wherein the temperature of the incubator is set to be 30 ℃. Extracting single bacterial colonies with different forms, culturing, and repeating for three times to obtain four purified bacteria.

(4) Four strains LV4', LV4' '', LV4 obtained by purification were inoculated into a pyridine removal medium (pyridine concentration 500 mg/L) having a salinity of 40g/L. Comprehensively considering the growth of each strain in a high-salt pyridine removal culture medium and the degradation effect on pyridine and COD, and screening and selecting LV4 bacteria as target bacteria.

The strain has the following phenotypic characteristics: the bacterial colony is milky white, round, convex, regular in edge, smooth in bacterial spot, slightly raised in bacterial body, smooth in surface, moist in texture and opaque; the strain LV4 has bright bacterial cells, rod shape and round ends; the individual arrangement of the cells of the cell line occurs, and the cell size is about 0.5-1.4 μm×0.2-0.4 μm. Positive under microscope after passing through gram stain.

The 16S rDNA gene sequence of the strain is characterized in that shown in a sequence table, and the length of a base sequence is 1432bp.

According to the form and GenBank database analysis of NCBI website, the Blast homology analysis shows that the strain LV4 has close relationship with rhodococcus and with the strainRhodococcus sp, strain YC-JH2 sequence homology is up to 100%, so that strain LV4 is determined to be rhodococcus bacteriaRhodococcus sp.）。

In addition, the invention also provides the salt-tolerant pyridine degradation strain (rhodococcus [ ]Rhodococcus sp.) bacteria LV 4) in the treatment of high-salt pyridine-containing wastewater.

The rhodococcus bacteria LV4 provided by the invention can efficiently degrade pyridine with the initial concentration of 500mg/L under the conditions of no salt and high salt with the salinity ranging from 10g/L to 60g/L, and when the salinity is not higher than 40g NaCl/L, the bacterial strain LV4 can completely degrade pyridine in 36h, so that the preferred salinity of the bacterial strain LV4 is selected to be 40g NaCl/L.

As described above, rhodococcus bacterium LV4 can tolerate pyridine up to 900mg/L under high salt condition of 40g NaCl/L and degrade quinoline completely within 72 hours, and after domestication, strain LV4 can tolerate pyridine with concentration up to 2100mg/L under high salt condition of 40g NaCl/L, and after 72 hours, the pyridine removal rate can reach 95.54%.

The rhodococcus LV4 can degrade pyridine in high salt condition of 40g NaCl/L and has excellent pyridine mineralizing capacity. Wherein the temperature of the wastewater is 25-40 ℃, preferably 30 ℃; the dissolved oxygen of the wastewater is 2.48-6.99 mg/L, preferably 4.69mg/L; the pH of the wastewater is 4-10, preferably 7.

The invention also provides the salt-tolerant pyridine degradation strain (rhodococcus @Rhodococcus sp.) bacteria LV 4) in the treatment of high-salt quinoline-containing wastewater.

The invention also provides the salt-tolerant pyridine degradation strain (rhodococcus @Rhodococcus sp.) bacteria LV 4) in the treatment of wastewater with high-salt pyridine and quinoline coexistence.

The rhodococcus LV4 can degrade not only pyridine or quinoline alone but also pyridine and quinoline simultaneously in a high-salt environment of 40g NaCl/L, and the coexistence of pyridine can promote the degradation of quinoline, which is more advantageous in the actual wastewater treatment process.

Compared with the prior art, the invention has the following beneficial effects:

the method is suitable for treating high-salt pyridine-or quinoline-containing wastewater, is also suitable for treating high-salt pyridine-and quinoline-containing wastewater, has wide application prospect, and has good practical application value and social benefit.

Drawings

FIG. 1 shows the pyridine degrading performance of four different strains (LV 4', LV4' '', LV 4).

FIG. 2a shows colony morphology of pyridine degrading strain LV 4.

FIG. 2b shows the cell morphology of pyridine degrading strain LV 4.

FIG. 3 shows a phylogenetic tree of pyridine degrading strain LV 4.

FIG. 4a shows the effect of different salinity on the growth of pyridine degrading strain LV 4.

FIG. 4b shows the effect of different salinity on pyridine degradation by pyridine degrading strain LV 4.

FIG. 5a shows the effect of different initial pyridine concentrations on strain LV4 high salt pyridine removal.

FIG. 5b shows the high salt pyridine removal performance of the domesticated strain LV 4.

FIG. 6 shows the effect of temperature on strain LV4 high salt pyridine removal.

FIG. 7 shows the effect of dissolved oxygen on strain LV4 high salt pyridine removal.

FIG. 8 shows the effect of initial pH on strain LV4 high salt pyridine removal.

FIG. 9a shows the cell growth and pyridine degradation characteristics of strain LV4 under high salt conditions with pyridine as the sole carbon nitrogen source.

FIG. 9b shows the pyridine and TOC removal rates of strain LV4 under high salt conditions with pyridine as the sole carbon nitrogen source.

FIG. 10a shows the cell growth and pyridine degradation characteristics of strain LV4 under high salt conditions with quinoline as the sole carbon nitrogen source.

FIG. 10b shows the removal of quinoline and TOC from strain LV4 under high salt conditions with quinoline as the sole carbon nitrogen source.

FIG. 11 shows the degradation performance of strain LV4 under high salt conditions for the coexistence of pyridine and quinoline.

Detailed Description

The invention is further illustrated below with reference to specific examples.

In the following examples, the methods are conventional, unless otherwise specified. In the examples, the pyridine and quinoline concentrations were measured by ultraviolet spectrophotometry at 254nm and 313nm, respectively, dissolved oxygen was measured by portable dissolved oxygen meter (HQ 30D, HACH), TOC was measured by combustion assay, pH was measured by pH meter (Seven 2Go pro, switzerland-tolido), OD ₆₀₀ Measurement was performed at a wavelength of 600nm using a visible spectrophotometer, and pH adjustment was performed using 2mol/L hydrochloric acid and 2mol/L NaOH. The various units used in the examples all adopt the national standard in a unified way.

Example 1

The pyridine degrading strain LV4 was screened as follows:

(1) Obtaining a sewage activated sludge water sample from an aeration tank of a coking wastewater treatment plant of Shanxi Taiyuan iron and steel company. Firstly, 10mL of water sample is taken and placed in a 250mL conical flask containing 90mL of enrichment medium, wherein the enrichment medium comprises 10g/L of peptone, 5g/L, naCl g/L of yeast extract and pH=7.0. The mouth was then sealed with a sterile breath sealing membrane and placed in a shaker at 30 ℃ and 120rpm to enrich the bacterial suspension. After 2d of culture, 5mL of the cell suspension from the enrichment medium was transferred to 100mL of sterile pyridine removal medium with an initial pyridine concentration of 500mg/L, the pyridine removal medium formulation was 0.5g/L, K pyridine ₂ HPO ₄ ·3H ₂ O 0.75g/L、NaH ₂ PO ₄ ·2H ₂ O 0.25g/L、MgSO ₄ ·7H ₂ O 0.05g/L、MnSO ₄ ·4H ₂ O 0.01g/L、FeSO ₄ ·7H ₂ O0.01 g/L, ph=7.0. And domesticating and culturing pyridine degrading bacteria at 30 ℃ and 120rpm for 2d.

The sludge is taken from an aeration tank (Shanxi province of China) of a coking wastewater treatment plant of a Taiyuan iron and steel company, 5g of the sludge is weighed and placed in 100mL of LB liquid culture medium, a bottle opening is tightly sealed by a sterile breathing sealing film, and the bottle opening is placed in a shaking table at 30 ℃ and 120rpm to enrich bacterial suspension, and the bottle opening is subjected to marked culture for 1-2 days. From the enriched medium, 5mL of the cell fungus suspension was transferred to 100mL sterile medium with an initial pyridine concentration of 500mg/L for cultivation under the above conditions. Continuously inoculating the liquid culture medium for domestication for 5 times.

(2) Transferring 1mL of the mixed bacterial suspension subjected to domestication five times for 10 times ⁻¹ ~10 ⁻⁹ Gradient dilution, respectively taking 10 ⁻³ 、10 ⁻⁶ And 10 ⁻⁷ Each 100. Mu.L of the bacterial liquid is coated on a solid pyridine removal culture medium containing a corresponding substrate (pyridine 500 mg/L), and the solid pyridine removal culture medium is placed in an incubator for 3-5 d, and the temperature of the incubator is set to be 30 ℃. When the solid plate is full of macroscopic colonies, single colonies with better growth vigor are picked up, single colonies with different forms are extracted on the solid agar plate for culture, and four purified bacteria (LV 4, LV4' ' ') are obtained by repeating three times. Finally, adopting two methods of ultralow temperature freeze thawing method and low temperature solid inclined plane preservation method to preserve the strain.

(3) Four purified strains LV4, LV4' ' ' were inoculated into 250mL Erlenmeyer flasks containing 100mL of pyridine removal medium (pyridine concentration 500 mg/L) and activated at 30℃and shaking at 120rpm until the strain grew to the logarithmic phase. 5mL of the activated bacterial suspension is taken and put into fresh pyridine removal culture medium (the pyridine concentration is 500 mg/L), the pyridine degradation performance of the bacterial suspension is studied under the conditions of 30 ℃ and 120rpm of shaking table rotation speed, and the pyridine and COD removal rates in different time periods are measured by a timing sampling mode. Meanwhile, the four strains LV4, LV4' ' and LV4' ' ' are inoculated into a pyridine removal culture medium with the salinity of 40g NaCl/L, and the growth condition of the strains in high-salt pyridine wastewater is examined. The experimental results are shown in figure 1. Under the condition of taking pyridine as the sole carbon and nitrogen source, the removal rates of four bacteria in 48h on pyridine are 52.92%, 47.73%, 47.81% and 100% respectively, and the maximum OD is the maximum ₆₀₀ The values are respectively 0.36, 0.33, 0.36 and 0.55, and only strain LV4 in four purified bacteria can normally grow in high-salt pyridine wastewater and degrade pyridine, and the results show that: among the four purified bacteria, only strain LV4 has the best effect on pyridine degradation and can tolerate a high-salt environment, and LV4 is selected as a target bacteria.

Example 2

The pyridine degrading strain LV4 was identified as follows:

pyridine degrading bacteria LV4 are inoculated on a solid agar plate culture medium, wherein the formula of the culture medium is agar 1.8%, peptone 10g/L, yeast extract 5g/L, naCl g/L, pH=7.0, and biochemical culture is carried out at 30 ℃ for 48 hours. The bacterial colony of the pyridine degrading strain LV4 is in a shape of milky white, round, convex, neat in edge, smooth in bacterial spot, slightly raised in bacterial body, smooth in surface, moist in texture and opaque as shown in FIG. 2 a; the cell morphology of the pyridine degrading strain LV4 is shown in figure 2b, and the thallus is bright, rod-shaped and round at two ends; the strain LV4 has single cell arrangement, and cell size of about 0.5-1.4 μm×0.2-0.4 μm.

Gram staining of pyridine degrading strain LV4 revealed that the stained cells turned purple in color, indicating that strain LV4 was a gram positive bacterium.

The 16S rDNA sequencing base sequence of the pyridine degradation strain LV4 is shown in the attached sequence table, the sequence is submitted to GenBank database analysis of NCBI website, and the Blast homology analysis shows that the strain LV4 has close relationship with rhodococcus and with the strain (figure 3)Rhodococcus sp, strain YC-JH2 sequence homology is up to 100%, so that strain LV4 is determined to be rhodococcus bacteriaRhodococcus sp.). The strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection) of microbiological culture Collection center, china academy of sciences of China, beijing, at 2022, 06 and 10 days, address: the registration number of the collection center is CGMCC No.25045, and the registration number of the North Chen Xili No. 1 and 3 in the Chaoyang area of Beijing city is CGMCC No. 25045.

Example 3

Quinoline degradation performance of different salinity on pyridine degradation strain LV4 is as follows:

the working solution was purified bacteria extracted from the preserved strain LV4 and activated in a 250mL Erlenmeyer flask containing 100mL (containing pyridine at 500 mg/L) of pyridine removal medium (same as in example 1) at 30℃and shaking speed of 120rpm until the strain grew to the logarithmic phase (OD ₆₀₀ ≈0.4）。

Preparation of pyridine-removed medium (same as in example 1) with initial concentrations of 500mg/L, having salinity of 0, 10g NaCl/L, 20g NaCl/L, 30g NaCl/L, 40g NaCl/L, 50g NaCl/L and 60g NaCl/L, the activated bacterial suspension was inoculated into sterile 100mL pyridine-removed medium at an inoculum size of 5% (v/v), cultured in a shaker at 30℃ C, pH =7 at 120rpm for 72 hours, sampled at 12 hours intervals, and growth of strain LV4 in medium of different salinity was measured (OD ₆₀₀ Indicated), pyridine and TOC. As shown in FIG. 4, the strain LV4 can grow in a salt-free environment and a salinity range of 10g/L to 60g/L and completely degrade pyridine. The increase in salinity caused a delay increase in strain LV4, when salinity was 0, 10g NaCl/L, 20g NaCl/L, 30g NaCl/L, 40g NaCl/L, 50g NaCl/L and 60g NaCl/L, respectively, strain LV4 entered the log phase of rapid growth after the delay of 12h, 12h, 12h, 24h, 48h and 72h, respectively (FIG. 4 a); at the same time, the pyridine content decreases rapidly (FIG. 4 b), further illustrating pyridine degradation and its overgrowthThe process is closely related. Although the growth and pyridine degradation of strain LV4 were different at different salinity, when the salinity was not higher than 40g NaCl/L, strain LV4 could completely degrade pyridine within 36h, so that a salinity of 40g NaCl/L was selected as the preferred salinity of strain LV 4.

Example 4

The high salt pyridine degradation experiments of strain LV4 at different initial pyridine concentrations were as follows:

the initial pyridine concentrations of the pyridine removal media (same as in example 1) were set to 100mg/L, 300mg/L, 500mg/L, 700mg/L and 900mg/L, respectively, with pyridine as the sole carbon source nitrogen source and with a salinity of 40g NaCl/L. In the experiment, pyridine is taken as the only carbon and nitrogen source, 5mL of working solution (the same as in example 3) is inoculated into the fresh 100mL culture medium, and the culture is carried out for 24 hours under the culture conditions of 30 ℃ and pH=7 and 120rpm, and the high-salt pyridine degradation condition of the strain under different initial pyridine concentrations is sampled and measured.

As shown in FIG. 5a, the results of the study showed that strain LV4 can completely degrade pyridine within 36 hours at a pyridine concentration of not more than 500mg/L, with the removal rate being 100%, whereas the pyridine degradation rate decreases with increasing pyridine concentration at a pyridine concentration of more than 500mg/L, and the removal rates of pyridine are 78.69% and 54.47% at initial pyridine concentrations of 700mg/L and 900mg/L, respectively. Meanwhile, when the initial concentrations of pyridine are respectively 100mg/L, 300mg/L, 500mg/L, 700mg/L and 900mg/L, TOC degradation rates are respectively 64.58%, 81.06%, 84.58%, 78.82% and 45.12%, which further indicate that the strain LV4 has good mineralization ability on pyridine in a high-salt environment. OD of Strain LV4 at 36h of culture ₆₀₀ This increases with increasing concentration, mainly because an increase in pyridine concentration provides strain LV4 with a more carbon and nitrogen source, which grows better after a delay. When the culture period was prolonged to 72 hours, the strain LV4 was able to completely degrade 700mg/L and 900mg/L of pyridine, with corresponding TOC removal rates of 85.45% and 78.09%, respectively (results not shown in the figures), indicating that the strain LV4 was able to achieve efficient degradation of pyridine in high salt environments.

Domesticating and culturing strain LV4 in pyridine removal medium (same as example 1) with salinity of 40g NaCl/L and initial pyridine concentration of 900mg/L to logarithmic phaseOD ₆₀₀ Approximately 0.4), the initial pyridine concentrations of the pyridine removal media (same as in example 1) were set to 900mg/L, 1300mg/L, 1600mg/L and 2100mg/L, respectively. Inoculating 5mL of domesticated bacterial suspension into the fresh 100mL of culture medium, culturing for 72 hours at 30 ℃ under the culture conditions of pH=7 and 120rpm, sampling and measuring the degradation condition of high-salt pyridine of the domesticated bacterial strain LV4, wherein the result is shown in a figure 5b, the pyridine tolerance concentration of the domesticated bacterial strain LV4 is improved to 2100mg/L, and the pyridine removal rate can reach 95.54% when culturing for 72 hours, thus indicating the superiority of the bacterial strain LV4 in treating high-salinity and high-concentration pyridine wastewater.

Example 5

The high-salt pyridine degradation experiment of the strain LV4 under different temperature conditions is as follows:

pyridine is taken as the only carbon and nitrogen source, pyridine removal culture medium (same as example 1) with the pyridine concentration of 500mg/L, pH =7, the rotation speed of a shaking table of 120rpm and the salinity of 40g NaCl/L is taken, 5mL of working solution (same as example 3) is inoculated into the 100mL of culture medium and sealed by a sealing film, the culture medium is placed in the shaking table, the temperature is respectively adjusted to 20 ℃, 25 ℃,30 ℃, 35 ℃, 40 ℃ and 45 ℃ for culturing for 24 hours, and the strain pyridine degradation condition at different temperatures is sampled and measured.

As shown in FIG. 6, the results of the study show that in a high salt environment with a salinity of 40g NaCl/L, the strain LV4 can grow at a temperature range of 25-40 ℃ by taking pyridine as the sole carbon-nitrogen source, wherein when the temperature is 30 ℃, the strain LV4 grows optimally, and the degradation rate of pyridine is 49.16% at the highest. The statistical analysis result of the degradation rate of the bacterial strain LV4 to pyridine under different temperature conditions at the time of culturing 24h shows that the bacterial strain LV4 has significant difference to the degradation capacity of pyridine at each culture temperature. Pyridine can be completely degraded by the strain LV4 in the whole 72h culture period under various culture temperature conditions, and has good mineralization effect, and TOC removal rate can reach 88% when the culture temperature is 30 ℃ (the result is not shown in the figure). In conclusion, the strain LV4 has a good degradation effect on high-salt pyridine wastewater in the temperature range of 25-40 ℃, and the optimal temperature is 30 ℃.

Example 6

The high-salt pyridine degradation experiment of the strain LV4 under different dissolved oxygen conditions is as follows:

pyridine is taken as the only carbon nitrogen source, pyridine removal culture medium (same as in example 1) with pyridine concentration of 500mg/L, pH =7 and temperature of 30 ℃ and salinity of 40g NaCl/L is inoculated into 100mL of culture medium, sealing is carried out by a sealing film, the culture medium is placed in a shaking table, the rotation speed of the shaking table is respectively regulated to 40rpm (DO 2.48 mg/L), 80rpm (DO 3.25 mg/L), 120rpm (DO 4.69 mg/L), 160rpm (DO 5.73 mg/L) and 200rpm (DO 6.99 mg/L), and the strain pyridine degradation condition under different dissolved oxygen is measured by sampling.

As shown in FIG. 7, in the high salt environment with a salinity of 40g NaCl/L, the growth rate of the strain, the degradation rate of pyridine and TOC were gradually increased with increasing rotation speed, and the strain growth was optimal at 120rpm (OD ₆₀₀ =0.57), at which point the pyridine and TOC degradation rates reached a maximum of 100% and 84.78%, respectively. Cell growth (OD) of strain LV4 at 160rpm compared to 120rpm ₆₀₀ =0.59), the difference between the pyridine and TOC degradation rates was not significant, at which time the pyridine and TOC degradation rates were 97.38% and 78.70%, respectively. The pyridine and TOC degradation rates of strain LV4 were reduced by 90.36% and 78.61%, respectively, as the rotation rate was increased to 200 rpm. In summary, it was found that an appropriate increase in the shaking speed promoted cell growth and pyridine degradation, and that 4.69mg/L of dissolved oxygen was selected as the preferred dissolved oxygen for strain LV4 in view of experimental economy.

Example 7

The high salt pyridine degradation experiments of strain LV4 at different initial pH conditions were as follows:

pyridine was used as the sole carbon nitrogen source, and the initial pH of the pyridine-removed medium (same as in example 1) at a pyridine concentration of 500mg/L and a salinity of 40g NaCl/L was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 using 2mol/L hydrochloric acid and 2mol/L NaOH. 5mL of the working solution (same as in example 3) was inoculated into 100mL of the above culture medium, sealed with a sealing film, placed in a shaking table at 30℃and cultured at 120rpm for 24 hours, and sampled and assayed for pyridine degradation at different pH values.

As shown in FIG. 8, the results of the study show that the strain LV4 can be used in the pH range of 4-10 under the high-salt condition with the salinity of 40g NaCl/LAnd pyridine is used as the only carbon and nitrogen source for growth. There was a significant difference in the pyridine degrading ability of strain LV4 (P < 0.05) at each initial pH, wherein at an initial pH of 7, the pyridine degrading effect of strain LV4 was best and the pyridine removal rate was 42.63%. It was found that strain LV4 was still able to grow with pyridine as the sole carbon nitrogen source at an initial pH of 4 or 10, and was cultured to OD at 72h ₆₀₀ And 0.42 and 0.29, respectively, with 100% and 15.77% pyridine degradation (not shown), indicating that the strain can grow and degrade pyridine in strong acid or alkali environments, but the more acidic or basic the more inhibitory to strain LV4 growth. The experimental result shows that the strain LV4 can survive in the pH range of 4-10 and completely degrade pyridine, and the pH value is selected to be 7, which is the preferable pH value of the strain LV 4.

Example 8

Cell growth and pyridine degradation experiments of strain LV4 under high salt conditions with pyridine as the sole carbon nitrogen source were as follows:

the experiment was performed in a pyridine removal medium (same as in example 1) with pyridine (500 mg/L) as sole carbon nitrogen source and a salinity of 40g NaCl/L. 5mL of the working solution (same as in example 3) was inoculated into the fresh 100mL of the above-mentioned medium, and the culture was carried out at 30℃under the culture conditions of pH=7 and 120rpm for 72 hours, and samples were taken at intervals to determine the growth and pyridine degradation of the strain LV4 at different periods.

As shown in FIG. 9a, strain LV4 was in a lag phase and cells grew slowly during the first 24h, at which time the pyridine content in the water was gradually reduced from initial 502.33mg/L to 349.14mg/L. After that, strain LV4 enters the logarithmic phase of rapid growth and OD ₆₀₀ The value rapidly increased from 0.18 at 24h to 0.57 at 36 h; the pyridine content of the strain in the logarithmic growth phase rapidly drops to zero, and the pyridine removal rate is 29.10 mg/(L.h), which indicates that the degradation of pyridine is closely related to cell growth. The TOC degradation trend in water is basically consistent with that of pyridine, and the TOC degradation rate is 88.36% when the water is finally stabilized at about 42.80mg/L after 72 hours of culture, which further proves that the mineralization effect of the strain LV4 on the pyridine is better.

As can be seen from FIG. 9b, with the ring-opening degradation of pyridine, the ammonia nitrogen content in water is continuously increased, the ammonia nitrogen content reaches the highest value up to 48 hours of cultivation, is about 73mg/L, and is slightly reduced and maintained at about 70mg/L, and the theoretical calculated value of the N content of the initial pyridine in water is about 77mg/L, which indicates that the N existing form after the ring-opening of pyridine exists in the form of ammonia nitrogen. The pH value in the water shows a change trend of firstly decreasing and then increasing in the whole experimental determination process, and the pH value in the water is gradually increased along with the increase of the ammonia nitrogen content released in the pyridine ring-opening process due to the fact that acidic substances are generated in the pyridine degradation process possibly due to the decrease of the pH value in the early stage.

In view of toxicity and durability of pyridine and lack of current salt-tolerant pyridine degradation microbial degradation research, the efficient pyridine degradation bacteria LV4 is used for carrying out pyridine degradation in a high salinity environment, and has very important practical significance and application value.

Example 9

Cell growth and quinoline degradation experiments of strain LV4 under high salt conditions with quinoline as the sole carbon and nitrogen source were as follows:

the experiment was performed in a quinoline removal medium with quinoline (100 mg/L) as the sole carbon and nitrogen source and a salinity of 40g NaCl/L, the quinoline removal medium formulation was quinoline 0.1g/L, K ₂ HPO ₄ ·3H ₂ O 0.75g/L、NaH ₂ PO ₄ ·2H ₂ O 0.25g/L、MgSO ₄ ·7H ₂ O 0.05g/L、MnSO ₄ ·4H ₂ O 0.01g/L、FeSO ₄ ·7H ₂ O0.01 g/L, ph=7.0. 5mL of the working solution (same as in example 3) was inoculated into the fresh 100mL of the above-mentioned medium, and the culture was carried out at 30℃under the culture conditions of pH=7 and 120rpm for 96 hours, and samples were taken at intervals to determine the growth of strain LV4 and the degradation of quinoline at different periods.

As shown in FIG. 10a, strain LV4 grows slowly in a lag phase within 36h of initial culture due to dual inhibition of high salinity and quinoline self-toxicity, and the quinoline degradation effect is not obvious. After 36h of cultivation, strain LV4 enters the logarithmic phase of rapid growth, to OD at 72h ₆₀₀ Reaches a maximum value of 0.23. Meanwhile, the quinoline content in water is rapidly reduced, and the quinoline is completely degraded after the final culture for 72 hours, and the removal rate can reach 100% (figure 10 b). In addition, the TOC content change trend and the quinoline content change trend in water are basically the sameMeanwhile, the TOC degradation rate was the largest at 84h and the largest degradation rate was 83.26% (FIG. 10 b), further demonstrating that the mineralization effect of strain LV4 was also better. Therefore, experiments show that the strain LV4 is closely related to quinoline degradation and growth, and can realize complete degradation of quinoline under high-salt conditions.

Example 10

The degradation experiment of the strain LV4 on the coexistence of pyridine and quinoline under the high-salt condition is as follows:

the experiment was performed in pyridine removal medium (same as in example 1) with pyridine (500 mg/L) and quinoline (100 mg/L) together and with a salinity of 40g NaCl/L. 5mL of the working solution (same as in example 3) was inoculated into the fresh 100mL of the above-mentioned medium, and the culture was carried out at 30℃under the culture conditions of pH=7 and 120rpm for 72 hours, and samples were taken at intervals to determine the strain LV4 growth, quinoline and pyridine degradation at different periods.

As shown in FIG. 11, when pyridine and quinoline coexist, strain LV4 completely degraded quinoline in a slow growth delay period, i.e., in 36 hours of initial culture, which is 24 hours earlier than when strain LV4 uses quinoline as the sole carbon and nitrogen source. The co-presence of pyridine with quinoline accelerates the degradation of quinoline by strain LV4, probably because the simultaneous degradation of both substrates causes competition for intracellular electron donors, whereas the first single oxidation of quinoline (to form 2-hydroxyquinoline) is always faster than the first single oxidation of pyridine (to form 2-hydroxypyridine). Along with the growth of the strain LV4, the pyridine content in water is gradually reduced, and pyridine in water is completely degraded when the strain LV4 is cultured for 48 hours, which further shows that the strain LV4 can degrade quinoline and pyridine substances simultaneously in a high-salt environment, provides good strain resources for the wastewater treatment of high-salt nitrogen-containing heterocyclic compounds, and has great potential in the field of the treatment of nitrogen heterocyclic pollutants in the high-salt wastewater.

The foregoing description is only illustrative of the invention and is not intended to limit the scope of the invention, and all changes and substitutions made herein without departing from the spirit and scope of the invention as defined by the appended claims.

<110> university of Tai principle engineering

<120> a salt-tolerant pyridine degradation strain and application thereof in high-salt pyridine wastewater

<160>1

<210>1

<211>1432

<212>DNA

<213> Rhodococcus (Rhodococcus sp.) bacterium LV4

<220>

<223> 16S rDNA of rhodococcus LV4

<400>1

GACGCTGGCG GCGTGCTTAA CACATGCAAG TCGAACGATG AAGCCCAGCT TGCTGGGTGG 60

ATTAGTGGCG AACGGGTGAG TAACACGTGG GTGATCTGCC CTGCACTCTG GGATAAGCCT 120

GGGAAACTGG GTCTAATACC GGATATGACC TCTTGCTGCA TGGCGAGGGG TGGAAAGTTT 180

TTCGGTGCAG GATGAGCCCG CGGCCTATCA GCTTGTTGGT GGGGTAATGG CCTACCAAGG 240

CGACGACGGG TAGCCGGCCT GAGAGGGCGA CCGGCCACAC TGGGACTGAG ACACGGCCCA 300

GACTCCTACG GGAGGCAGCA GTGGGGAATA TTGCACAATG GGCGCAAGCC TGATGCAGCG 360

ACGCCGCGTG AGGGATGACG GCCTTCGGGT TGTAAACCTC TTTCACCCAT GACGAAGCGC 420

AAGTGACGGT AGTGGGAGAA GAAGCACCGG CCAACTACGT GCCAGCAGCC GCGGTAATAC 480

GTAGGGTGCG AGCGTTGTCC GGAATTACTG GGCGTAAAGA GCTCGTAGGC GGTTTGTCGC 540

GTCGTCTGTG AAATCCCGCA GCTCAACTGC GGGCTTGCAG GCGATACGGG CAGACTCGAG 600

TACTGCAGGG GAGACTGGAA TTCCTGGTGT AGCGGTGAAA TGCGCAGATA TCAGGAGGAA 660

CACCGGTGGC GAAGGCGGGT CTCTGGGCAG TAACTGACGC TGAGGAGCGA AAGCGTGGGT 720

AGCGAACAGG ATTAGATACC CTGGTAGTCC ACGCCGTAAA CGGTGGGCGC TAGGTGTGGG 780

TTTCCTTCCA CGGGATCCGT GCCGTAGCCA ACGCATTAAG CGCCCCGCCT GGGGAGTACG 840

GCCGCAAGGC TAAAACTCAA AGGAATTGAC GGGGGCCCGC ACAAGCGGCG GAGCATGTGG 900

ATTAATTCGA TGCAACGCGA AGAACCTTAC CTGGGTTTGA CATGTACCGG ACGACTGCAG 960

AGATGTGGTT TCCCTTGTGG CCGGTAGACA GGTGGTGCAT GGCTGTCGTC AGCTCGTGTC 1020

GTGAGATGTT GGGTTAAGTC CCGCAACGAG CGCAACCCTT GTCCTGTGTT GCCAGCACGT 1080

GATGGTGGGG ACTCGCAGGA GACTGCCGGG GTCAACTCGG AGGAAGGTGG GGACGACGTC 1140

AAGTCATCAT GCCCCTTATG TCCAGGGCTT CACACATGCT ACAATGGTCG GTACAGAGGG 1200

CTGCGATACC GTGAGGTGGA GCGAATCCCT TAAAGCCGGT CTCAGTTCGG ATCGGGGTCT 1260

GCAACTCGAC CCCGTGAAGT CGGAGTCGCT AGTAATCGCA GATCAGCAAC GCTGCGGTGA 1320

ATACGTTCCC GGGCCTTGTA CACACCGCCC GTCACGTCAT GAAAGTCGGT AACACCCGAA 1380

GCCGGTGGCC TAACCCCTTG TGGGAGGGAG CCGTCGAAGG TGGGATCGGC GA 1432

Claims

1. A salt-resistant pyridine degradation strain is characterized by being classified and named as rhodococcus genus #Rhodococcussp.) bacteria LV4, which are preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 25045.

2. A salt tolerant pyridine degrading strain according to claim 1, wherein the 16S rDNA gene sequence of salt tolerant pyridine degrading strain LV4 is SEQ ID NO:1.

3. the use of the salt-tolerant pyridine degradation strain according to claim 1 in the treatment of high-salt pyridine-containing wastewater, wherein the salinity of the wastewater is calculated as NaCl, and the salinity of the wastewater is in the range of 10g/L to 60g/L.

4. Use of a salt-tolerant pyridine degrading strain according to claim 3 in the treatment of high-salt pyridine-containing wastewater, wherein the initial concentration of pyridine in the wastewater is not higher than 2100mg/L.

5. The application of the salt-tolerant pyridine degradation strain in the treatment of high-salt pyridine-containing wastewater, according to claim 3, wherein the temperature of the wastewater is 25-40 ℃, the dissolved oxygen of the wastewater is 2.48-6.99 mg/L, and the pH of the wastewater is 4-10.

6. Use of a salt-tolerant pyridine degrading strain according to claim 3 in the treatment of high salt pyridine-containing waste water, wherein the salinity of the waste water is 40g/L, the temperature of the waste water is 30 ℃, the dissolved oxygen of the waste water is 4.69mg/L, and the pH of the waste water is 7.

7. Use of a salt-tolerant pyridine degrading strain according to claim 1 for the treatment of high salt quinoline-containing wastewater, wherein the wastewater salinity is calculated as NaCl, the wastewater salinity being 40g/L.

8. Use of a salt tolerant pyridine degrading strain according to claim 1 in the treatment of high salt pyridine and quinoline co-existing wastewater, wherein the wastewater salinity is calculated as NaCl, the wastewater salinity being 40g/L.