CN114940961A

CN114940961A - Salt-tolerant pyridine degrading strain and application thereof in high-salt pyridine wastewater

Info

Publication number: CN114940961A
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: 2022-08-26
Anticipated expiration: 2042-06-21
Also published as: CN114940961B

Abstract

The invention discloses a salt-tolerant pyridine degrading strain and application thereof in high-salt pyridine wastewater; the strain is Rhodococcus (A), (B)Rhodococcussp.) bacterium LV4 with the preservation number of CGMCC NO.25045, the strain can grow and completely degrade pyridine by taking pyridine as a unique carbon-nitrogen source under the conditions of no salt and high salt with the salinity range of 10 g/L-60 g/L, and can completely degrade the pyridine with the concentration of 900mg/L under the condition of high salt with 40g NaCl/L, the tolerance concentration of the strain to the pyridine is increased to 2100mg/L after domestication, and the removal efficiency of the pyridine can reach 95.54% within 72 h; the strain can grow and completely degrade quinoline by taking quinoline as a unique carbon-nitrogen source under the condition of high salt, and can degrade pyrazine simultaneouslyPyridine and quinoline coexist, and the degradation of quinoline is accelerated by the coexistence of pyridine and quinoline, which is of great significance to the treatment of high-salt wastewater containing pyridine and quinoline.

Description

Salt-tolerant pyridine degrading 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 degrading 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 enterprises, coal gasification enterprises and the like, and is also an important basic raw material for the production of pesticides, medicines, dyes, disinfectants and other daily chemicals. Pyridine, although simple in chemical structure, has remarkable teratogenic, carcinogenic and mutagenic properties, such as being discharged without proper disposal, which can be harmful to human health and the ecosystem. Therefore, it is necessary to find an effective removal method.

The removal method of pyridine is mainly classified into a physical method, a chemical method and a biological method. The physical method is mainly divided into extraction method, adsorption method, coagulation method, etc., and the chemical method is mainly oxidation method. Compared with physical methods and chemical methods, the biological method has the advantages of low investment, high efficiency, mild operating environment, no secondary pollution and the like, but the biological degradability of pyridine molecules is greatly reduced due to the 'pi electron deficient structure' in the pyridine molecules, and the pyridine molecules are difficult to be utilized by common microorganisms, so that the conventional biological wastewater treatment method has poor treatment effect. Therefore, exploring and developing a technology for efficiently processing pyridine is the key for realizing green sustainable development of related industries.

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

Research shows that screening halophilic bacteria can be used for improving the treatment effect of high-salt organic wastewater. Yellow and the like, 9 halotolerant bacteria screened from different bacteria sources are prepared into a compound halotolerant microbial inoculum to continuously operate and treat the actual reverse osmosis concentrated brine in the coal chemical industry, and the removal rate of organic matters can reach about 30% (2021, 72(9): 4881-4891, reported in chemical industry). Mehdi et al used 3 halotolerant bacteria screened from petrochemical wastewater to construct halotolerant bacteria, and added into SBR reactor for actual treatment of the halotolerant wastewater, and the removal rate of organic matters in the SBR strengthening reactor can reach as high as 78.7% (Journal of Environmental Management, 2017, 191: 198-208.). Chen et al uses salt-tolerant petroleum-degrading bacteria and surfactant-producing bacteria to construct a complex flora, which has a degradation rate of 95.8% in 10 days and can effectively degrade crude oil in the ranges of pH (4-10) and salinity (0-120 g/L) (International Biodetermination & Biodetermination, 2020, 154: 105047.). Therefore, aiming at high-salt pyridine wastewater, screening and obtaining of salt-tolerant pyridine-degrading bacteria are probably the key points of biologically strengthening treatment of the wastewater, but most of the pyridine-degrading microorganisms reported in the literature at present are carried out in a salt-free or low-salt environment, and pyridine degradation in the high-salt environment is rarely reported. Therefore, the development of microorganisms having pyridine degradation performance and capable of tolerating high salinity is very important for realizing the effective treatment of high-salinity pyridine wastewater.

Disclosure of Invention

Aiming at the defects in the actual high-salt pyridine wastewater treatment, the invention provides the salt-tolerant pyridine degrading strain, the strain can grow and completely degrade pyridine under the conditions of no salt and high salt with the salinity ranging from 10g/L to 60g/L, and the strain can grow and completely degrade quinoline by taking quinoline as a unique carbon-nitrogen source under the condition of high salt, and can also degrade pyridine and quinoline simultaneously, and the coexistence of the pyridine and the quinoline accelerates the degradation of the quinoline, so that the salt-tolerant pyridine degrading strain has important significance for the treatment of the high-salt pyridine and quinoline-containing wastewater.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a salt-tolerant pyridine-degrading strain belonging to RhodococcusRhodococcus sp.) bacterium LV4, deposited at 10.06.2022 in the China general microbiological culture Collection center, address: xilu No. 1 Hospital No. 3, the institute of microbiology, China academy of sciences, Beijing, Chaoyang(ii) a The preservation number is CGMCC No. 25045.

The salt-tolerant pyridine degrading strain (A)Rhodococcus sp.) method for screening LV4, as follows:

(1) and acquiring a water sample from a biochemical aerobic section of a coking wastewater treatment plant, firstly carrying out enrichment culture, and carrying out shake culture at 30 ℃ and 120rpm for 2 d.

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

(3) And (3) carrying out 10 on the mixed bacterial liquid in the step (2) ⁻¹ ~10 ⁻⁹ And (3) performing gradient dilution, namely coating the diluent on a solid pyridine removal culture medium with the pyridine concentration of 500mg/L, and then placing the solid pyridine removal culture medium in an incubator for 3-5 days, wherein the temperature of the incubator is set to be 30 ℃. Extracting single colonies with different forms for culturing, and repeating for three times to obtain four purified strains.

(4) The four purified strains LV4 ', LV 4' ', LV 4' '', LV4 were inoculated into a pyridine-free medium having a salinity of 40g/L (pyridine concentration: 500 mg/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 strain as a target strain.

The strain has the following phenotypic characteristics: the bacterial colony is milky white, round, convex, neat in edge, smooth in bacterial points, slightly raised in thallus, smooth in surface, moist in texture and opaque; bacterial strain LV4 has bright bacterial cells, rod-shaped and blunt ends; the somatic cells are singly arranged, and the cell size is about 0.5-1.4 μm multiplied by 0.2-0.4 μm. Positive under the microscope after gram staining.

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

According to the morphology and the GenBank database analysis of NCBI website, Blast homology analysis shows that the strain LV4 is closely related to Rhodococcus and is also related to the strainRhodococcus sequence homology of sp, strain YC-JH2 was up to 100%, thus strain LV4 was determined to be a Rhodococcus bacterium (R.)Rhodococcus sp.）。

In addition, the invention also provides the salt-tolerant pyridine-degrading strain (Rhodococcus: (A) (B)Rhodococcus sp.) bacteria LV 4) in the treatment of high-salt pyridine-containing wastewater.

The Rhodococcus bacterium LV4 can efficiently degrade pyridine with initial concentration of 500mg/L under the conditions of no salt and high salinity ranging from 10g/L to 60g/L, and strain LV4 can completely degrade pyridine within 36h when the salinity is not higher than 40g NaCl/L, so that the selected salinity of 40g NaCl/L is the preferred salinity of strain LV 4.

The Rhodococcus bacterium LV4 can tolerate pyridine of up to 900mg/L under the high-salt condition of 40g NaCl/L and completely degrade quinoline within 72h, the domesticated strain LV4 can tolerate pyridine of up to 2100mg/L under the high-salt condition of 40g NaCl/L, and the removal rate of pyridine can reach 95.54% after 72 h.

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

The invention also provides the salt-tolerant pyridine-degrading strain (Rhodococcus: (R) (R))Rhodococcus sp.) bacteria LV 4) in the treatment of high-salt quinoline-containing waste water.

The invention also provides the salt-tolerant pyridine-degrading strain (Rhodococcus: (R) (R))Rhodococcus sp.) bacteria LV 4) in the treatment of wastewater containing high-salt pyridine and quinoline.

The Rhodococcus bacterium 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 promotes 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 the high-salt pyridine or quinoline-containing wastewater and also suitable for treating the high-salt pyridine and quinoline coexisting 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 ', LV 4' ', LV 4' '', LV 4).

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

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

FIG. 3 shows a phylogenetic tree of the 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 the degradation of pyridine by pyridine-degrading strain LV 4.

FIG. 5a shows the effect of different initial pyridine concentrations on high salt pyridine removal by strain LV 4.

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

FIG. 6 shows the effect of temperature on high salt pyridine removal by strain LV 4.

FIG. 7 shows the effect of dissolved oxygen on the removal of high salt pyridine by strain LV 4.

FIG. 8 shows the effect of initial pH on high salt pyridine removal by strain LV 4.

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

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

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

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

Detailed Description

The present invention is further illustrated by the following specific examples.

The following embodimentsIn the examples, the methods are all conventional ones unless otherwise specified. In the examples, the concentrations of pyridine and quinoline were measured by UV spectrophotometry at 254nm and 313nm, respectively, dissolved oxygen was measured by a portable dissolved oxygen meter (HQ 30D, HACH), TOC was measured by combustion measurement, pH was measured by a pH meter (Seven 2Go pro, Mettler-Toolido, Switzerland), OD ₆₀₀ The measurement was carried out at a wavelength of 600nm using a visible spectrophotometer, and pH was adjusted using 2mol/L hydrochloric acid and 2mol/L NaOH. The various units used in the examples are all in accordance with national standards.

Example 1

The pyridine-degrading strain LV4 was screened as follows:

(1) and 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 put into a 250mL conical flask containing 90mL of enrichment medium, wherein the enrichment medium is composed of 10g/L of peptone, 5g/L, NaCl 10g/L of yeast extract and pH = 7.0. The mouth of the bottle was then sealed with a sterile breathing seal film and placed in a shaker at 30 ℃ and 120rpm to enrich the bacterial suspension. After 2 days of culture, 5mL of 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 being 0.5g/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. The pyridine-degrading bacteria were acclimatized and cultured at 30 ℃ and 120rpm for 2 d.

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

(2) Transferring and taking 1mL of mixed bacterial suspension after domestication of the five bacteria for 10 ⁻¹ ~10 ⁻⁹ Diluting with gradient, respectively taking 10 ⁻³ 、10 ⁻⁶ And 10 ⁻⁷ And (3) coating 100 mu L of each bacterial liquid on a solid pyridine removal culture medium containing a corresponding substrate (500 mg/L of pyridine), and culturing in an incubator for 3-5 days, wherein the temperature of the incubator is set to be 30 ℃. When the solid plate is full of macroscopic colonies, single colonies with better growth are picked, single colonies with different forms are extracted from the solid agar plate for culture, and four purified strains (LV 4, LV4 ', LV 4' 'and LV 4' '') are obtained by repeating three times. And finally, preserving the strain by an ultralow temperature freeze thawing method and a low-temperature solid slant preservation method.

(3) The four purified strains LV4, LV4 ', LV 4' 'and LV 4' '' were inoculated into a 250mL Erlenmeyer flask containing 100mL pyridine removal medium (pyridine concentration 500 mg/L) and activated at 30 ℃ with a shaker speed of 120rpm until the strain grew to the log phase. 5mL of the activated bacterial suspension is inoculated into a fresh pyridine removal culture medium (the pyridine concentration is 500 mg/L), the pyridine degradation performance of the suspension is studied under the conditions of 30 ℃ and 120rpm of the shaking table rotation speed, and the removal rate of pyridine and COD in different time periods is determined by a timing sampling mode. Meanwhile, the four strains LV4, LV4 ', LV 4' 'and LV 4' '' are inoculated into a pyridine removal medium with the salinity of 40g NaCl/L, and the growth condition of the strains in high-salinity pyridine waste water is examined. The experimental results are shown in figure 1. Under the condition of taking pyridine as the only carbon-nitrogen source, the removal rates of the four strains to the pyridine within 48h are respectively 52.92%, 47.73%, 47.81% and 100%, and the maximum OD is ₆₀₀ The values are 0.36, 0.33, 0.36 and 0.55 respectively, and only the strain LV4 in the four purified strains can normally grow and degrade pyridine in high-salt pyridine wastewater, and the results show that: only the strain LV4 in the four purified strains has the best effect on degrading pyridine and can tolerate a high-salt environment, and LV4 is selected as a target strain.

Example 2

The pyridine-degrading strain LV4 was identified as follows:

the pyridine degrading bacteria LV4 are inoculated on a solid agar plate culture medium, the formula of the culture medium is 1.8% of agar, 10g/L of peptone and 5g/L, NaCl 10g/L of yeast extract, the pH is =7.0, and biochemical culture is carried out for 48h at 30 ℃. The colony morphology of the pyridine degrading strain LV4 is shown in figure 2a, the colony is milky white, round, convex, neat in edge, smooth in bacterial points, slightly raised in thallus, smooth in surface, moist in texture and opaque; the cell morphology of the pyridine degrading strain LV4 is shown in figure 2b, the thallus is bright and rod-shaped, and the two ends are blunt; the strain LV4 has somatic cells which are singly arranged and the cell size is about 0.5-1.4 μm multiplied by 0.2-0.4 μm.

The gram staining result of the pyridine degrading strain LV4 shows that the color of the stained thallus is changed into purple, which indicates that the strain LV4 is a gram positive bacterium.

The 16S rDNA sequencing base sequence of the pyridine degrading strain LV4 is shown in the attached sequence table, the sequence is submitted to GenBank database analysis of NCBI website, Blast homology analysis shows (figure 3), the strain LV4 is closely related to Rhodococcus, and is closely related to the strainRhodococcus sequence homology of sp, strain YC-JH2 was up to 100%, thus strain LV4 was determined to be a Rhodococcus bacterium (R.)Rhodococcus sp.). The strain was deposited in the general microbiological culture collection center of the China Committee for culture Collection of microorganisms, located at the institute of microbiology, China academy of sciences, Beijing, 10.06 months and 2022, address: the No. 3 Xilu Beijing province of Chaoyang, the registration number of the preservation center is CGMCC number 25045.

Example 3

Quinoline degradation performance of pyridine-degrading strain LV4 by different salinity was as follows:

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

Preparing a pyridine removal medium with an initial concentration of 500mg/L and with a salinity of 0, 10g NaCl/L, 20g NaCl/L, 30g NaCl/L, 40g NaCl/L, 50g NaCl/L and 60g NaCl/L (same as example 1), inoculating the activated bacterial suspension into sterile 100mL of pyridine removal medium at an inoculum size of 5% (v/v), and incubating at 30 ℃In-shaker at pH =7 and 120rpm for 72h, samples were taken at 12h intervals, and the growth (OD) of strain LV4 in media of different salinity was determined separately ₆₀₀ Indicated), degradation of pyridine and TOC. As shown in figure 4, the strain LV4 can grow in a salt-free environment and in a salinity range of 10-60 g/L and completely degrade pyridine. The salinity rise can cause the lag phase of the strain LV4 to increase, when the salinity is 0, 10g NaCl/L, 20g NaCl/L, 30g NaCl/L, 40g NaCl/L, 50g NaCl/L and 60g NaCl/L respectively, the strain LV4 enters the logarithmic phase of rapid growth after 12h, 24h, 48h and 72h lag phases respectively (figure 4 a); at the same time, the pyridine content decreased rapidly (fig. 4 b), which further illustrates that pyridine degradation is closely related to its growth process. Although strain LV4 was growing and pyridine degradation at different salinity varied, strain LV4 could completely degrade pyridine within 36h when the salinity was not higher than 40g NaCl/L, so the selected salinity of 40g NaCl/L was 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 medium (same as in example 1) were set to 100mg/L, 300mg/L, 500mg/L, 700mg/L and 900mg/L, respectively, using pyridine as a sole carbon and nitrogen source and a salinity of 40g NaCl/L. Experiment in the use of pyridine as the only carbon nitrogen source, 5mL of the working solution (same as example 3) was inoculated into the above fresh 100mL of culture medium, cultured at 30 ℃, pH =7, 120rpm for 24h, and sampled to determine the degradation of high-salt pyridine of the strain at different initial pyridine concentrations.

As shown in FIG. 5a, the results of the study showed that strain LV4 could completely degrade pyridine within 36h at pyridine concentration not higher than 500mg/L, with 100% removal rate, whereas pyridine degradation rate decreased with increasing pyridine concentration at pyridine concentration higher than 500mg/L, and pyridine removal rate was 78.69% and 54.47% at initial pyridine concentration of 700mg/L and 900mg/L, respectively. Meanwhile, when the initial concentrations of pyridine were 100mg/L, 300mg/L, 500mg/L, 700mg/L, and 900mg/L, respectively, the TOC degradation rates were 64.58%, 81.06%, 84.58%, 78.82%, and 45.12%, respectively, further indicating that strain LV4 was in strain LV4Has good mineralization ability to pyridine in high-salt environment. OD of Strain LV4 when cultured for 36h ₆₀₀ The concentration increased, mainly because the increased pyridine concentration provided more carbon and nitrogen source for strain LV4, which grew better after the lag phase. When the culture period is prolonged to 72h, 700mg/L and 900mg/L of pyridine can be completely degraded by the strain LV4, the removal rates of corresponding TOC are 85.45% and 78.09%, respectively (the results in the figure are not shown), and the strain LV4 can realize effective degradation of pyridine in a high-salt environment.

Strain LV4 was acclimatized and cultured to log phase (OD) in a pyridine removal medium (same as example 1) having a salinity of 40g NaCl/L and an initial pyridine concentration of 900mg/L ₆₀₀ Approximatively, 0.4), the initial pyridine concentrations of the pyridine removal medium (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 72h under the culture conditions of 30 ℃, pH =7 and 120rpm, and sampling to determine the degradation condition of high-salt pyridine of the domesticated strain LV4, wherein the result is shown in figure 5b, the tolerance concentration of the domesticated strain LV4 pyridine is increased to 2100mg/L, and the removal rate of the pyridine can reach 95.54% when the domesticated strain LV4 is cultured for 72h, which indicates the superiority of the strain LV4 in treating high-salinity and high-concentration pyridine wastewater.

Example 5

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

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

As shown in FIG. 6, the research result shows that in a high-salt environment with the salinity of 40g NaCl/L, the strain LV4 can grow at the temperature range of 25-40 ℃ by using pyridine as the only carbon and nitrogen source, wherein when the temperature is 30 ℃, the strain LV4 grows best, and the pyridine degradation rate is highest and is 49.16%. Statistical analysis results of the degradation rate of the strain LV4 to pyridine under different temperature conditions during 24h culture show that the strain LV4 has significant difference in pyridine degradation capability at each culture temperature. Pyridine can be completely degraded by the strain LV4 under each culture temperature condition in the whole 72-hour culture period, the pyridine has a good mineralization effect, and the 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 within the temperature range of 25-40 ℃, and the optimal temperature is 30 ℃.

Example 6

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

pyridine is used as the only carbon nitrogen source, pyridine removal culture medium with the concentration of 500mg/L, pH =7, the temperature of 30 ℃ and the salinity of 40g NaCl/L (same as example 1) is inoculated into the 100mL culture medium by taking 5mL working solution (same as example 3), the culture medium is sealed by a sealing film, a shaking table is arranged, the rotating speed of the shaking table is respectively adjusted 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) for culturing for 36h, and the degradation condition of the strain pyridine under different dissolved oxygen is sampled and determined.

As shown in FIG. 7, in a high salt environment with a salinity of 40g NaCl/L, the growth rate, the pyridine degradation rate and the TOC degradation rate of the strain are gradually increased along with the increase of the rotation speed, and the strain grows best (OD) when the rotation speed is 120rpm ₆₀₀ = 0.57) when 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 pyridine to TOC degradation rates were less distinct, at which point the pyridine to TOC degradation rates were 97.38% and 78.70%, respectively. The pyridine and TOC degradation rates of strain LV4 decreased to 90.36% and 78.61% when the rpm was increased to 200 rpm. In conclusion, it can be seen that the cell growth and the degradation of pyridine can be promoted by appropriately increasing the shaking speed, and in view of the experimental economy, 4.69mg/L of dissolved oxygen was selected as the preferred dissolved oxygen for strain LV 4.

Example 7

High salt pyridine degradation experiments of strain LV4 under different initial pH conditions were as follows:

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

As shown in FIG. 8, the research result shows that under the high-salt condition with the salinity of 40g NaCl/L, the strain LV4 can grow by taking pyridine as the only carbon and nitrogen source in the pH range of 4-10. The strain LV4 has significant difference (P is less than 0.05) on pyridine degradation capacity under each initial pH condition, wherein when the initial pH is 7, the pyridine degradation effect of the strain LV4 is best, and the pyridine removal rate is 42.63%. It can be easily found that the strain LV4 can still grow by using pyridine as the only carbon and nitrogen source when the initial pH is 4 or 10, and the OD is increased to 72h when the strain is cultured ₆₀₀ 0.42 and 0.29, respectively, corresponding to pyridine degradation rates of 100% and 15.77% (not shown), indicating that the strain can grow and degrade pyridine in a strong acid or strong base environment, but the stronger the acid or base, the more growth inhibition is exerted on strain LV 4. The experimental results show that the strain LV4 can survive in the pH range of 4-10 and completely degrade pyridine, and the pH value of 7 is selected as the preferable pH value of the strain LV 4.

Example 8

The cell growth and pyridine degradation experiments of the strain LV4 under high salt conditions with pyridine as the only carbon and nitrogen source are as follows:

the experiment was carried out in a pyridine removal medium (same as in example 1) having a salinity of 40g NaCl/L and pyridine (500 mg/L) as the sole carbon and nitrogen source. 5mL of the working solution (same as in example 3) was inoculated into 100mL of the fresh medium, cultured at 30 ℃ and pH =7 at 120rpm for 72 hours, sampled at intervals, and tested for growth of strain LV4 and degradation of pyridine at different stages.

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

As can be seen from FIG. 9b, the ammonia nitrogen content in water continuously increases with the ring-opening degradation of pyridine, and reaches the highest value of about 73mg/L when the ammonia nitrogen content is cultured for 48 hours, and then slightly decreases and maintains about 70mg/L, while 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 mostly exists in the form of ammonia nitrogen. The pH value in water shows a change trend of firstly decreasing and then increasing in the whole experimental determination process, the early-stage pH value decrease is probably caused by acidic substances generated in the pyridine degradation process, and the pH value in water gradually increases along with the increase of the content of released ammonia nitrogen in the pyridine ring opening process.

In view of the toxicity and the persistence of pyridine and the lack of the current research on the degradation of salt-tolerant pyridine-degrading microorganisms, the utilization of the efficient pyridine-degrading bacteria LV4 to degrade pyridine in a high-salinity environment has very important practical significance and application value.

Example 9

The cell growth and quinoline degradation experiments of the strain LV4 under high salt conditions with quinoline as the only carbon-nitrogen source are as follows:

the experiment is carried out in a quinoline removal medium with the salinity of 40g NaCl/L and the quinoline (100 mg/L) as the only carbon-nitrogen source, wherein the formula of the quinoline removal medium is 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 100mL of the fresh medium, and cultured at 30 ℃ and pH =7 at 120rpm for 96 hours,samples were taken at intervals and growth and quinoline degradation of strain LV4 were determined at different stages.

As shown in FIG. 10a, due to the double inhibition effect of high salinity and quinoline toxicity, the strain LV4 is in the lag phase in 36h of initial culture and grows slowly, and the quinoline degradation effect is not obvious. Strain LV4 entered the fast-growing log phase after 36h of culture, OD by 72h ₆₀₀ A maximum value of 0.23 is reached. Meanwhile, the quinoline content in water is rapidly reduced, and finally, the quinoline is completely degraded after being cultured for 72h, and the removal rate can reach 100% (fig. 10 b). In addition, the variation trend of the TOC content in water is basically the same as that of the quinoline content, the TOC degradation rate is the largest when reaching 84h, and the maximum degradation rate is 83.26% (fig. 10 b), further explaining that the mineralization effect of the strain LV4 is also better. Therefore, the experiment shows that the strain LV4 is closely related to the growth of quinoline, and can realize the complete degradation of quinoline under the condition of high salt.

Example 10

The strain LV4 was subjected to the degradation experiment of the coexistence of pyridine and quinoline under the high-salt condition as follows:

the experiment was carried out in a pyridine removal medium (same as in example 1) in which pyridine (500 mg/L) and quinoline (100 mg/L) were present and the salinity was 40g NaCl/L. 5mL of the working solution (same as example 3) was inoculated into 100mL of the fresh medium, cultured at 30 ℃ and pH =7 and 120rpm for 72h, sampled at intervals, and tested for growth of strain LV4 and degradation of quinoline and pyridine at different stages.

As shown in FIG. 11, when pyridine coexisted with quinoline, strain LV4 completely degraded quinoline in the slow-growing lag phase, i.e., within 36h of the initial culture, which was 24h earlier than that of strain LV4 which had quinoline as the sole carbon and nitrogen source. The presence of pyridine and quinoline accelerated the degradation of quinoline by strain LV4, probably because the simultaneous degradation of both substrates caused competition by the intracellular electron donors, whereas the first mono-oxidation of quinoline (to 2-hydroxyquinoline) was always faster than the first mono-oxidation of pyridine (to 2-hydroxypyridine). The pyridine content in water is gradually reduced along with the growth of the strain LV4, and the pyridine in the water is completely degraded when the strain is cultured for 48 hours, which further shows that the strain LV4 can simultaneously degrade quinoline and pyridine substances in a high-salt environment, provides good strain resources for the treatment of high-salt nitrogen heterocyclic compound wastewater, and has great potential in the field of the treatment of nitrogen heterocyclic pollutants in the high-salt wastewater.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications and equivalents of the technical solutions of the present invention using the contents of the present specification and the drawings are included in the scope of the present invention.

<110> Tai principals university

<120> salt-tolerant pyridine degrading 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 bacterium 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-tolerant pyridine-degrading strain is characterized by being classified and named Rhodococcus (R.)Rhodococcussp.) bacterium LV4, deposited in China general microbiological culture Collection center (CGMCC), with the preservation number of CGMCC 25045.

2. The halopyridine degrading strain of claim 1, wherein the 16S rDNA gene sequence of halopyridine degrading strain LV4 is SEQ ID NO: 1.

3. the halopyridine degrading strain of claim 1, wherein said halopyridine degrading strain LV4 has a phenotypic characteristic that: the bacterial colony is milky white, round, convex, neat in edge, smooth in bacterial points, slightly raised in thallus, smooth in surface, moist in texture and opaque; the thallus is bright and rod-shaped, and two ends of the thallus are blunt and round; the somatic cells are singly arranged, and the cell size is about 0.5-1.4 mu m multiplied by 0.2-0.4 mu m; positive under the microscope after gram staining.

4. The use of the salt-tolerant pyridine-degrading strain of claim 1 in the treatment of high-salt pyridine-containing wastewater.

5. The application of the salt-tolerant pyridine-degrading strain in the treatment of high-salt pyridine-containing wastewater according to claim 4, wherein the salinity of the wastewater is calculated by NaCl and is 10-60 g/L.

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

7. The application of the salt-tolerant pyridine-degrading strain in the treatment of high-salt pyridine-containing wastewater according to claim 4, 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.

8. The use of the salt-tolerant pyridine-degrading strain according to claim 4 in the treatment of high-salt pyridine-containing wastewater, wherein the salinity of the wastewater is 40g/L, the temperature of the wastewater is 30 ℃, the dissolved oxygen of the wastewater is 4.69mg/L, and the pH of the wastewater is 7.

9. The use of the salt-tolerant pyridine-degrading strain of claim 1 in the treatment of high-salt quinoline-containing wastewater.

10. The use of the salt-tolerant pyridine-degrading strain of claim 1 in the treatment of wastewater containing high-salt pyridine and quinoline.