Method for improving acid-eating bacterium tolerance and degrading high-concentration pyridine

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for improving acid-eating bacterium tolerance and degrading high-concentration pyridine. According to the specific technical scheme, the method for improving the tolerance and pyridine degradation capacity of the acidovorax sp. Comprises the following steps: extracting a corncob medullary layer leaching solution, and adding the corncob medullary layer leaching solution into a growth environment of the acidovorax sp. According to the invention, it is found for the first time that the corncob medullary layer leachate can effectively improve the tolerance and high-concentration pyridine degradation capability of acidovorax. By adding the corncob medullary layer leachate in the culture environment of the acidovorax bacteria, the acidovorax bacteria can degrade 99% or more of about 2700 mg / L of pyridine after being treated for about 84 hours. The method provided by the invention can effectively improve the high-concentration pyridine degradation effect of the acidovorax LD-B, the method is simple, and the used corncob is an agricultural waste, is wide in source and low in price, has both environmental friendliness and sustainability, greatly reduces the application cost, and has huge popularization value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for improving the tolerance and degradation of high-concentration pyridine by acidophilic bacteria. Background Art

[0002] Coking wastewater contains highly toxic and refractory organic compounds, and its sustainable treatment is challenging. In coking wastewater, characteristic pollutants include high-concentration inorganic and organic pollutants such as ammonia, sulfide, phenol, nitrogen-containing heterocyclic compounds, and polycyclic aromatic hydrocarbons. Pyridine is a typical nitrogen-containing heterocyclic compound among them, with significant biological toxicity, teratogenicity, and mutagenicity, posing a serious threat to the ecosystem and human health. Pyridine combines chemical inertness and environmental persistence. Traditional physical and chemical treatment methods have problems such as high treatment costs, complex processes, high risks of secondary pollution, and inability to completely degrade pollutants. While the biodegradation method has the advantages of environmental friendliness, economic efficiency, simple process, and easy maintenance, it can overcome the problems of high energy consumption and secondary pollution of physical and chemical methods while efficiently removing pyridine.

[0003] Although the biodegradation method has many advantages, the molecular structure of pyridine is highly stable, and it has strong resistance to most microorganisms. The inventor's research group used pyridine as the target pollutant and screened a new microorganism: acidophilic bacteria (Paracidovorax avenae) LD-B. The acidophilic bacteria LD-B was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 4, 2023, with the deposit number: CGMCC NO. 28102; the acidophilic bacteria LD-B was disclosed in patent CN117264831 A. This microorganism has extremely strong adaptability in pyridine and coking wastewater and can efficiently degrade pyridine. However, studies have found that as the concentration of pyridine increases, the ability of this microorganism to degrade pyridine gradually decreases. When the pyridine concentration rises above 2500 mg / L, high-concentration pyridine inhibits the growth of microorganisms, and the ability to degrade pyridine drops significantly.

[0004] Therefore, if a method can be provided to help improve the tolerance and degradation ability of acidophilic bacteria LD-B to high-concentration pyridine, it will have important research value and application value. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for improving the tolerance and degradation of high-concentration pyridine by acidophilic bacteria.

[0006] To achieve the above-mentioned invention purpose, the technical solution adopted by the present invention is: a method for improving the tolerance and degradation ability of acidophilic bacteria to pyridine, the method comprising: extracting the leaching solution of corn cob pith layer and adding it to the growth environment of acidophilic bacteria.

[0007] Correspondingly, a method for degrading pyridine or treating coking wastewater is to jointly apply acidovorax bacteria and the leaching solution of corn cob medullary layer to the environment where pyridine is to be degraded.

[0008] Preferably, in the growth environment of acidovorax bacteria, the addition concentration of the leaching solution of corn cob medullary layer is 500 mg / L.

[0009] Preferably, the acidovorax bacteria is Acidovorax avenae LD-B, which was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on August 4, 2023, and the deposit number is: CGMCC NO. 28102.

[0010] Preferably, the preparation method of the leaching solution of corn cob medullary layer includes: taking corn cobs, cleaning, drying, separating the epidermal layer, medullary layer and central layer structures of the corn cob, taking the medullary layer and crushing it, and soaking the medullary layer in water. The drying temperature is 60 - 100 °C. The soaking time is 36 - 60 h.

[0011] The present invention has the following beneficial effects: The present invention discovers for the first time that the leaching solution of corn cob medullary layer can effectively improve the ability of acidovorax bacteria to tolerate and degrade high-concentration pyridine. By adding the leaching solution of corn cob medullary layer to the culture environment of acidovorax bacteria, the acidovorax bacteria can degrade more than 99% of about 2700 mg / L of pyridine after about 84 h of treatment.

[0012] The method provided by the present invention can effectively improve the effect of acidovorax bacteria LD-B in degrading high-concentration pyridine. The method is simple, and the corn cob used is an agricultural waste, with a wide source, low price, environmental friendliness and sustainability, greatly reducing the application cost, and having great popularization value. Description of the Drawings

[0013] Figure 1 Schematic diagrams of different structures of corn cobs;

[0014] Figure 2 Pyridine standard curve. Detailed Embodiments

[0015] The present invention provides a method that can improve the tolerance and degradation of high-concentration pyridine by Acidovorax avenae LD-B. The method includes: extracting the leaching solution of corn cob medullary layer, adding it to the growth environment of acidovorax bacteria LD-B, and using the leaching solution of corn cob medullary layer as a co-metabolic substrate to promote the degradation of high-concentration pyridine by acidovorax bacteria.

[0016] The preferred solution is: in the growth environment of acidophilic bacterium LD-B, the addition concentration of the leaching solution of corn cob pith layer is 500 mg / L. The research group of the present invention tested a variety of different corn varieties and found that the corn variety has no significant influence on the results; in the examples of the present invention, the corn variety used is Chuandan 99.

[0017] An optional method for preparing the leaching solution of corn cob pith layer is: rinse the surface impurities of the corn cob with deionized water, dry at 60-100 °C, separate the epidermis layer, pith layer and central layer structures of the corn cob, take the pith layer and crush it. After separating the corn cob structure, the epidermis layer, pith layer and central layer structures are respectively as Figure 1 shown. Immerse the pith layer in deionized water, and the mass-volume ratio (mg / L) of the pith layer to deionized water is 1:20-60, preferably 1:40, soak at room temperature for 36-60 h, filter with a 0.45 μm water-based filter head, and sterilize at high temperature and high pressure.

[0018] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. If not specifically specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The obtained data are all the averages obtained after at least 3 repetitions, and all the repetitions obtained are valid data.

[0019] The various reagents and culture media involved in the examples are as follows:

[0020] 1. LB medium (enrichment medium): peptone 10 g, NaCl 10 g, yeast powder 5 g, water 1000 mL, pH 7.0-7.2, sterilize at 121 °C for 20 min.

[0021] 2. Inorganic salt medium: MgSO4·7H2O 0.2 g, K2HPO4 4.26 g, KH2PO4 2.65 g, CaCl2 0.02 g, 1 mL trace element solution, pyridine at the required concentration, 1000 mL distilled water, pH = 7.

[0022] 3. Trace element solution: KI 0.005 g, MnSO4·4H2O 0.2 g, CuSO4·2H2O 0.02 g, ZnSO4·7H2O 0.2 g, Na2MoO4·2H2O 0.25 g, H3BO3 0.008 g, FeCl·6H2O 0.1 g, add water to 100 mL.

[0023] The detection methods involved in the examples are as follows:

[0024] 1. Pyridine determination method: The absorbance of the cell-free culture solution at 256 nm was measured using a microplate reader, and the concentration of pyridine was determined through a pyridine standard curve to calculate the degradation rate. The degradation rate η of pyridine was calculated using the following formula:

[0025]

[0026] C0: Initial pyridine concentration (mg / L); C t : Pollutant concentration (mg / L) after degradation time t hours.

[0027] 2. Method for establishing pyridine standard curve: By performing a full-wavelength ultraviolet scan of a single substance, pyridine, using a laboratory microplate reader, the characteristic absorption peak wavelength of this organic substance was determined to be 256 nm. The pyridine standard curve is as Figure 2 shown. The linear relationship between pyridine concentration C and absorbance A is: y = 0.0161x + 0.0629.

[0028] Example 1: Comparison of the effects of different biomass materials on the degradation of pyridine by Acidovorax sp. LD-B

[0029] The strain LD-B was inoculated into LB medium and cultured with shaking at 30 °C and 150 r / min for 2 d until the late logarithmic phase. Then, it was centrifuged at 5000 r / min for 10 min using a centrifuge, washed 3 times with sterilized inorganic salt medium, and the OD 600 was adjusted to 1.0 to obtain the seed solution. Unless otherwise specified, the preparation method of the strain LD-B seed solution is the same as that described here.

[0030] Rice husks, peanut shells, wheat straw, corn straw, and corn cobs were washed, dried at 45 °C, and crushed respectively. The biomass materials and deionized water were mixed at a mass-to-volume ratio of 1:40 (g / mL). Each biomass material was immersed in deionized water and soaked at room temperature for 48 h. The obtained seed solution was inoculated into an inorganic salt medium containing a high concentration of pyridine supplemented with the leachate of each biomass material (addition amount: 2% (v / v)) at an inoculation amount of 5% (v / v). The initial concentration of pyridine was about 2700 mg / L. The culture temperature was controlled at 30 °C, the rotation speed was 150 r / min, and the culture was carried out with shaking. Samples were taken after culturing for 12 - 96 h respectively. After centrifuging at 15000 r / min for 15 min, the absorbance of the supernatant (cell-free culture solution) at a wavelength of 256 nm was measured to calculate the degradation rate. At the same time, a blank control group was set up with the same conditions except that the leachate of the biomass material was not added, and deionized water equal to the amount of the leachate of the biomass material was added to the inorganic salt medium. The degradation results are shown in Table 1. It should be noted that: during the experiment, not only the biomass materials listed in Table 1 were tested. Due to space limitations, only some materials with more ideal effects are shown here.

[0031] Table 1 Table of the situation of LD-B degrading high-concentration pyridine after adding different biomass materials

[0032]

[0033]

[0034] The results show that corncobs have the most obvious promoting effect on the degradation of high-concentration pyridine by acidotrophic bacterium LD-B. Therefore, corncobs are selected for subsequent examples.

[0035] Example 2: Influence of the concentration of corncob leachate on the pyridine degradation effect

[0036] Wash the corncobs, dry them at 45 °C, and crush them. The dosage relationship between corncobs and deionized water is: mass-volume ratio 1:40 (mg / L). Immerse the corncobs in deionized water and soak them at room temperature for 48 h to obtain corncob leachate.

[0037] Inoculate the acidotrophic bacterium LD-B seed solution into the inorganic salt medium added with corncob leachate at different concentrations according to an inoculation amount of 5% (v / v). In the inorganic salt medium, additionally add pyridine, and the initial concentration of pyridine is about 2645 mg / L. Control the culture temperature at 30 °C, the rotation speed at 150 r / min, and perform shaking culture. Sample and measure the absorbance of the cell-free culture solution at a wavelength of 256 nm at 24 h, 48 h, 72 h, and 96 h respectively, and calculate the pyridine degradation rate. At the same time, set a blank control group with the same other conditions, without adding corncob leachate, and add deionized water equal to the corncob leachate in the inorganic salt medium. The degradation results are shown in Table 2.

[0038] Table 2 Table of the situation of LD-B degrading high-concentration pyridine after adding different concentrations of corncobs

[0039]

[0040] Example 3: Influence of different structures of corncobs on the pyridine degradation effect

[0041] Rinse the surface impurities of the corncobs with deionized water, dry them at 80 °C, separate the epidermis layer, medulla layer, and central layer structures of the corncobs by physical means, and crush each component to 18 meshes with a crusher. Immerse the epidermis layer, medulla layer, and central layer of the corncobs in deionized water respectively. The dosage relationship between the materials and deionized water is: according to the mass-volume ratio 1:40 (mg / L), soak them at room temperature for 48 h to obtain the leachate of each material.

[0042] The strain LD-B seed liquid was inoculated into the inorganic salt medium containing pyridine supplemented with the leachate of the epidermis layer, medulla layer, and central layer of corncob at an inoculation amount of 5% (v / v). The concentration of pyridine was about 2700 g / L, and the addition concentration of each component of the corncob was 500 mg / L. The culture temperature was controlled at 30 °C, the rotation speed was 150 r / min, and the culture was carried out with shaking. Samples were taken after culturing for 12 - 96 h respectively to measure the absorbance of the cell-free culture solution at a wavelength of 256 nm, and the degradation rate was calculated. At the same time, a blank control group was set up, with the same other conditions, without adding the corncob leachate, and adding the same amount of deionized water as the biomass material leachate to the inorganic salt medium. The degradation results are shown in Table 3.

[0043] Table 3 Degradation of high-concentration pyridine by LD-B after adding different corncob structures

[0044]

[0045] The results showed that the medulla layer of the corncob had a more ideal effect on promoting the degradation of high-concentration pyridine by the strain LD-B. It might contain the main active components that promoted the strain LD-B, or there were certain substances that could play a synergistic role with the strain LD-B.

[0046] Example 4: Influence of the state of the corncob medulla layer on the pyridine degradation effect

[0047] The leachate of the corncob medulla layer was obtained in the manner of Example 3. At the same time, the corncob medulla layer was obtained in the manner of Example 3 and pulverized for standby.

[0048] The strain LD-B seed liquid was inoculated into the inorganic salt medium containing pyridine supplemented with the corncob medulla layer and the leachate of the corncob medulla layer at an inoculation amount of 5% (v / v). The initial concentration of pyridine in the medium was controlled at about 2700 mg / L, and the concentrations of the corncob medulla layer solid and the medulla layer leachate in the inorganic salt medium were both 500 mg / L. The culture temperature was controlled at 30 °C, the rotation speed was 150 r / min, and the culture was carried out with shaking. Samples were taken after culturing for 12 - 96 h respectively to measure the absorbance of the cell-free culture solution at a wavelength of 256 nm, and the degradation rate was calculated. At the same time, a blank control group was set up, with the same other conditions, without adding the corncob leachate, and adding the same amount of deionized water as the biomass material leachate to the inorganic salt medium. The degradation results are shown in Table 4.

[0049] Table 4 Degradation of high-concentration pyridine by LD-B after adding the corncob medulla layer solid and the leachate

[0050]

[0051] The results showed that both the solid corn cob medulla layer and the leachate of the medulla layer could significantly improve the ability of strain LD-B to degrade high-concentration pyridine. This indicates that the adsorption of the corn cob medulla layer is not the main reason for helping LD-B degrade pyridine. More likely, certain water-soluble active components in the corn cob medulla layer played a synergistic role with LD-B.

[0052] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, variations, modifications, and substitutions made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for improving the pyridine tolerance of acidophilic bacteria, characterized in that: The method includes: extracting the leaching solution of the core medulla layer of corn cob and adding it to the growth environment of acidotrophic bacteria.

2. A method for improving the ability of acidophilic bacteria to degrade pyridine, characterized in that: The method includes: extracting the leaching solution of the core medulla layer of corn cob and adding it to the growth environment of acidotrophic bacteria.

3. A method for degrading pyridine, characterized in that: The acidotrophic bacteria and the leaching solution of the core medulla layer of corn cob are jointly applied to the environment to be degraded of pyridine.

4. A method for treating coking wastewater, characterized in that: The acidotrophic bacteria and the leaching solution of the core medulla layer of corn cob are jointly applied to the environment to be degraded of pyridine.

5. The method according to any one of claims 1 to 4, characterized in that: In the growth environment of acidotrophic bacteria, the addition concentration of the leaching solution of the core medulla layer of corn cob is 500 mg / L.

6. The method according to any one of claims 1 to 4, characterized in that: The acidotrophic bacteria is Acidotrophic bacteria (Paracidovorax avenae) LD-B, which was deposited on August 4, 2023 at the General Microbiology Center of the China Microbial Culture Collection Center, and the deposit number is: CGMCC NO. 28102.

7. According to the method described in any one of claims 1 to 4, characterized in that: The preparation method of the leaching solution of the core medulla layer of corn cob includes: taking corn cob, cleaning, drying, separating the epidermal layer, medulla layer and central layer structures of the corn cob, taking the medulla layer and crushing it, and soaking the medulla layer in water.

8. The method according to claim 7, wherein: The drying temperature is 60-100 °C.

9. The method according to claim 7, characterized in that: The soaking time is 36-60 h.

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