A method for improving the tolerance and degradation of high-concentration pyridine by acid-fast bacteria

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a method for improving the tolerance and degradation of high-concentration pyridine of acid-fast bacteria. The specific technical solution comprises: a method for improving the tolerance and degradation of pyridine of acid-fast bacteria, and extracting a corncob pith leachate and adding the same to the growth environment of the acid-fast bacteria. It is found for the first time that the corncob pith leachate can effectively improve the tolerance and degradation of high-concentration pyridine of the acid-fast bacteria. After the addition of the corncob pith leachate to the culture environment of the acid-fast bacteria, the acid-fast bacteria can degrade more than 99% of 2700 mg / L or so of pyridine after about 84 h of treatment. The method provided by the present application can effectively improve the effect of the acid-fast bacteria LD-B in degrading high-concentration pyridine, the method is simple, the corncob used is an agricultural waste, is widely available, is low in price, is environmentally friendly and sustainable, greatly reduces the application cost, and has great popularization value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for improving the tolerance of acid-eating bacteria and degrading high concentrations of pyridine. Background Technology

[0002] Coking wastewater contains highly toxic and recalcitrant organic pollutants, making its sustainable treatment challenging. Characteristic pollutants in coking wastewater include high concentrations of inorganic and organic pollutants such as ammonia, sulfides, phenols, nitrogen-containing heterocyclic compounds, and polycyclic aromatic hydrocarbons. Pyridine is a typical nitrogen-containing heterocyclic compound with significant biotoxicity, teratogenicity, and mutagenicity, posing a serious threat to ecosystems and human health. Pyridine possesses both chemical inertness and environmental persistence. Traditional physicochemical treatment methods suffer from high costs, complex processes, high risks of secondary pollution, and inability to completely degrade pollutants. In contrast, biodegradation methods offer advantages such as environmental friendliness, cost-effectiveness, simple processes, and ease of maintenance, achieving efficient removal of pyridine while overcoming the high energy consumption and secondary pollution problems of physicochemical methods.

[0003] While biodegradation methods offer numerous advantages, pyridine's highly stable molecular structure makes it resistant to most microorganisms. The inventors' research group screened and obtained a new microorganism, *Paracidovoraxavenae* LD-B, targeting pyridine as the pollutant. This *Paracidovoraxavenae* LD-B was deposited on August 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.28102; it is also disclosed in patent CN117264831 A. This microorganism exhibits strong adaptability to pyridine and coking wastewater, and can efficiently degrade pyridine. However, studies have found that the microorganism's ability to degrade pyridine gradually decreases with increasing pyridine concentration. When the pyridine concentration exceeds 2500 mg / L, high concentrations of pyridine inhibit microbial growth, and the pyridine degradation ability drops significantly.

[0004] Therefore, if a method can be provided to help improve the LD-B tolerance and ability of acid-eating bacteria to degrade high concentrations of pyridine, it will have significant research and application value. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving the tolerance of acid-eating bacteria and degrading high concentrations of pyridine.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is: a method for improving the tolerance and pyridine degradation ability of acid-eating bacteria, the method comprising: extracting corn cob medulla extract and adding it to the growth environment of acid-eating bacteria.

[0007] Correspondingly, a method for degrading pyridine or treating coking wastewater involves applying acid-eating bacteria and corn cob medullary leachate together to the environment in which the pyridine is to be degraded.

[0008] Preferably, in an environment conducive to the growth of acid-producing bacteria, the concentration of the corn cob medullary extract is 500 mg / L.

[0009] Preferably, the acid-eating bacteria is Paracidovoraxavenae LD-B, which was deposited on August 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.28102.

[0010] Preferably, the method for preparing the corn cob medullary extract includes: taking corn cobs, washing, drying, separating the epidermis, medullary layer, and central layer of the corn cob, taking and breaking the medullary layer, and soaking the medullary layer in water. The drying temperature is 60–100℃. The soaking time is 36–60 hours.

[0011] This invention has the following beneficial effects: For the first time, it has been discovered that corn cob medullary extract can effectively improve the ability of acid-eating bacteria to tolerate and degrade high concentrations of pyridine. By adding corn cob medullary extract to the culture environment of acid-eating bacteria, the bacteria can degrade more than 99% of approximately 2700 mg / L of pyridine after about 84 hours of treatment.

[0012] The method provided by this invention can effectively improve the degradation effect of high-concentration pyridine by acid-producing bacteria LD-B. The method is simple, and the corn cobs used are agricultural waste, which are widely available and inexpensive. It is both environmentally friendly and sustainable, greatly reducing the application cost and has great promotional value. Attached Figure Description

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

[0014] Figure 2 This is the standard curve for pyridine. Detailed Implementation

[0015] This invention provides a method for improving the tolerance of Paracidovoraxavenae to LD-B and its degradation of high concentrations of pyridine. The method includes: extracting a corn cob medulla extract and adding it to the growth environment of Paracidovoraxavenae LD-B, using the corn cob medulla extract as a co-metabolic substrate to promote the degradation of high concentrations of pyridine by the Paracidovoraxavenae.

[0016] The preferred method is to add the corn cob medulla extract at a concentration of 500 mg / L in the LD-B growth environment of acidophilic bacteria. Our research group tested various corn varieties and found that the variety had no significant impact on the results; in this embodiment, the corn variety used was Chuandan 99.

[0017] An optional method for preparing corn cob medullary extract is as follows: The surface of the corn cob is rinsed with deionized water to remove impurities; the cob is dried at 60–100°C; the epidermis, medulla, and mantle are separated; the medulla is collected and broken up. After separation, the epidermis, medulla, and mantle structures are as follows: Figure 1 As shown. The medulla is immersed in deionized water at a mass-to-volume ratio (mg / L) of 1:20-60, preferably 1:40, at room temperature for 36-60 hours, filtered through a 0.45μm water filter, and then sterilized under high temperature and high pressure.

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All obtained data are average values ​​obtained after at least three repetitions, and each repetition yields valid data.

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

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

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

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

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

[0024] 1. Pyridine Determination Method: The absorbance of the culture medium after cell removal was measured at 256 nm using a microplate reader. The concentration of pyridine was determined using a pyridine standard curve, and the degradation rate was calculated. The degradation rate η of pyridine was calculated using the following formula:

[0025]

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

[0027] 2. Method for establishing the pyridine standard curve: Using a laboratory microplate reader, the wavelength of the characteristic absorption peak of pyridine was determined to be 256 nm using full-wavelength ultraviolet scanning. The pyridine standard curve is shown below. Figure 2 As 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 acid-eating bacteria LD-B

[0029] Strain LD-B was inoculated into LB medium and cultured at 30℃ and 150 rpm for 2 days with shaking until the late logarithmic phase. After centrifugation at 5000 rpm for 10 min, the culture was washed three times with sterilized inorganic salt medium, and the OD was adjusted. 600 =1.0, to obtain the seed culture. Unless otherwise specified, the preparation method of the LD-B seed culture is the same as described here.

[0030] Rice husks, peanut shells, wheat straw, corn straw, and corn cobs were washed, dried at 45℃, and pulverized. Biomass materials and deionized water were added at a mass-to-volume ratio of 1:40 (g / mL). Each biomass material was then immersed in deionized water and soaked at room temperature for 48 hours. The resulting seed solution was inoculated at a rate of 5% (v / v) into an inorganic salt medium containing a high concentration of pyridine, supplemented with the extract of each biomass material (2% (v / v)). The initial concentration of pyridine was approximately 2700 mg / L. The culture temperature was controlled at 30℃, and the rotation speed was 150 r / min with shaking. Samples were taken after 12–96 hours of culture, centrifuged at 15000 r / min for 15 min, and the absorbance of the supernatant (culture medium after removing bacterial cells) at 256 nm was measured to calculate the degradation rate. A blank control group was also set up, with all other conditions the same, but without the addition of biomass material extract, and with an equal volume of deionized water added to the inorganic salt medium. The degradation results are shown in Table 1. It should be noted that the experiment did not only test the biomass materials listed in Table 1. Due to space limitations, only some of the materials with more ideal results are shown here.

[0031] Table 1. Degradation of high-concentration pyridine by LD-B after adding different biomass materials.

[0032]

[0033]

[0034] The results showed that corn cob had the most significant promoting effect on the degradation of high concentrations of pyridine by the acid-producing bacteria LD-B. Therefore, corn cob was selected for subsequent examples.

[0035] Example 2: Effect of corn cob leachate concentration on pyridine degradation

[0036] The corn cobs were washed, dried at 45℃, and crushed. The ratio of corn cobs to deionized water was 1:40 (mg / L). The corn cobs were then soaked in deionized water at room temperature for 48 hours to obtain corn cob extract.

[0037] The LD-B seed culture of *Acidobacterium glutamicum* was inoculated at a rate of 5% (v / v) into inorganic salt media containing different concentrations of corn cob extract. Pyridine was additionally added to the inorganic salt media, with an initial concentration of approximately 2645 mg / L. The culture was maintained at 30℃ and a shaking speed of 150 rpm. Samples were taken at 24 h, 48 h, 72 h, and 96 h to measure the absorbance of the culture medium after cell removal at 256 nm, and the pyridine degradation rate was calculated. A blank control group was also included, with all other conditions identical except for the addition of corn cob extract and the addition of an equal volume of deionized water to the inorganic salt media. The degradation results are shown in Table 2.

[0038] Table 2. Degradation of high-concentration pyridine by LD-B after adding different concentrations of corn cob.

[0039]

[0040] Example 3: The effect of different corn cob structures on pyridine degradation

[0041] The corn cob was rinsed with deionized water to remove surface impurities and dried at 80°C. The outer layer, medulla, and core layer of the corn cob were separated by physical means, and each component was crushed to 18 mesh using a crusher. The outer layer, medulla, and core layer of the corn cob were then immersed in deionized water at a mass-to-volume ratio of 1:40 (mg / L) for 48 hours at room temperature to obtain leachates for each material.

[0042] The seed culture of strain LD-B was inoculated at a rate of 5% (v / v) into pyridine-containing inorganic salt medium supplemented with extracts of the corn cob epidermis, medulla, and mantle. The concentration of pyridine was approximately 2700 g / L, and the concentration of each corn cob component was 500 mg / L. The culture was maintained at 30℃ and 150 rpm with shaking. Samples were taken after 12–96 h of culture to measure the absorbance of the culture medium after cell removal at 256 nm, and the degradation rate was calculated. A blank control group was also included, with all other conditions identical except for the addition of corn cob extract and the addition of an equal volume of deionized water to the inorganic salt medium containing the biomass extract. The degradation results are shown in Table 3.

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

[0044]

[0045] The results showed that the corn cob medulla was more effective in promoting the degradation of high concentrations of pyridine by strain LD-B, possibly because it contains the main active ingredient that promotes the degradation of strain LD-B, or because it contains certain substances that can synergistically work with strain LD-B.

[0046] Example 4: The effect of corn cob medullary layer state on pyridine degradation efficiency

[0047] A corn cob medulla extract was obtained according to the method described in Example 3. Simultaneously, the corn cob medulla was obtained according to the method described in Example 3 and pulverized for later use.

[0048] The seed culture of strain LD-B was inoculated at a rate of 5% (v / v) into pyridine-containing inorganic salt medium supplemented with corn cob medulla and corn cob medulla extract, respectively. The initial concentration of pyridine in the medium was controlled at approximately 2700 mg / L, and the concentrations of corn cob medulla solids and medulla extract in the inorganic salt medium were both 500 mg / L. The culture was maintained at 30℃ and 150 r / min with shaking. Samples were taken after 12–96 h of culture to measure the absorbance of the culture medium after cell removal at 256 nm to calculate the degradation rate. A blank control group was also included, with all other conditions identical except that corn cob extract was not added, and an equal volume of deionized water (equal to the amount of biomass extract) was added 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 the addition of corn cob medullary solids and leachate.

[0050]

[0051] The results showed that both the solid corn cob medulla and the medulla extract significantly enhanced the ability of strain LD-B to degrade high concentrations of pyridine. This indicates that the adsorption effect of the corn cob medulla is not the main reason for LD-B's ability to degrade pyridine; rather, it is more likely that certain water-soluble active ingredients in the corn cob medulla exert a synergistic effect with LD-B.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for improving the pyridine tolerance of acid-eating bacteria, characterized in that: The method includes: extracting a corn cob medulla extract and adding it to the growth environment of acid-eating bacteria; the concentration of pyridine in the environment is above 2500 mg / L; the acid-eating bacteria is classified as Paracidovorax sp. LD-B and was deposited at the China General Microbiological Culture Collection Center on August 4, 2023, with the accession number CGMCC NO. 28102.

2. A method for improving the ability of acid-consuming bacteria to degrade pyridine, characterized in that: The method includes: extracting a corn cob medulla extract and adding it to the growth environment of acid-eating bacteria; the concentration of pyridine in the environment is above 2500 mg / L; the acid-eating bacteria is classified as Paracidovorax sp. LD-B and was deposited at the China General Microbiological Culture Collection Center on August 4, 2023, with the accession number CGMCC NO. 28102.

3. A method for degrading pyridine, characterized in that: The acid-eating bacteria and corn cob medullary extract were jointly applied to the environment containing pyridine to be degraded; the concentration of pyridine in the environment was above 2500 mg / L; the acid-eating bacteria was classified as Paracidovorax sp. LD-B and was deposited at the China General Microbiological Culture Collection Center on August 4, 2023, with accession number CGMCC NO. 28102.

4. A method for treating coking wastewater, characterized in that: The acid-eating bacteria and corn cob medullary extract were jointly applied to the environment containing pyridine to be degraded; the concentration of pyridine in the environment was above 2500 mg / L; the acid-eating bacteria was classified as Paracidovorax sp. LD-B and was deposited at the China General Microbiological Culture Collection Center on August 4, 2023, with accession number CGMCC NO. 28102.

5. The method according to any one of claims 1 to 4, characterized in that: In an environment conducive to the growth of acid-loving bacteria, the concentration of the corn cob medullary extract added is 500 mg / L.

6. The method according to any one of claims 1 to 4, characterized in that: The method for preparing the corn cob medullary extract includes: taking corn cobs, washing, drying, separating the corn cob epidermis, medullary layer and central layer structure, taking the medullary layer and breaking it, and soaking the medullary layer in water.

7. The method according to claim 6, characterized in that: The drying temperature is 60-100℃.

8. The method according to claim 6, characterized in that: Soaking time is 36–60 hours.

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