A method for degrading p-hydroxybenzoic acid, a degrading bacterial agent and application thereof

Abstract

This invention relates to a method for degrading p-hydroxybenzoic acid (p-hydroxybenzoic acid), a degrading microbial agent, and its applications. The microbial agent is a fermentation broth or cell preparation of Acinetobacter JS-1. The degradation method involves inoculating Acinetobacter JS-1 into a culture medium containing p-hydroxybenzoic acid for fermentation, thereby degrading p-hydroxybenzoic acid. This method achieves a 97% degradation rate of 0.75 g / L p-hydroxybenzoic acid within 12 hours and almost completely degrades 1.5 g / L p-hydroxybenzoic acid within 48 hours, demonstrating high degradation efficiency. This microbial agent can be used to degrade p-hydroxybenzoic acid in soil and promote plant growth. Using potted cucumbers as experimental subjects, application of the microbial agent effectively alleviated the autotoxicity of cucumbers, increasing the aboveground plant height by approximately 34.8% and the underground root length by approximately 31.9%. This has significant value in alleviating continuous cropping obstacles caused by p-hydroxybenzoic acid and improving the soil environment.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology and ecological restoration technology, specifically to a method for degrading p-hydroxybenzoic acid, a degrading agent, and its application. Background Technology

[0002] p-Hydroxybenzoic acid (PHBA or 4-HBA) possesses an ideal structure of benzene ring, carboxyl group, and hydroxyl group, and has been widely used in the production of preservatives, dyes, pharmaceuticals, food, and cosmetics. The large-scale production and use of p-hydroxybenzoic acid has led to its continuous accumulation in the environment, posing potential risks to plants, animals, and humans (Li Yi-Xi, Lin Wei, Yong-He et al. Biodegradation of p-hydroxybenzoic acid in Herbaspirillum aquaticum KLS-1 isolated from tailing soil: Characterization and molecular mechanism[J]. Journal of Hazardous Materials, 2023, 456: 131669). p-Hydroxybenzoic acid is also one of the main autotoxic substances produced by plant allelopathic processes, entering the soil through root exudation and the decomposition of plant residues, thereby affecting plants, soil microorganisms, and soil nutrient cycling. The accumulation of p-hydroxybenzoic acid in the soil causes continuous cropping obstacles, significantly reducing soil fertility and crop yield. Numerous studies have confirmed that phenolic acids are the most prevalent autotoxic substances in common greenhouse vegetables and fruits, with p-hydroxybenzoic acid being the most prevalent and highly toxic in the root exudates of strawberries and cucumbers. (Isolation and application of the autotoxic substance p-hydroxybenzoic acid degrading bacteria ZH2, Journal of Agronomy, 2021, 11(7): 84-91) P-hydroxybenzoic acid causes crop rotation obstacles, such as in cucumbers, tomatoes, strawberries, and tobacco, which has seriously affected the growth of crop seedlings and damaged the economic benefits of crops (Wu Fenghui, An Yan-Qiu, An Yanrong et al. Acinetobacter calcoaceticus CSY-P13 Mitigates Stress of Ferulic and p-Hydroxybenzoic Acids in Cucumber by Affecting Antioxidant Enzyme Activity and Soil Bacterial Community[J]. Frontiers in Microbiology, 2018, 9: 1262.). Meanwhile, p-hydroxybenzoic acid in the soil also stimulates the growth of rhizosphere pathogens, leading to an imbalance in the rhizosphere microbial community structure, exacerbating soil-borne infectious diseases, and seriously restricting the sustainable development of soil ecology.

[0003] Currently, chemical degradation and bioremediation are the main methods for treating p-hydroxybenzoic acid (p-hydroxybenzoic acid) pollution. Microorganisms can convert p-hydroxybenzoic acid into protocatechuic acid through the action of monooxygenases. Finally, under the action of a series of hydrolases, hydratases, and aldolases, it is metabolized into acetyl-CoA and succinyl-CoA, which enter the citric acid cycle. In the entire metabolic process, the activity of key enzymes affects the degradation efficiency of p-hydroxybenzoic acid. For example, studies have shown that the catechol ring cleavage products cis and cis mucoconazole formed during the degradation of p-hydroxybenzoic acid inhibit the utilization of p-hydroxybenzoic acid. Specifically, different strains have different degradation abilities and substrate tolerances for p-hydroxybenzoic acid. For example, Acinetobacter K7 can completely degrade 0.1 g / L p-hydroxybenzoic acid within 6 h at 30 ℃ and pH 7 (Li Min, Qu Huan, Liu Jianli, et al., Degradation efficiency of Acinetobacter K7 for phenolic acid compounds [J]. Journal of Nanjing Agricultural University, 2019, 42(4): 689-696.). Acinetobacter calcoaceticus WH-B2 can completely degrade approximately 0.4 g / L of p-hydroxybenzoic acid within 16 hours (Yang Pengfei, Biological characteristics of Acinetobacter calcoaceticus WH-B2 and its degradation of benzoic acid, an autotoxin of peach [D]. Huazhong Agricultural University, 2018.). Acinetobacter johnsonii FZ-5 can degrade approximately 71.04% of 2 g / L of p-hydroxybenzoic acid under anaerobic conditions for 72 hours (Lu Peng, Huang Huiying, Sun Yixiao et al. Biodegradation of 4-hydroxybenzoic acid by Acinetobacter johnsonii FZ-5 and Klebsiella oxytoca FZ-8 under anaerobic conditions [J]. Biodegradation, 2022, 33: 17-31). However, current research on the microbial degradation of p-hydroxybenzoic acid mostly focuses on concentrations below 1 g / L. Obviously, obtaining strains that can tolerate high concentrations of substrate and have high degradation efficiency has greater potential application value. Therefore, screening strains with high tolerance to high concentrations and high degradation efficiency of p-hydroxybenzoic acid and applying them to soil remediation for crop continuous cropping obstacles can provide a scientific basis for improving the soil environment for crop cultivation and increasing crop yield and quality, and has important application value. Summary of the Invention

[0004] The primary objective of this invention is to provide a p-hydroxybenzoic acid degrading bacterial agent.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0006] A p-hydroxybenzoic acid degrading bacterial agent, wherein the bacterial agent is the fermentation broth or cell preparation of Acinetobacter JS-1;

[0007] The bacterial preparation is obtained by centrifuging the fermentation broth to remove the supernatant and then resuspending the bacterial cells in water.

[0008] The Acinetobacter JS-1 accession number is CCTCC M 20221600.

[0009] In a preferred embodiment, the Acinetobacter bacillus is fermented in LB medium to obtain a fermentation broth.

[0010] In a preferred embodiment, the bacterial preparation is used after being diluted to OD600 = 1.

[0011] The second objective of this invention is to provide the application of the above-mentioned microbial agent in the degradation of p-hydroxybenzoic acid in soil and in promoting plant growth; wherein the plant is a crop that secretes p-hydroxybenzoic acid during its growth.

[0012] In a preferred embodiment, the microbial agent is applied to the soil at a ratio of 200-300 mg / kg soil.

[0013] In a preferred embodiment, the microbial agent is applied to the soil together with sodium bicarbonate.

[0014] In a preferred embodiment, the amount of sodium bicarbonate used is 200-300 mg / kg soil.

[0015] Furthermore, the microbial agent of the present invention can also be applied to the preparation of microbial fertilizers for the remediation of p-hydroxybenzoic acid soil.

[0016] A third objective of this invention is to provide a method for degrading p-hydroxybenzoic acid, comprising inoculating Acinetobacter JS-1 into a culture medium containing p-hydroxybenzoic acid for fermentation culture to degrade p-hydroxybenzoic acid;

[0017] The Acinetobacter JS-1 accession number is CCTCC M 20221600.

[0018] In a preferred embodiment, the culture medium is a BSM medium using sodium nitrate, urea, or yeast extract as the nitrogen source.

[0019] In a preferred embodiment, sodium pyruvate is added to the culture medium as an external carbon source. Adding 0.2 g / L sodium bicarbonate, sodium pyruvate, or sodium succinate as external carbon sources all promote the degradation of p-hydroxybenzoic acid (1 g / L). Among these, the effects of adding sodium pyruvate and sodium bicarbonate are more significant, achieving degradation rates of 15.66% and 13.60% for p-hydroxybenzoic acid (1 g / L), respectively, at 15°C, pH 7, and 12 hours.

[0020] The method of this invention is highly efficient in degrading p-hydroxybenzoic acid (0.25-2.0 g / L). It exhibits excellent degradation effects on p-hydroxybenzoic acid (1.5 g / L) at 30°C within 48 hours, achieving a degradation rate of 99.33%. Within 12 hours, the degradation rate of 0.75 g / L p-hydroxybenzoic acid reaches 97%. Adding 0.2 g / L sodium bicarbonate, sodium pyruvate, or sodium succinate as external carbon sources can all promote the degradation of 1 g / L p-hydroxybenzoic acid. Considering the cost of external carbon sources, sodium bicarbonate is preferred. The live bacteria preparation prepared by this invention can be used to degrade p-hydroxybenzoic acid-related autotoxic substances produced by cucumbers in soil, alleviate continuous cropping obstacles, and promote cucumber seedling growth. Attached Figure Description

[0021] Figure 1 This is the standard curve for p-hydroxybenzoic acid.

[0022] Figure 2 The degradation rate of p-hydroxybenzoic acid by strain JS-1 at different concentrations is shown.

[0023] Figure 3 The effect of nitrogen source on the degradation of p-hydroxybenzoic acid by strain JS-1.

[0024] Figure 4 The effect of pH on the degradation of p-hydroxybenzoic acid by strain JS-1.

[0025] Figure 5 The effect of temperature on the degradation of p-hydroxybenzoic acid by strain JS-1.

[0026] Figure 6 The effect of NaCl concentration on the degradation of p-hydroxybenzoic acid by strain JS-1.

[0027] Figure 7 The effect of an external carbon source on the degradation of p-hydroxybenzoic acid by strain JS-1.

[0028] Figure 8 The effects of different treatment conditions on cucumber seedling growth. Detailed Implementation

[0029] Reagent and culture medium formulations:

[0030] LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L.

[0031] LB solid medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, agar 20 g / L.

[0032] BSM fermentation medium: NaNO3 2g / L, NaCl 10g / L, CaCl2 0.1g / L, K2HPO4 2.8g / L, KH2PO4 0.5g / L, MgSO4 0.1g / L, FeSO4 0.01g / L.

[0033] The strain JS-1 involved in the examples has been disclosed in the applicant's prior application CN116240134A, and the accession number of strain JS-1 is CCTCC M 20221600.

[0034] Example 1

[0035] This example illustrates the plotting of the standard curve for p-hydroxybenzoic acid.

[0036] A standard solution of p-hydroxybenzoic acid was prepared using BSM culture medium, with a concentration range of 0.000-0.01 mg / mL. A blank tube was used, and the absorbance was measured at 255 nm using a quartz cuvette. A standard curve was plotted with absorbance on the ordinate and p-hydroxybenzoic acid concentration on the abscissa. Figure 1 The absorbance values ​​at 255 nm for different concentrations of p-hydroxybenzoic acid are shown in Table 1.

[0037] Table 1 Absorbance values ​​of p-hydroxybenzoic acid at different concentrations

[0038]

[0039] Example 2

[0040] This example illustrates the tolerance and degradation ability of strain JS-1 to p-hydroxybenzoic acid.

[0041] A single colony of JS-1 was picked and inoculated into LB liquid medium and cultured at 37℃ and 200 rpm for 12 h to obtain a seed culture. 1 mL of the seed culture was inoculated into BSM fermentation medium, and 0.25, 0.5, 0.75, 1.00, 1.25, 1.5 g / L, and 2 g / L of p-hydroxybenzoic acid were added as carbon sources, respectively. The medium was then incubated in a 30℃ shaker at 200 rpm, and samples were taken every 3 h to measure the OD of the strain. 600 Value and degradation rate of p-hydroxybenzoic acid. Experimental results are as follows: Figure 2As shown, when the carbon source concentration was 0.25 g / L, the degradation rate of strain JS-1 reached its highest level, approximately 97.51%, at 6 hours. Strain JS-1 achieved a 97% degradation rate of 0.75 g / L p-hydroxybenzoic acid at 12 hours of fermentation. At 48 hours, the degradation rate of 1.5 g / L p-hydroxybenzoic acid reached 99.33%. At a p-hydroxybenzoic acid concentration of 2 g / L, strain JS-1 achieved a degradation rate of approximately 79.8% at 72 hours. The efficiency of strain JS-1 in degrading p-hydroxybenzoic acid is significantly higher than that of Acinetobacter K7, Acinetobacter calcium acetate WH-B2, and Acinetobacter riyoger FZ-5, making it the only reported hemolytic Acinetobacter strain with p-hydroxybenzoic acid degradation capabilities. Furthermore, strain JS-1's p-hydroxybenzoic acid degradation does not rely on external supplementation of growth factors (yeast extract), making its culture conditions more flexible and convenient compared to Acinetobacter K7. Furthermore, the degradation of p-hydroxybenzoic acid by Acinetobacter riyoger FZ-5 requires anaerobic conditions, which are difficult to achieve in practice. Complex processes or expensive reagents may be needed to effectively address p-hydroxybenzoic acid contamination under anaerobic conditions. Therefore, compared to most reported Acinetobacter species, strain JS-1 is more efficient at degrading p-hydroxybenzoic acid and has practical application value.

[0042] Example 3

[0043] This example illustrates the effect of nitrogen sources on the degradation of p-hydroxybenzoic acid by strain JS-1.

[0044] A single colony of JS-1 was picked and inoculated into LB liquid medium and cultured at 37℃ and 200 rpm for 12 h to obtain a seed culture. 1 mL of the seed culture was inoculated into BSM medium containing 0.75 g / L p-hydroxybenzoic acid as a carbon source, with 2 g / L each of ammonium chloride, ammonium sulfate, sodium nitrate, disodium hydrogen phosphate, urea, and yeast extract added as nitrogen sources. The medium was then incubated in a 30℃ shaker at 200 rpm. The OD of the strain was measured at 6 h. 600 Value and degradation rate of p-hydroxybenzoic acid. Experimental results are as follows: Figure 3 As shown, strain JS-1 can utilize sodium nitrate, urea, and yeast extract as nitrogen sources to grow and degrade p-hydroxybenzoic acid, with urea being the optimal nitrogen source.

[0045] Example 4

[0046] This example illustrates the effect of different initial pH conditions on the degradation of p-hydroxybenzoic acid by strain JS-1.

[0047] 1 mL of JS-1 seed culture was inoculated into BSM medium containing 0.75 g / L p-hydroxybenzoic acid as a carbon source and 2 g / L urea as a nitrogen source. The pH of the medium was adjusted to 5-9 using NaOH and hydrochloric acid, respectively. The medium was incubated at 30°C in a shaker at 200 rpm. The OD of the strain was measured at 6 h. 600 Value and degradation rate of p-hydroxybenzoic acid. Experimental results are as follows: Figure 4 As shown, strain JS-1 can grow under conditions ranging from pH 6 to 9; among them, when pH = 7, the OD of strain JS-1 is... 600 The value was 0.388, and the degradation rate was 33%; at pH=8, the OD of strain JS-1 was... 600 The value was 0.416, and the degradation rate was 31.49%; under alkaline conditions at pH 9, the OD of the strain was... 600 Both the value and degradation rate decreased.

[0048] Example 5

[0049] This example illustrates the degradation of p-hydroxybenzoic acid by strain JS-1 under different temperature conditions.

[0050] 1 mL of JS-1 seed culture was inoculated into BSM medium (pH = 7) containing 0.75 g / L p-hydroxybenzoic acid and 2 g / L urea, and cultured in constant temperature shakers at 15℃, 20℃, 25℃, 30℃, 35℃, and 40℃. The OD of the strain was measured at 6 h, 12 h, and 24 h. 600 Value and degradation rate of p-hydroxybenzoic acid. Experimental results are as follows: Figure 5 As shown, strain JS-1 can utilize p-hydroxybenzoic acid as a carbon source for growth under conditions ranging from 15 to 40℃, and the degradation rate can reach 99.8% within 24 hours. Therefore, strain JS-1 has a relatively wide temperature adaptability and can metabolize p-hydroxybenzoic acid under ambient temperatures ranging from 15 to 40℃, with the metabolic rate increasing at higher temperatures.

[0051] Example 6

[0052] This example illustrates the degradation of p-hydroxybenzoic acid by strain JS-1 under different salt concentrations.

[0053] 1 mL of seed culture was inoculated into BSM medium (pH = 7) containing 0.75 g / L p-hydroxybenzoic acid and 2 g / L urea. The NaCl concentration of the medium was adjusted to 0-40 g / L. The culture was placed in a 30°C constant temperature shaker and incubated at 200 rpm. The OD of the strain was measured at 6 h. 600 Value and degradation rate of p-hydroxybenzoic acid. Experimental results are as follows: Figure 6 As shown, when the salt concentration is 10 g / L, the OD of the strain... 600The value was 0.352, the degradation rate was 31.85%, and the OD value of the strain was [not specified] when the salt concentration was higher than 20 g / L. 600 Both the degradation value and the degradation rate decreased, and the growth of the strain was significantly inhibited.

[0054] Example 7

[0055] This example illustrates the effect of an external carbon source on the degradation of p-hydroxybenzoic acid by strain JS-1.

[0056] A single colony of JS-1 was picked and inoculated into LB liquid medium and cultured at 37℃ and 200 rpm for 12 h to obtain a seed culture. 1 mL of the seed culture was inoculated into BSM medium containing 1 g / L p-hydroxybenzoic acid and 2 g / L urea. 0.2 g / L sodium bicarbonate, sodium pyruvate, sodium succinate, and sodium acetate were added as external carbon sources. A p-hydroxybenzoic acid degradation experiment was conducted at 15℃, pH 7, and a NaCl concentration of 10 g / L. The OD of the strain was measured after 12 h of culture. 600 Value and degradation rate. Multiple comparisons of degradation rates were performed using the LSD method (P < 0.05), and the experimental results are as follows: Figure 7 As shown, the addition of sodium bicarbonate and sodium succinate did not significantly promote the degradation of p-hydroxybenzoic acid by strain JS-1, while the addition of sodium pyruvate had a more significant effect, with the degradation rate of p-hydroxybenzoic acid reaching 15.66%.

[0057] Example 8

[0058] This example illustrates the preparation of a suspension of strain JS-1.

[0059] Single colonies of JS-1 were picked and inoculated onto LB liquid medium. After incubation at 37°C and 200 rpm for 12 hours to obtain the seed culture, the supernatant was removed by centrifugation, and the culture was resuspended in sterile water until the bacterial concentration reached OD500. 600 The value is 1 (2×10 8 A suspension of strain JS-1 was obtained by preparing cfu / mL.

[0060] Example 9

[0061] This example illustrates the application effect of strain JS-1 in the degradation of p-hydroxybenzoic acid in cucumber cultivation.

[0062] Cucumber seeds were sown in sterilized potting soil and placed in a light-incubated incubator. On the 7th day, the cucumber plants were transplanted. Each pot contained 120g of potting soil (60g of sterilized dry potting soil mixed with 60mL of sterile water or treatment solution). After transplanting, the pots were placed in a greenhouse at 25℃ with a light intensity of 5000 lux, and watered with 5mL of sterile water daily.

[0063] The following treatment groups were set up: Treatment 1 consisted of cucumbers grown in normal nutrient soil, serving as a blank control. Treatment 2 included OD245... 600 The final concentration of the JS-1 bacterial suspension with a value of 1 was 220 mg / kg soil. In treatment three, p-hydroxybenzoic acid was applied, resulting in a p-hydroxybenzoic acid content of 220 mg / kg soil in the nutrient soil. In treatment four, p-hydroxybenzoic acid and OD were applied... 600 JS-1 bacterial suspension with a value of 1 was applied at 220 mg / kg soil. In treatment five, 220 mg / kg soil of p-hydroxybenzoic acid and OD were applied. 600 JS-1 bacterial suspension (value 1) at 220 mg / kg soil and sodium bicarbonate at 220 mg / kg soil were used. Five replicates were set up for each treatment group. After 15 days of growth in a light incubator, cucumber seedling height, stem diameter, root number, maximum leaf transverse and longitudinal diameters, and leaf color were recorded. Growth characteristics of each group are shown below. Figure 8 Growth data are shown in Table 2.

[0064] The cucumber seedlings in the blank control group (treatment 1) had smaller stem diameters and leaf transverse and longitudinal diameters than those in treatment 2, which included the addition of bacterial strain JS-1. After applying JS-1 bacterial suspension, the aboveground plant height increased by approximately 34.8%, and the underground root length increased by approximately 31.9%, indicating that applying JS-1 bacterial suspension to the cultivation soil can promote the growth of cucumber seedlings. The mechanism may be that JS-1 bacterial suspension degrades p-hydroxybenzoic acid, an autotoxic substance synthesized during cucumber growth. After adding 220 mg / kg of p-hydroxybenzoic acid to the soil (treatment 3), the toxic effects on cucumber seedlings were mainly manifested as stunted growth and yellowing of leaf edges. After applying JS-1 bacterial suspension to the soil with added p-hydroxybenzoic acid (treatment 4), the cucumber seedlings grew more robustly, with greener leaves, increased stem diameter, and larger leaves, further demonstrating that JS-1 bacterial suspension can reduce the toxic effects of p-hydroxybenzoic acid in the soil on cucumber seedlings and promote their growth. Based on treatment four, treatment five additionally added 220 mg / kg of sodium bicarbonate to the soil. The cucumber seedling height, maximum leaf transverse diameter, longitudinal diameter, and root length were all higher in treatment group than in other treatment groups, indicating that the weakly alkaline environment created by the low-concentration sodium bicarbonate solution enhances the growth-promoting properties of JS-1 bacterial suspension. These results show that applying JS-1 bacterial agent to the soil in cucumber seedling cultivation can alleviate the autotoxic effects of cucumber growth, mitigate or eliminate the soil toxicity of p-hydroxybenzoic acid, and promote the growth of cucumber seedlings.

[0065] Table 2. Measurement of plant height, number of roots, transverse stem, longitudinal stem, and stem diameter of cucumber seedlings.

[0066]

Claims

1. The application of a p-hydroxybenzoic acid degrading bacterial agent in the degradation of p-hydroxybenzoic acid in soil and in promoting plant growth, characterized in that, The plant is a cucumber that secretes p-hydroxybenzoic acid during its growth; the bacterial agent is the fermentation broth or bacterial preparation of Acinetobacter hemolyticus JS-1; The bacterial preparation is obtained by centrifuging the fermentation broth to remove the supernatant and then resuspending the bacterial cells in water. The accession number for the Acinetobacter hemolyticus JS-1 is CCTCC M 20221600.

2. The application according to claim 1, characterized in that, The hemolytic Acinetobacter was fermented in LB medium to obtain the fermentation broth.

3. The application according to claim 1, characterized in that, The bacterial preparation is used after being diluted to OD600=1.

4. The application according to claim 1, characterized in that, The microbial agent is applied to the soil at a ratio of 200-300 mg / kg soil.

5. The application according to claim 1, characterized in that, The bacterial agent was applied to the soil together with sodium bicarbonate.

6. The application according to claim 5, characterized in that, The amount of sodium bicarbonate used is 200-300 mg / kg soil.

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