Method for degrading organochlorine compounds by using ginseng endophytic bacteria

Abstract

The invention discloses a method for degrading organochlorine pesticide residues by using endophytic bacteria separated from healthy ginseng. According to the method, an endophytic bacterium Pseudomonas poae nH121 obtained from healthy ginseng is utilized, and the strain can degrade quintozene and hexachlorobenzene after being fermented and can be used for removing quintozene and hexachlorobenzene compounds in an old ginseng field polluted by pesticides. The method is easy to operate, green, safe, efficient and economical, and a new method can be provided for removing organochlorine pesticide residues in the environment.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for degrading organochlorine pesticide residues pentachloronitrobenzene and hexachlorobenzene by utilizing ginseng endophytic bacteria fermentation liquid. Background Art

[0002] Currently, approximately one-third of agricultural products are produced using pesticides. According to statistics, 4.6 million tons of chemical pesticides are sprayed into the environment annually worldwide. Due to low pesticide utilization, most pesticides are released into non-target soil, water, and the atmosphere, ultimately being absorbed by organisms. In the past, organochlorine pesticides were used extensively and irrationally, posing a significant potential threat to the ecological environment and human health. Therefore, finding efficient methods to degrade organochlorine pesticide residues is crucial. Research has shown that organochlorine pesticides have the following characteristics: 1) They are chemically stable, have long half-lives, and are difficult to degrade; 2) they have low polarity and can accumulate in adipose tissue, exhibiting bioaccumulation and biomagnification; 3) they are highly toxic, with teratogenic effects, potentially damaging the endocrine, immune, hematopoietic, digestive, and reproductive systems. 4) they can migrate long distances through various environmental media, becoming widely distributed in water, air, soil, and other environmental media, as well as in some organisms.

[0003] Pentachloronitrobenzene (PCNB), a typical organochlorine protective fungicide, has been widely used in my country for some time. It is a common seed dressing and soil treatment agent, particularly for the prevention and control of soilborne diseases. It has a broad spectrum of activity, showing significant efficacy against various fungal diseases, including damping-off, damping-off, anthracnose, and sclerotinia. Hexachlorobenzene, a byproduct of its production process, is even more toxic. PCNB has a long residual life, with a soil half-life of approximately 4 to 10 months. It can also volatilize into the atmosphere and has a photolytic half-life of 2200 days. In the environment, it metabolizes into various toxic metabolites, such as pentachloroaniline and methyl pentachlorophenyl sulfide. These products are highly toxic, carcinogenic, persistent, and bioaccumulative. They not only pollute the environment but can also cause immune and reproductive system problems in humans and animals, and can even induce cancer. Currently, remediation methods for organochlorine pesticide contamination can be categorized into physical, chemical, and biological methods. Microbial remediation, a bioremediation technique, utilizes naturally occurring or cultured functional microbial communities to promote or enhance microbial metabolism under suitable environmental conditions, thereby reducing the activity of toxic pollutants or degrading them into non-toxic substances. This method offers advantages such as ease of operation, environmental friendliness, and a low production of toxic byproducts. It is currently recognized as a safe and effective way to remove residual pentachloronitrobenzene and its products. However, due to the high toxicity of organochlorine pesticides, it is currently difficult to isolate microorganisms that can effectively degrade them. Existing reports indicate that microorganisms such as Paenibacillus polymyxa, Arthrobacter nicotianae, Achromobacter xylosoxidans, Bacteroides distasonis, Nocardioides sp., and Labrys portucalensis, or complex bacterial agents composed of bacteria, fungi, and actinomycetes, can degrade pentachloronitrobenzene. However, these degradation efficiencies are low and still cannot meet current production needs, requiring further research. Therefore, the continued research and discovery of effective microbial remediation bacteria for pentachloronitrobenzene and its byproducts is a key focus of microbial remediation technology in this field.

[0004] Ginseng (Panax ginseng CA Meyer) is a major traditional Chinese medicinal herb in my country. The Compendium of Materia Medica records that its medicinal part is the root, which is sweet, slightly cold, and non-toxic, making it a top-grade tonic. Ginseng is a perennial herb that is susceptible to various diseases during the seedling stage, such as ginseng damping-off, ginseng damping-off, ginseng Fusarium root rot, and ginseng rust. During its growth, pests and diseases also frequently occur. Currently, pesticide application is the primary method for control, but regulations regarding the type, frequency, and dosage of pesticides are unscientific, and pesticide residues have become a major exogenous pollutant of ginseng. In the past, organochlorine compounds were widely used to control ginseng pests and diseases. Research on the risk profile of pesticide residues in ginseng has shown that the main pesticide residues in ginseng are organochlorine pesticides such as tetrachloronitrobenzene, pentachloronitrobenzene (PCNB), and hexachlorobenzene. As people's living standards improve, the demand for the safety of traditional Chinese medicines is also increasing. The 2020 Chinese Pharmacopoeia stipulates that the residue limit for pentachloronitrobenzene and hexachlorobenzene in ginseng must not exceed 0.10 mg / mL. Due to their long half-life and difficulty in degradation, organochlorine pesticides have become a major obstacle to ginseng exports. This is especially true in countries like Japan, South Korea, and the European Union, where ginseng is classified as food and the pesticide residue types and limits are more stringent than in China. The long-term residue of PCNB in ​​the soil of ginseng cultivation sites in my country poses a serious obstacle to the reuse of old ginseng fields and is a significant factor affecting ginseng quality. Degrading PCNB in ​​the soil is one of the key technologies for restoring the function of ginseng fields.

[0005] Pseudomonas poae has been isolated from grassland phyllospheres, ginseng roots, seeds of Panax notoginseng (a closely related wild species of Panax notoginseng), tomatoes, cabbage, and young radish. It is a multifunctional biocontrol strain with great potential. Pseudomonas poae JA01, an endophytic bacterium from five-year-old ginseng roots, has been reported to exhibit strong antifungal activity against plant pathogenic fungi such as Pythium ultimum, Phytophthora capsici, and Rhizoctonia solani. Pseudomonas poae also promotes the colonization of Pseudostellaria heterophylla roots and inhibits Fusarium wilt mediated by Fusarium oxysporum. Under drought stress, Pseudomonas poae S61 promotes root growth and the accumulation of active secondary metabolites in Astragalus membranaceus. Furthermore, Pseudomonas poae has been used as an aerobic denitrifier in rural wastewater treatment. However, there are currently no reports on its use in the degradation of organochlorine pesticide residues. The fermentation bacteria Pseudomonaspoae nH121 in this invention is isolated from healthy ginseng. Therefore, using this bacterium to degrade pentachloronitrobenzene and hexachlorobenzene through fermentation is an environmentally friendly and effective method for degrading organochlorine pesticide residues, and has potential application in degrading organochlorine pesticide residues in aged ginseng fields. Summary of the Invention

[0006] The present invention discloses a method for degrading organochlorine pesticide residues using an endophytic bacterium isolated from healthy ginseng. The method utilizes the endophytic bacterium, Pseudomonas poae nH121, isolated from healthy ginseng, as a fermentation bacterium. Pentachloronitrobenzene and hexachlorobenzene are used as raw materials to study the strain's degradation efficiency against these two organochlorine pesticide residues.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for degrading organochlorine pesticide residues utilizes endophytic bacterial fermentation to degrade the organochlorine pesticide residues.

[0009] Preferably, the endophytic bacteria are ginseng endophytic bacteria.

[0010] Preferably, the ginseng endophytic bacterium is Pseudomonas poae, preferably Pseudomonas poae nH121.

[0011] Preferably, the endophytic bacteria are endophytic bacteria isolated from biennial healthy ginseng roots.

[0012] Preferably, the organochlorine pesticide residues are pentachloronitrobenzene and hexachlorobenzene.

[0013] The beneficial effects of the present invention are as follows: when the endophytic bacteria are used to ferment in a fermentation broth containing pentachloronitrobenzene and hexachlorobenzene, pentachloronitrobenzene is substantially completely degraded on the 14th day of fermentation, and hexachlorobenzene is substantially completely degraded on the 21st day of fermentation, achieving significant effects of efficient degradation and environmental friendliness. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention is described below with reference to the accompanying drawings, but the present invention is not limited thereto. In the accompanying drawings:

[0015] Figure 1 Figure 1 shows the peaks of HPLC analysis of organochlorine pesticide residues fermented by Pseudomonas poae nH121. A shows the peaks of standard pentachloronitrobenzene and hexachlorobenzene. From left to right, the first peak represents the pentachloronitrobenzene standard, and the second peak represents the hexachlorobenzene standard. Figures B, C, and D represent the peaks of HPLC analysis of the products of organochlorine pesticide residues fermented by Pseudomonas poae nH121 using the method provided by the present invention after 0 days, 14 days, and 21 days of fermentation, respectively. In the figure, nH121 represents Pseudomonas poae nH121, PCNB represents pentachloronitrobenzene, HCB represents hexachlorobenzene, and 0d, 14d, and 21d represent 0, 14, and 21 days of fermentation, respectively. DETAILED DESCRIPTION

[0016] In order to more clearly understand the present invention, the present invention is described in detail below by using preferred embodiments in conjunction with the accompanying drawings, but the present invention is not limited thereby.

[0017] It should be noted that the scientific terms and test methods mentioned in the present invention but not explained or described in detail have the same meanings and contents as those understood by those skilled in the art.

[0018] The molecular biology reagents used in the following preferred examples were mainly purchased from Shanghai Bioengineering Co., Ltd., and specific operations were performed according to the reagent or kit instructions. Molecular biology procedures were mainly performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0019] Example 1

[0020] Materials and methods

[0021] The sequencing of DNA products was assisted by the Major Platform Center of the Chinese Academy of Agricultural Sciences. Healthy two-year-old ginseng root plant materials were collected from the cultivation site of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences in the winter of 2022. PDA solid culture medium contains 200 g / L potatoes, 20 g / L glucose, and 20 g / L agar. PDB culture medium contains 200 g / L potatoes and 20 g / L glucose. In terms of mass volume percentage, the fermentation medium contains the following components: (NH4)2SO4 0.10%, KH2PO4 0.05%, K2HPO40.15%, NaCl 0.10% and MgSO4·7H2O 0.01%. The pH of the fermentation medium is 6.5-7.5, and it is sterilized at 121°C for 20 minutes. Main experimental steps:

[0022] 1. Isolation and identification of strains

[0023] The steps for extraction, separation and smear culture of endophytic bacteria of healthy ginseng are as follows:

[0024] 1.1 Material cleaning

[0025] Fresh ginseng roots, washed with tap water, were placed in 50 mL sterile centrifuge tubes. Each tube was soaked in 30 mL of 5% sodium hypochlorite solution (g / mL) (from Meiyuan Water Purification Materials Co., Ltd.) for approximately 25 minutes. Ultrasound was used during this soaking, and the tubes were inverted intermittently. On a sterile operating table (from Guangzhou Ruizhi Purification Equipment Co., Ltd.), the roots were rinsed with 70% ethanol for 30 seconds and then washed three times with sterile water. During this period, the roots were transferred using tweezers that had been cooled after burning.

[0026] 1.2 Isolation of endophytic bacteria

[0027] Use a sterile blade to cut the washed and disinfected fresh medicinal materials into small cubes with a side length of about 0.3 cm, and inoculate them on a PDA plate. Seal with a sterile sealing film and culture at 29°C for 7 days. Pick a typical single colony on the plate and inoculate it into PDB culture medium, and culture it at 29°C and 190r / min for 20 hours. Dilute the obtained bacterial solution 100 times, inoculate it on a PDA culture plate, culture it at 29°C for 24 hours, and isolate pure colonies. Inoculate the pure cultured bacteria into PDB culture medium, then add an equal volume of 30% glycerol (from Beijing Beihua Fine Chemicals Co., Ltd.) and store it in a -20°C refrigerator. One of the endophytic bacteria was named nH121. 1.3 Identification of endophytic bacteria nH121

[0028] Morphological identification of nH121: nH121 exhibited turbid growth in PDB medium, with a yellowish-white bacterial suspension. After inoculation onto a PDA plate and incubation at 29°C for 24 hours, individual colonies formed, which were round, slightly raised, smooth, and yellowish-white in diameter, 0.1-0.2 cm in diameter, lacked hyphae, and were easily removed, consistent with bacterial colony morphology.

[0029] The strain was further identified using molecular biological methods. The main steps included: extracting genomic DNA of the strain using the CTAB method, designing 16s rDNA primers based on the conserved regions of the gene sequence, and designing Pseudomonas-specific primers ileS and gyrB. The primer sequences are as follows:

[0030] 27f: 5'-AGAGTTTGATCMTGGCTCAG-3';

[0031] 1492r: 5'-GGTTACCTTGTTACGACTT-3';

[0032] ileS-F: 5'-TTCCCAATGAARGCCGGCCTGCC-3';

[0033] ileS-R: 5'-GGGGTGGTGGTCCAGATCACG-3';

[0034] gyrB-F: 5'-GGTGGTCGATAACTCCATCG-3';

[0035] gyrB-R: 5'-CGCTGAGGAATGTTGTTGGT-3';

[0036] The above three primers were used to perform PCR amplification on the genomic DNA of ginseng endophyte nH121.

[0037] The PCR amplification system is as follows:

[0038]

[0039] The PCR amplification procedure is as follows:

[0040] The 16S rDNA amplification protocol consisted of 5-minute denaturation at 94°C, 35 temperature cycles (each cycle consisting of 30-second denaturation at 94°C, 30-second annealing at 43°C, and 50-second extension at 72°C), followed by a 10-minute incubation at 72°C. For gyrB, the PCR annealing temperature was set at 51°C. For ileS, the PCR annealing temperature was set at 56°C.

[0041] The amplified product was recovered and sent to the company for sequencing. The resulting sequences are shown in SEQ ID NOs. 1, 2, and 3 in the sequence listing. Homology analysis of the 16s rDNA, gyrB, and ileS sequences was performed using the BLAST program in the GenBank database. The results revealed that nH121 shared 99.50% identity with the 16s rDNA sequence, 99.10% identity with the gyrB sequence, and 97.70% identity with the ileS sequence of Pseudomonas poae. Based on these results, strain nH121 was identified as Pseudomonas poae.

[0042] 2. Degradation of organochlorine pesticide residues pentachloronitrobenzene and hexachlorobenzene using ginseng endophytic bacteria Pseudomonas poae nH121

[0043] benzene

[0044] 2.1 Fermentation experiment

[0045] Pseudomonas poae nH121 was inoculated into PDB medium, activated and cultured at 29°C for 24 hours, shaken at 160-180 rpm, and centrifuged at 3000 rpm for 8-10 minutes at 4-10°C. The bacterial precipitate was collected and resuspended in an equal volume of sterile fermentation medium aqueous solution to obtain an nH121 bacterial suspension. Pentachloronitrobenzene and hexachlorobenzene were added to the fermentation medium at 0.05-0.1 g / L, respectively, and the inoculum size of the fermentation strain was 5%-10%. The fermentation temperature was 25-29°C, the shaker speed was 140-160 rpm, and the total fermentation time was 21 days. After fermentation, the fermentation broth was centrifuged at 4000 rpm for 5 minutes, and the supernatant was extracted three times with equal volumes of n-hexane. The supernatant extract was centrifuged at 8000 rpm for 5 minutes at 4°C. The supernatant extract was collected and concentrated to dryness to obtain a dry product containing degradation products of pentachloronitrobenzene and hexachlorobenzene.

[0046] 2.2 High Performance Liquid Chromatography (HPLC) Detection of Changes in Organochlorine Pesticide Residues

[0047] Organochlorine pesticide residue standards (containing pentachloronitrobenzene and hexachlorobenzene) and the dried sample containing pentachloronitrobenzene and hexachlorobenzene degradation products prepared in 2.1 above were dissolved in methanol (Thermo Fisher, chromatography grade) and filtered through a 0.22 μm microporous filter membrane (from Tianjin Jinteng Experimental Equipment Co., Ltd.) before HPLC analysis. HPLC analysis was performed using an Agilent ZORBAX SB-Phenyl 250×4.6 mm, 5 μm column, an injection volume of 20 μL, a flow rate of 0.8 mL / min, a column temperature of 30°C, and a UV detector at 245 nm. Isocratic elution was performed using a mobile phase of methanol:water = 90:10 (volume ratio).

[0048] 2.3 Identification results

[0049] like Figure 1 As shown, the fermentation products obtained after fermentation for different times were compared and analyzed, and the retention times of the fermentation products were compared with those of pentachloronitrobenzene and hexachlorobenzene standards to analyze the changes in the contents of pentachloronitrobenzene PCNB (peak 1) and hexachlorobenzene HCB (peak 2). The degradation rates of pentachloronitrobenzene and hexachlorobenzene under the method provided by the present invention were calculated. The data results are shown in Table 1 below.

[0050] Table 1. Changes in organochlorine pesticide residues before and after fermentation of strain nH121

[0051]

[0052] This demonstrates that the ginseng endophyte Pseudomonas poae nH121 strain has the ability to degrade pentachloronitrobenzene and hexachlorobenzene. After 14 days of fermentation, the degradation efficiency for pentachloronitrobenzene reached 99.36%, and for hexachlorobenzene reached 85.94%. By the 21st day of fermentation, the degradation efficiency for both organochlorine pesticide residues exceeded 90%.

[0053] Industrial applicability

[0054] The present invention isolated an endophytic bacterium, Pseudomonas poae nH121, from healthy biennial ginseng. It was found that the strain can efficiently degrade pentachloronitrobenzene and hexachlorobenzene, organochlorine pesticide residues with long residual periods, and can be used for soil improvement and environmental management, etc., and is an environmentally friendly strain.

[0055] Sequence Listing

[0056] Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences

[0057] A method for degrading organochlorine compounds using ginseng endophytic bacteria

[0058] DNA

[0059] >1 nH121-16s rDNA

[0060]

[0061] >2 nH121-gyrB

[0062] AAAAAAATTTGTGAAGCACAACAGGTTTTCGTTGAAGCTGTCGTTCCATTGCAGGGCGATTTCCACGCCGATGCCGTCTTCACGCTGGATGTTGAAGTGGAACACCTGGTTGACCGCAGTCTTGTTGGTGTTCAGGTATTCAACGAATGCACGCAGGCCGCCTTCGTACTTGAACAGCTCTTCCTTACCGCTGCGCTCATCCTTGAGGACGATACCCACACCGGAGTTGAGGAAGGACAGTTCACGAATCCGCTTGGCCAGGATGTCCCAGCTGAAGTGGATGTTCTTGAAGGTTTCCGCCGAGGGCTTGAAGTGGATCTGCGTACCGGTGGATTCACTGTCGCCAACGATTTTCATCGGCTCTTGTGGAACACCGTGGACGTAGGTCTGTTCCCAGATCTTGCCGCTACGGCGCACAGTCAGGATCAGCTCTTCGGACAGCGCGTTCACCACCGACACACCTACACCGTGCAAACCGCCGGAGACTTTATAGGAGTTGTCGTCGAACTTACCGCCGGCGTGGAGCACGGTCATGATGACCTCTGCCGCCGAAACGCCTTCTTCTTTGTGCACGTCTACCGGAATGCCGCGGCCGTTGTCGCGCACAGTGATGGACTCATCCGGGTGGATGATGATGCTGATGTCGTCGCAGTGACCGGCCAGAGTTCGTGTTTTTTTTTTTTAACCCCCCCC

[0063] >3 nH121-ileS

[0064] CTTTTTTTTTCCCAAAAAAAAAAACGCCTGCCACAGCGCGAAACGAAGATCCTGCAGCGCTGGGACAGCATTGGCCTGTACGGAAAGTTGCGCGAAATTGGCAAGGATCGACCGAAGTTCGTCCTGCACGACGGCCCTCCTTATGCCAACGGCACGATTCACATCGGTCATGCGCTGAACAAGATTCTCAAGGACATGATCCTTCGCTCGAAGACCCTGTCGGGCTTCGACGCGCCGTATGTCCCGGGTTGGGACTGCCACGGCCTGCCCATCGAACACAAAGGCGAAGAGACCTACGGCAAGAACCTGGACGCGGATAAAACCCGCGAACTGAGCCGTGCCTACGCTACCGAGCAGATCGAAGGGAAAAAGTCCGAATTCATCCGCCTGGAGGTGCTCGGCGAGGGGGACAACCCGTACAAGACCATGAACTTCAAGAACGAGGCCGCCGAAATCCGTGCCTTGGCCGAAATCGTCAAGGGCGGTTTTGTGTTCAAGGGCCTCAAGCCCGTGAACTGGTGCTTCGACTGCGGTTCGGCCCTGGCTGAAGCGGAGGTCGAGTACGAAGAGAAAAAGTCCTCGACCATCGACGTCGCCTTCCCGATCGCCGACGACGCCAAGCTGGCCGAGGCCTTTGGCCTGGCAAACCTGAGCAAGCCGGCAGCCATCGTGATCTGCC。

Claims

1. A method for degrading organochlorine pesticide residues, which utilizes endophytic bacterial fermentation to degrade organochlorine pesticide residues.

2. The method according to claim 1, wherein the endophytic bacteria are ginseng endophytic bacteria.

3. The method according to claim 1, wherein the ginseng endophytic bacteria is Pseudomonas poae, preferably Pseudomonas poae nH121.

4. The method according to claim 1, wherein the endophytic bacteria are endophytic bacteria isolated from biennial healthy ginseng roots.

5. The method according to any one of claims 1 to 4, wherein the organochlorine pesticide residues are pentachloronitrobenzene and hexachlorobenzene.

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