Atrazine degrading strain and application thereof

Abstract

The invention discloses an atrazine degrading strain and application thereof, the strain is a strain HB172176, the taxonomic name of the strain HB172176 is Paenibacillus Danchanensis, the preservation number of the strain HB172176 is CGMCC (China General Microbiological Culture Collection Center) NO.1. 16820, and the preservation date of the strain HB172176 is September 9, 2021. The invention further discloses application of the atrazine degrading strain in preparation of atrazine degrading products. The strain is separated from mangrove forest habitat with changeable environmental conditions, has atrazine degradation activity, has wide adaptability to pH and salinity, and can be applied in more environments.

Description

Technical Field

[0001] The invention belongs to the technical field of marine microorganisms, and particularly relates to an atrazine-degrading bacterial strain and application thereof. Background Art

[0002] Atrazine, a widely used triazine herbicide worldwide, poses a serious threat to ecosystems and human health due to its high residual properties, biotoxicity, and long-term environmental persistence. Its residues in soil and water not only cause damage to subsequent crops (e.g., growth inhibition of soybeans and wheat), but can also accumulate through the food chain, causing risks such as neurotoxicity and endocrine disruption. Traditional physical and chemical remediation methods are limited by high costs and secondary pollution. Microbial remediation, with its high efficiency and environmental benefits, has become a core strategy for atrazine contamination remediation.

[0003] In recent years, significant progress has been made in the screening and application of highly efficient atrazine-degrading strains. Examples include Pseudomonas, Arthrobacter, and Achromobacter. Furthermore, salt-tolerant strains such as Klebsiella oxytoca NTA-4 and Arthrobacter PSC have demonstrated degradation potential in high-salt environments (≤30 g / L NaCl), broadening their application. Currently, most atrazine-degrading strains have been isolated from terrestrial soil habitats, with few reports on degrading strains from marine environments. The marine environment is complex and diverse, harboring a rich and unique microbial resource. The development of marine bacterial species is bound to further broaden their application.

[0004] The unique high-salt, high-organic matter environment of the mangrove ecosystem gives it efficient degradation capabilities, and it can function stably in complex polluted environments without the need for additional carbon sources.

[0005] Therefore, the present invention intends to isolate a new strain with atrazine degradation activity from mangrove sediments. Summary of the Invention

[0006] The object of the present invention is to provide an atrazine-degrading bacterial strain, which has atrazine-degrading activity.

[0007] The present invention also aims to provide the use of the above-mentioned strain in the preparation of atrazine-degrading products.

[0008] The first object of the present invention can be achieved by the following technical solution: an atrazine-degrading strain, the strain is strain HB172176, the taxonomic name of the strain HB172176 is Paenibacillus DanchangensisThe deposit number is CGMCC NO.1.16820, the deposit date is September 9, 2021, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101.

[0009] The present invention isolated an atrazine-degrading strain from the sediment of the mangroves in Wenchang, Hainan, and identified the strain. Paenibacillus Danchangensis The deposit number of HB172176 is CGMCC NO.1.16820, the depositor is the General Microbiology Center of China Culture Collection Committee, the deposit date is September 9, 2021, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101.

[0010] The above-mentioned second object of the present invention can be achieved by the following technical solution: use of the above-mentioned atrazine-degrading strain in the preparation of atrazine-degraded products.

[0011] The invention has the following advantages: the strain HB172176 is isolated from a mangrove habitat with changeable environmental conditions, has atrazine degradation activity, is widely adaptable to pH and salinity, and can be applied in more environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 OD600 values ​​of the eight strains in Example 1 after culturing in an inorganic salt culture medium containing atrazine as a substrate for 48 h; Figure 2 This is a transmission electron micrograph of the strain HB172176 in Example 1 after being cultured in 2216E medium at 28°C for 48 hours; Figure 3 This is the phylogenetic tree of strain HB172176 constructed based on the 16S rDNA sequence in Example 2; Figure 4 This is the atrazine degradation ability curve of strain HB172176 in Example 3; Figure 5 This is the degradation effect of strain HB172176 in Example 3 on atrazine in soil. DETAILED DESCRIPTION

[0013] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0014] In the following examples, if no specific techniques or conditions are specified, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments used without manufacturer specified were all conventional products that can be purchased through regular channels. Example 1

[0015] (1) Enrichment and isolation of strains 5-10 cm sediments were collected from the mangroves in Wenchang, Hainan. 500 g of sediments were added to 500 ml of sterilized 50% aged seawater, and glass beads were added and shaken continuously for 15 min. Then, the sediments were inoculated into enrichment medium (0.1 g / L atrazine was added to the inorganic salt medium, the pH was adjusted to neutral, and sterilized at 121°C for 30 min. 1 L of inorganic salt medium included the following composition: K2HPO4 1.6 g / L, KH2PO4 0.4 g / L, MgSO4•7H2O 0.4 g / L, NaCl 0.1 g / L, glucose 3 g / L, and artificial seawater as the balance) for enrichment culture. After five transfers, the enriched bacterial solution was cultured at 10 -1 , 10 -2 , 10 -3 and 10 -4 Dilute the solution to a 500-fold concentration and spread evenly onto a 2216E solid medium plate. Place the 2216E solid medium in a constant temperature incubator at 28°C for 2-3 days. Once colonies have grown on the solid plate, pick a colony and re-inoculate it onto the 2216E solid medium. Repeat streak culture several times until a purified strain is obtained.

[0016] (2) Screening of atrazine-degrading bacteria The purified degradation strain was inoculated back into an inorganic salt culture medium (atrazine 0.1 g / L) with atrazine as the substrate. After 48 hours, the culture medium became turbid. The specific operation is as follows: Add 250 μL of inorganic salt culture medium with atrazine as the substrate (same as above, atrazine 0.1 g / L) to a 96-well plate, and inoculate the isolated strain into the culture medium with an inoculum volume of 25 μL. Make a parallel group each time you inoculate, and add 25 μL of sterilized phosphate buffer solution to the remaining 2 wells as a blank control group. Before the start of culture, use a microplate reader to measure the OD600 value in each well on the 96-well plate. Incubate the 96-well plate at 28 ° C for 48 h, and measure the OD600 value. A total of 8 strains were screened out, and the OD600 value increased significantly, among which the strain HB172176 had the highest OD600 value, as shown in Figure 2. Figure 1 As shown in . Example 2

[0017] (1) Cultivation and microbial characteristics of atrazine-degrading strain HB172176 (1.1) Morphology and culture characteristics The strain HB172176 screened in Example 1 was observed morphologically using a transmission electron microscope. It was found that the bacteria were rod-shaped. Figure 2 shown.

[0018] Strain HB172176 grows rapidly on 2216E agar and R2A medium, forming circular colonies approximately 2.0–3.5 mm in diameter after 24 hours of incubation at 30°C. These colonies are off-white or grayish-yellow, with neat, translucent edges and a moist, smooth surface. On nutrient agar, colonies form within 24 hours, but are smaller, with diameters of 1.0–1.5 mm, off-white, translucent, and a smooth, rounded surface. Specific characteristics are shown in Table 1 below: Table 1 Culture characteristics of strain HB172176 culture medium Colony color Colony size Growth status Nutrient agar Off-white Smaller Poor <![CDATA[R2A agar]]> Off-white-grayish yellow Small better 2216E agar Off-white Small better .

[0019] (1.2) Physiological and biochemical characteristics Among the 12 carbon sources in API 20 NE, strain HB172176 could utilize maltose and malic acid relatively well, but could not utilize glucose, gluconate, mannose, D-mannitol, arabinose, N-acetyl-glucosamine, capric acid, oxalic acid, citric acid, and phenylacetic acid.

[0020] Strain HB172176 can strongly produce starch hydrolases and cytochrome oxidases, as well as β-glucosidase and β-galactosidase, but has weak H2O2 production. It does not produce proteases, cellulases, nitrate reductases, arginine dihydrolases, or ureases, nor does it produce indole acid or acidify glucose. See Table 2 below for details:

[0021] Table 2 Physiological and biochemical characteristics of strain HB172176 pilot projects Reaction / enzyme result <![CDATA[NO3]]> <![CDATA[NO3–NO2、NO3– N2]]> - TRP Indole - GLU acidification - ADH Arginine dihydratase - URE Urease - ESC Hydrolysis (b-glucosidase) + GEL Hydrolysis (Protease) - PNPG β-galactosidase + GLU Assimilation of glucose - ARA Assimilation of arabinose - MNE Assimilation of mannose - MAN Assimilated mannitol - NAG Assimilation of N-acetyl-glucosamine - MAL Assimilating maltose + GNT Assimilation of gluconate - CAP anabolic acid - ADI Assimilation of oxalic acid - MLT Assimilation of malic acid + CIT Assimilated citric acid - PAC Assimilation of phenylacetic acid - OX Cytochrome oxidase + .

[0022] Growth pH range: The strain was inoculated onto R2A solid medium with a pH gradient of 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 and inverted at 28°C for 7-28 days. The results showed that strain HB172176 grew over a pH range of 4-10, with an optimum pH of 7. This indicates that the strain has a wide pH range of adaptability, particularly in acidic environments. This is due to the strain's long-term adaptation to the acidic mangrove habitat, where it originates.

[0023] Growth temperature range: The strain was inoculated on R2A solid culture medium and cultured inverted at 4°C, 14°C, 16°C, 20°C, 28°C, 37°C, 40°C, 45°C, and 50°C for 28 days. The results showed that the temperature growth range of strain HB172176 was 14-40°C, and the optimal growth temperature was 28°C.

[0024] Salt tolerance: The strain was inoculated on R2A solid culture medium with a salt concentration of 0%-9%, and cultured upside down in a 28°C incubator for 28 days. The results showed that the salt concentration growth range of strain HB172176 was 0%-4%, and the optimal growth salt concentration was 2%.

[0025] (2) 16S rDNA sequencing of atrazine-degrading strain HB172176 Pick a single colony of the strain HB172176 purified in Example 1, place it in 2216E medium and culture it in a shaker at 28 ° C and 150 rpm / min. Take 2 μL of bacterial solution in a 1.5 mL centrifuge tube, add 30 μL of sterile water, mix well and centrifuge quickly to prevent the liquid from sticking to the wall, and place the centrifuge tube in a 100 ° C metal bath for incubation for 10 min. Centrifuge at 12000 rpm for 1 minute, take 2 μL of supernatant as template DNA to amplify and sequence the 16S rRNA of the strain to be tested. The 16S rDNA sequence of the purified strain was amplified by PCR using bacterial universal primers, with primers 27f and 1492r. The 16S rDNA sequence was submitted to the EzBioCloud model strain database (https: / / www.ezbiocloud.net / ) for phylogenetic similarity comparison. The results showed that it was similar to the strain Paenibacillus paeoniae M4BSY-1 T The two strains are closely related, with a 16S rDNA homology of 95.98%.

[0026] in: The 16S rDNA sequence of strain HB172176 is shown in SEQ ID NO. 1; The sequence of primer 27f is shown below: 5′-AGAGTTTGAT CMTGCCTCAG-3′, as shown in SEQ ID NO. 2; The sequence of primer 1492r is shown below: 5′-TACGGYTACCTTGTTACGACTT-3′, as shown in SEQ ID NO.3.

[0027] The alignment results were used to perform multiple sequence alignment using Clustal_W in MEGA 7 software, the evolutionary distance was calculated using the Kimura2-Parameter Distance model, and the Neighbor-Joining method was used to construct a phylogenetic tree to analyze the evolutionary status of the strains. The results are shown in Figure 2. Figure 3 As shown, from Figure 3 It can be seen that strain HB172176 is separated from other strains and clustered into a separate branch. The strain was analyzed by combining 16S rDNA sequence similarity and cluster analysis tree and was confirmed to be a new species.

[0028] Combined with the results of physiological and biochemical experiments, it was further shown that the bacterium belongs to the genus Paenibacillus of the family Paenibacillaceae and is a new species with atrazine degradation function. Paenibacillus Danchangensis .

[0029] The strain HB172176 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on September 9, 2021. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, and the deposit number is CGMCC NO.1.16820. Example 3

[0030] (1) Determination of the ability of strains to degrade atrazine A single colony of the atrazine-degrading bacteria HB172176 purified in Example 1 was cultured in liquid. After the strain reached the logarithmic phase, it was inoculated into an inorganic salt medium containing 100 mg / L atrazine at a 1% inoculum dose. 12 h, 24 h, 36 h, and 48 h after inoculation, the culture solution was extracted with an equal volume of CHCl3 and an appropriate amount of NaCl solution. The lower organic phase was removed, concentrated using a rotary evaporator at 45 °C, and fixed to 1 mL with methanol.

[0031] The residual amount of atrazine was determined by liquid chromatography: the detection conditions were a C18 reverse-phase silica gel column (column length 250 mm × inner diameter 4.6 mm), methanol and water (V / V=80 / 20) as the mobile phase, a flow rate of 1 mL / min, a column temperature of 25°C, an injection volume of 5 μL, and a detection wavelength of 213 nm.

[0032] The calculation of atrazine degradation rate refers to the formula of YANG et al.: ;

[0033] Where, X is the degradation rate of atrazine, C X is the final content of atrazine (mg / L), C CKis the original atrazine content (mg / L).

[0034] The degradation ability curve of strain HB172176 on atrazine is as follows Figure 4 As shown in the figure, strain HB172176's atrazine degradation efficiency was slow during the initial culture period (0-12 hours) due to the low bacterial cell content in the culture medium. The degradation rate began to accelerate between 12 and 36 hours. After 48 hours of culture, strain HB172176 achieved an atrazine degradation efficiency of 94% ± 3%, exceeding 90%, making it an ideal, highly efficient degradation strain.

[0035] Therefore, strain HB172176 has a high ability to degrade atrazine. Within 48 hours, the atrazine degradation rate of this strain is 94%±3%, which is higher than 90%, making it an ideal and efficient degradation strain.

[0036] (2) Degradation effect of strain HB172176 on atrazine in soil Weigh 10 g of sterile soil without atrazine into a 9 cm diameter sterile Petri dish. Evenly add 500 μL of atrazine methanol solution (2 mg / mL) to a final atrazine concentration of 100 mg / kg. After the methanol evaporates, add 2 mL of bacterial suspension (refer to Example 2 for bacterial suspension preparation steps). An uninoculated soil sample serves as a negative control. Each treatment is replicated three times. Place the dish in a 28°C incubator and regularly spray with sterile water to keep the soil moist. Samples are collected on days 5, 10, 15, 20, 25, 30, 35, 40, and 45 to determine atrazine degradation. Extract the 10 g soil sample with 25 mL of CHCl₃ and an appropriate amount of NaCl solution. Detect residual atrazine in the soil as described in Example 3.

[0037] The degradation effect of strain HB172176 on atrazine in soil is as follows Figure 5 As shown in the figure, after 45 days of simulated soil remediation testing, the residual atrazine in the inoculated soil was significantly reduced compared to the uninoculated soil. The atrazine concentration in the soil decreased from 100 mg / kg to 19.1 mg / kg, with a degradation rate of 81%. This indicates that strain HB172176 has a strong ability to degrade atrazine in the soil, with the degradation efficiency being even higher within the first 30 days. In the uninoculated control experiment, the atrazine concentration in the soil decreased from 100 mg / kg to 98.4 mg / kg, with a natural degradation rate of only 1.6%. This indicates that strain HB172176 has a strong ability to remediate atrazine residues in the soil.

[0038] It should be pointed out that the above embodiments are only further explanations of the present invention, rather than limitations. Any adjustments or changes by those skilled in the art within the meaning and scope equivalent to the technical solutions of the present invention should be considered to be included in the scope of protection of the present invention.

Claims

1. An atrazine-degrading strain, characterized in that: The strain is strain HB172176, and the taxonomic name of the strain HB172176 is Paenibacillus Danchangensis , the deposit number is CGMCC NO.1.16820, and the deposit date is September 9, 2021.

2. Use of the atrazine-degrading strain according to claim 1 in the preparation of atrazine-degrading products.

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