Atrazine-degrading bacterial strain and application thereof

Abstract

The application discloses an atrazine-degrading strain and application thereof, the strain is strain HB172176, the taxonomic name of the strain HB172176 is Paenibacillus Danchangensis , the preservation number is CGMCC NO.1.16820, and the preservation date is September 9, 2021, and the application of the atrazine-degrading strain in preparation of an atrazine-degrading product is also disclosed. The strain is separated from a mangrove habitat with variable environmental conditions, has atrazine-degrading 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;

[0013] 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;

[0014] Figure 3 This is the phylogenetic tree of strain HB172176 constructed based on the 16S rDNA sequence in Example 2;

[0015] Figure 4 This is the atrazine degradation ability curve of strain HB172176 in Example 3;

[0016] Figure 5 This is the degradation effect of strain HB172176 in Example 3 on atrazine in soil. DETAILED DESCRIPTION

[0017] 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.

[0018] The following examples are not specified technical or conditions, according to the literature described in the art, or according to the product description. The reagents or instruments are not specified manufacturer, are available through regular channels of business to buy conventional products. Example 1

[0019] (1) enrichment and isolation of strains

[0020] 5-10 cm sediment from Hainan Wenchang mangrove, take 500 g sediment into 500 ml sterilized 50% old seawater, and put into glass beads for continuous shaking for 15 min, then inoculate into enrichment medium (add atrazine 0.1 g / L in inorganic salt medium, pH to neutral, 121 ℃ sterilization 30 min, 1 L inorganic salt medium includes the following composition: K2HPO41.6 g / L, KH2PO40.4 g / L, MgSO4•7H2O 0.4 g / L, NaCl 0.1 g / L, glucose 3 g / L, artificial seawater balance) for enrichment culture, after 5 times of transfer, the enriched bacterial solution is diluted by 10 -1 , 10 -2 , 10 -3 and 10 -4 times, and evenly coated on 2216E medium solid plate. Put the 2216E solid medium into the incubator and incubate at 28℃ for 2-3 days. After colonies grow on the solid plate, pick the colonies and re-inoculate into 2216E solid medium, and continuously streak culture for several times until the purified strain is obtained.

[0021] (2) screening of atrazine-degrading bacteria

[0022] The purified degrading strain is back-inoculated into inorganic salt medium with atrazine as substrate (atrazine 0.1 g / L), and after 48 h, the culture solution appears turbid; the specific operation is as follows:

[0023] In 96-well plate, add 250 μL inorganic salt medium with atrazine as substrate (as above, atrazine 0.1 g / L), inoculate the isolated strain into the medium with 25 μL inoculum, each time do a parallel group, the remaining 2 holes add 25 μL sterilized phosphate buffer solution as blank control group. Before the start of culture, use the enzyme marker to measure the OD600 value of each hole in the 96-well plate. Incubate the 96-well plate at 28℃ for 48 h, and measure the OD600 value. A total of 8 strains are screened out, and the OD600 value increases significantly, among which the OD600 value of strain HB172176 is the highest, as shown in Figure 1 . Example 2

[0024] (1) Cultivation and microbial characteristics of atrazine-degrading strain HB172176

[0025] (1.1) Morphology and culture characteristics

[0026] 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.

[0027] 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:

[0028] Table 1 Culture characteristics of strain HB172176

[0029] Culture medium Colony color Colony size Growth condition Nutrient agar Off-white Smaller Poor <![CDATA[R2A琼脂]]> Off-white to grayish yellow Small Better 2216E agar Off-white Small Better .

[0030] (1.2) Physiological and biochemical characteristics

[0031] 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.

[0032] 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:

[0033] Table 2 Physiological and biochemical characteristics of strain HB172176

[0034] Test item Reaction / enzyme Result <![CDATA[NO3]]> <![CDATA[NO3–NO2、NO3– N2]]> - TRP Indole - GLU Oxidation - ADH Arginine dihydrolase - URE Urease - ESC Hydrolysis (b-glucosidase) + GEL Hydrolysis (protease) - PNPG ß-galactosidase + GLU Assimilation of glucose - ARA Assimilation of arabinose - MNE Assimilation of mannose - MAN Assimilation of mannitol - NAG Assimilation of N-acetyl-glucosamine - MAL Assimilation of maltose + GNT Assimilation of gluconate - CAP Assimilation of caproic acid - ADI Assimilation of oxalate - MLT Assimilation of malate + CIT Assimilation of citrate - PAC Assimilation of phenylacetic acid - OX Cytochrome oxidase + .

[0035] Growth pH range: The strain was inoculated on R2A solid medium with pH gradient of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and incubated at 28 °C for 7-28 d, and the results were obtained: the pH growth range of the strain HB172176 was 4-10, and the optimum pH was 7. It can be seen that the strain has a wide range of adaptation to pH, especially in acidic environment, which is related to the habitat of the strain from mangrove forest, and the strain has long been adapted to the acidic habitat of mangrove forest.

[0036] Growth temperature range: The strain was inoculated on R2A solid medium, and incubated at 4 °C, 14 °C, 16 °C, 20 °C, 28 °C, 37 °C, 40 °C, 45 °C, 50 °C for 28 d, and the results were obtained: the temperature growth range of the strain HB172176 was 14-40 °C, and the optimum growth temperature was 28 °C.

[0037] Salt tolerance: The strain was inoculated on R2A solid medium with salt concentration of 0%-9%, and incubated at 28 °C for 28 d, and the results were obtained: the salt concentration growth range of the strain HB172176 was 0%-4%, and the optimum growth salt concentration was 2%.

[0038] (2) 16S rDNA sequencing of atrazine-degrading strain HB172176

[0039] The single colony of the strain HB172176 purified in Example 1 was picked and placed in 2216E medium, and incubated at 28 °C in a shaker at a speed of 150 rpm / min. 2 μL of bacterial liquid was taken into a 1.5 mL centrifuge tube, 30 μL of sterile water was added, mixed well, and quickly centrifuged to prevent liquid wall hanging. The centrifuge tube was incubated in a 100 °C metal bath for 10 min. 12000 rpm centrifugation for 1 min, 2 μL of supernatant was taken as template DNA for amplification and sequencing of the 16S rRNA of the strain to be tested. The 16S rDNA sequence of the purified strain was amplified by PCR using universal bacterial primers, and the primers were 27f and 1492r. The 16S rDNA sequence was submitted to EzBioCloud model strain database (https: / / www.ezbiocloud.net / ) for phylogenetic similarity comparison. The results showed that it was most closely related to the strain Paenibacillus paeoniae M4BSY-1 T The 16S rDNA homology was 95.98%.

[0040] Among them:

[0041] The 16S rDNA sequence of the strain HB172176 is shown as SEQ ID NO. 1;

[0042] The sequence of primer 27f is shown below:

[0043] 5′-AGAGTTTGAT CMTGCCTCAG-3′, as shown in SEQ ID NO. 2;

[0044] The sequence of primer 1492r is shown below:

[0045] 5′-TACGGYTACCTTGTTACGACTT-3′, as shown in SEQ ID NO.3.

[0046] 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.

[0047] 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 .

[0048] 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

[0049] (1) Determination of the ability of strains to degrade atrazine

[0050] 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.

[0051] 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.

[0052] The calculation of atrazine degradation rate refers to the formula of YANG et al.: ;

[0053] Where, X is the degradation rate of atrazine, C X is the final content of atrazine (mg / L), C CK is the original atrazine content (mg / L).

[0054] 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.

[0055] 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.

[0056] (2) Degradation effect of strain HB172176 on atrazine in soil

[0057] 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.

[0058] The degradation effect of strain HB172176 on atrazine in soil is as follows Figure 5As 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.

[0059] 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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