An atrazine-degrading bacterium and its application

Abstract

The present invention belongs to the technical field of environmental remediation microorganisms, and relates to an atrazine-degrading bacterium and its application. Aiming at the technical problems in the prior art that the atrazine-degrading bacterium has a low tolerance to atrazine, and the most suitable growth temperature and pH during degradation are not suitable for popularization and use, the present application provides an atrazine-degrading bacterium, Bordetella sp. strain BG-34, which is taxonomically named Bordetella sp. , and was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on November 9, 2022, with the deposit number of CGMCC No. 26091. It can tolerate atrazine with a concentration as high as 1500 mg / L, and is suitable for degrading atrazine in high-temperature seasons and low-latitude regions. The present application also provides an application of an atrazine-degrading bacterium. The most suitable growth temperature is 30-35 °C, the pH value is close to neutral, and it can grow and reproduce using atrazine as the sole nitrogen source, avoiding the inhibition of the growth of strain BG-34 by other nitrogen sources.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental remediation microorganisms, and specifically relates to an atrazine-degrading bacterium and its application. Background Art

[0002] A series of problems such as environmental pollution, ecological imbalance, and food safety caused by the use of pesticides or herbicides are becoming increasingly serious. Atrazine (chemical name: 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine, molecular formula: C8H 14 ClN5), also known as atrazen, belongs to the s-triazine herbicides, and is widely used to control annual gramineous weeds in corn fields, sorghum fields, sugarcane fields, orchards, nurseries, woodlands, etc., and also has a certain inhibitory effect on perennial weeds. It has become one of the most effective and typical agricultural diffusion herbicides. Due to its stable structure and difficult degradation, it has broad-spectrum toxicity and bioaccumulation, and is a typical "three-causing" pollutant containing chlorine element, which can cause teratogenesis, carcinogenesis, and mutagenesis. Therefore, it is crucial to effectively remove atrazine. How to quickly degrade the residues of atrazine in the soil and repair the polluted soil, so as to protect human health and realize the sustainable development of crop economy, is a problem widely concerned by governments and environmental scientists of various countries. From the current research status and development trend at home and abroad, the bioremediation technology with bioremediation materials as the core has become a hot spot in soil pollution remediation. Microbial remediation is a remediation method with low cost, high efficiency and environmental friendliness.

[0003] Regarding atrazine-degrading bacteria, bacteria of various genera have been reported. Most atrazine-degrading bacteria are easily inhibited in the presence of multiple nitrogen sources. The Chinese invention patent authorization announcement number CN104962494B, with the application date of June 25, 2015, and the name: A Shewanella strain with good degradation effect on the herbicide atrazine, discloses an atrazine-degrading strain YJY4 screened from a corn field in Nancha District, Yichun City, Heilongjiang Province, which has been long-term applied with atrazine by using the enrichment culture method. This strain can degrade 100 mg / L of atrazine into non-toxic cyanuric acid within 32 hours. This strain can grow with atrazine as the sole nitrogen source, solving the problem that the growth of atrazine-degrading bacteria is inhibited and the degradation result is affected when the nitrogen source is not unique. However, this strain only reports that the tolerance concentration to atrazine is 100 mg / L, and its practicability is not strong in soils that have been applied with atrazine for many years or some water areas that are prone to enrich high concentrations of atrazine.

[0004] The Chinese invention patent application publication number CN105567597A, with the application date of January 15, 2016, and the name: A highly efficient atrazine-degrading bacterium and its application and screening method, discloses a strain of Arthrobacter genus ( Arthrobacter)ZXY-2. This strain can also grow using atrazine as the sole carbon and nitrogen source and can completely degrade atrazine with an initial concentration of 100 mg / L within 14 h. However, the optimal growth pH value of this strain is 8.0 - 9.0. In the natural environment where the pH value is around neutral, this strain cannot exert its maximum degradation ability in practical applications. Chinese Patent Application Publication No. CN102492637A, with an application date of November 21, 2011, and a title of: A Strain of Atrazine-Degrading Bacteria, discloses a strain of Acinetobacter ( Acinetobacter sp. )strain DNS32. This strain DNS32 has a strong tolerance to salt. Under the condition of a salinity of 1 - 4%, the degradation rate is higher than 60% within 36 h, and it is suitable for the remediation of atrazine pollution in high-salt environments. However, the optimal temperature range of this strain DNS32 is 25 - 30 °C, and its use is restricted in summer high temperatures and some southern regions. Summary of the Invention

[0005] 1. Technical Problems to be Solved by the Invention

[0006] Aiming at the technical problems that the atrazine-degrading bacteria in the prior art have a low tolerance to atrazine and the most suitable growth temperature and pH during degradation are not suitable for popularization and use, this application provides a strain of atrazine-degrading bacteria, Bordetella sp. strain BG-34, classified and named as Bordetella sp. ,which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 09, 2022, with the deposit number CGMCC No. 26091. It can tolerate atrazine with a concentration as high as 1500 mg / L and is suitable for the degradation of atrazine in high-temperature seasons and low-latitude regions. This application also provides the application of a strain of atrazine-degrading bacteria. The most suitable growth temperature is 30 - 35 °C, the pH value is close to neutral, and it can grow and reproduce using atrazine as the sole nitrogen source.

[0007] 2. Technical Solutions

[0008] To achieve the above object, the technical solutions provided are as follows:

[0009] A strain of atrazine-degrading bacteria of the present invention, Bordetella sp. strain BG-34, classified and named as Bordetella sp. ,which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 09, 2022, with the deposit number CGMCC No. 26091.

[0010] Furthermore, the base sequence of the 16S rDNA of the strain is as shown in SEQ ID NO.1.

[0011] The strain was identified by 16S rDNA, and the results were compared for homology through the Blast program. The homology of this strain with Bordetella sp. strain BAB-6428 reached 99%.

[0012] Furthermore, the morphological identification of the strain was as follows: Gram-negative bacteria, coccobacilli, without spores, and non-motile.

[0013] Furthermore, the colonies of the strain on the LB solid plate medium were yellow, semi-transparent, round, convex on the surface, smooth, and with regular edges.

[0014] The morphology of the strain and colonies conformed to the general characteristics of the genus Bordetella. It was obligately aerobic and chemoheterotrophic. It could also grow at 37°C. It could not reduce nitrate to nitrite, was positive for oxidase, positive for catalase, and negative for urease.

[0015] Furthermore, the strain could grow using atrazine as the sole carbon source or nitrogen source.

[0016] Among most of the reported genera that can degrade atrazine, they can utilize atrazine as the sole nitrogen source for growth, avoiding the inhibitory effect of other nitrogen sources on the growth of the strain.

[0017] Furthermore, the tolerance of the strain to atrazine reached 1500 mg / L.

[0018] The strain of the present application has a high tolerance to atrazine and is suitable for soils or waters that have been contaminated with atrazine for many years.

[0019] The application of an atrazine-degrading bacterium, applying the strain to the degradation of triazine herbicides.

[0020] Triazine herbicides, also known as triazobenzene herbicides, have been widely used for pre-emergence or post-emergence weeding since the 1950s to control the growth of annual gramineous plants and broad-leaved weeds. This type of herbicide belongs to selective herbicides and targets the D1 protein of photosystem II (PSII), and exerts its herbicidal effect by inhibiting plant photosynthesis. The strain of the present application can be used for the degradation of this type of herbicide, thereby achieving the purpose of bioremediation.

[0021] Furthermore, applying the strain to the degradation of atrazine.

[0022] Furthermore, applying the strain to a microbial inoculant for degrading atrazine.

[0023] Preferably, the microbial inoculant is in liquid, powder or granular form. The concentration of the inoculant is 6*10 6 ~9*10 8 CFU / mL.

[0024] Furthermore, the temperature for degradation is 30~35 °C and the pH value is 6~8.

[0025] The strain of the present application is suitable for the degradation of atrazine in high-temperature seasons and regions, and the optimal degradation pH value is close to neutral. Compared with the strains reported in the prior art, it is more practical and more suitable for popularization and use in polluted water bodies.

[0026] Biological material preservation information:

[0027] A strain of atrazine-degrading bacterium, Bordetella sp. strain BG-34, classified and named as Bordetella sp. was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on November 09, 2022, and the deposit number is CGMCC No. 26091.

[0028] 3. Beneficial effects

[0029] Adopting the technical solution provided by the present invention, compared with the existing well-known technologies, it has the following beneficial effects:

[0030] (1) A strain of atrazine-degrading bacterium of the present invention is a rare atrazine-degrading bacterium of the genus Bordetella, which combines a high tolerance to atrazine (up to 1500 mg / L), a suitable higher degradation environmental temperature (the degradation ability is the strongest at 30~35 °C and it can also grow at 37 °C), and an optimal degradation pH value close to neutral (the degradation effect is the best at pH 7, and there is also a good degradation ability at the end point values of pH 5~10) in the same strain, and it can use atrazine as the sole carbon and nitrogen source, suitable for areas long-term polluted by atrazine, as well as high-temperature in summer or high-temperature areas at low latitudes, for the degradation of atrazine.

[0031] (2) The application of a strain of atrazine-degrading bacterium of the present invention, when applying the strain to triazine herbicides, especially to the degradation of atrazine, as an unmodified non-engineered bacterium and as a domesticated strain added to atrazine-polluted soil or water bodies, will not be excluded and inhibited by indigenous bacteria, can colonize quickly, and rapidly enter the exponential growth stage. It starts to enter the logarithmic growth phase at 12 h, and enters the stationary growth phase after 40 h. The degradation rate of atrazine can reach 90% within 36 h, and the atrazine degradation rate can reach 93.8% after 4 d. It realizes the purpose of quickly degrading atrazine and repairing the environment. Description of the drawings

[0032] Figure 1 For the strain Bordetella sp.Neighbor-joining tree of BG-34 based on 16S rDNA sequences and its closest 16S rDNA match in GenBank.

[0033] Figure 2 For strain Bordetella sp. Growth curve of strain BG-34.

[0034] Figure 3 For strain Bordetella sp. Curve of atrazine degradation by strain BG-34.

[0035] Figure 4 For strain Bordetella sp. Atrazine degradation graph of strain BG-34 at different temperatures.

[0036] Figure 5 For strain Bordetella sp. Atrazine degradation graph of strain BG-34 under different pH conditions.

[0037] Figure 6 For strain Bordetella sp. Atrazine degradation graph of strain BG-34 under different shaker speeds.

[0038] Figure 7 For strain Bordetella sp. Degradation graph of strain BG-34 under different initial atrazine concentrations. Detailed implementation manner

[0039] The present invention will be further described below in conjunction with specific embodiments.

[0040] The technical solutions in 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 a part of the embodiments of the present invention, rather than all of the embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0041] Example 1

[0042] 1. Soil sampling method

[0043] The soil samples were collected from the surface soil of a corn field in the demonstration area of Gongzhuling City, Changchun City, Jilin Province, where pesticides and herbicides have been applied for many years. The soil is typical black soil in the north.

[0044] Using the diagonal sampling method, set 5 sampling points. For each sampling point: Select 3 fields for sampling as 3 sub-samples. For each sub-sample, take 3 - 5 points along an S-shaped path and put them into a large bag, with a distance of more than 10 meters between each point. Sampling is carried out uniformly in the narrow rows between the wide and narrow rows, avoiding the fertilization ditch. The sampling depth is 0 - 15 cm (the marker pen indicates 14 cm, and 0 - 15 cm is the plough layer). When sampling, remove the surface straw residues, and use a soil drill to take soil samples at 0 - 15 cm simultaneously; after taking the 3 sub-samples, wear disposable gloves and mix them thoroughly (mix evenly in the field), and use the quartering method for multiple divisions until the soil sample remains 1 kg, then bag it and take it back to the laboratory.

[0045] 2. Culture Medium

[0046] The composition of the basic medium (MSM) is: MgSO4·7H2O (0.1 g), K2HPO4 (1 g), KH2PO4 (1 g), FeSO4·7H2O (0.05 g), NaCl (1 g), glucose (1 g), distilled water (1 L), pH 7.0.

[0047] Inorganic salt medium: MgSO4·7H2O (0.1 g), K2HPO4 (1 g), KH2PO4 (1 g), FeSO4·7H2O (0.05 g), NaCl (1 g), atrazine (1 g / L methanol solution) 10 mL.

[0048] LB medium: peptone (10 g), yeast powder (5 g), NaCl (5 g), distilled water (1 L), pH 7.0 - 7.2.

[0049] The above media are all sterilized at 121 °C and 0.1 MPa steam for 20 min. 20 g of agar powder is added to the solid medium.

[0050] 3. Strain Enrichment and Screening

[0051] Place 10 g of the soil sample in step 1 into the basic medium, without adding atrazine first (the purpose is to allow indigenous microorganisms to survive and grow under nutrient conditions). After culturing for 7 d, transfer the soil sample to the inorganic salt medium and culture it at a constant temperature of 30 °C with a constant shaking speed of 160 r / min.

[0052] Inoculate into the newly prepared inorganic salt medium at a ratio of 10% (v / v) per week and continue culturing, gradually increasing the concentration of atrazine. After continuous enrichment and domestication culture for 2 months, until the atrazine concentration increases to 1500 mg / L.

[0053] Dilute the enriched culture by 10 -1 ~10-9 , then take 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , each take 0.1 mL and coat them on the inorganic salt plates added with corresponding atrazine concentrations respectively, and place them in a constant temperature incubator at 30 °C for 5 - 7 d.

[0054] After the cultivation is completed, pick the single colonies with transparent degradation zones on the inorganic salt plates. The degradation zone can be used as a sign to preliminarily determine that the strain has the degradation ability to atrazine. Pick the single colonies for bacterial purification culture, and repeat it more than 3 times until the single colonies with the same morphological size appear on the plate.

[0055] After multiple separation and purification, select the strain that can grow with atrazine as the sole carbon and nitrogen source; finally, pick the single colonies and streak them onto the slant solid medium and culture for 2 - 3 d, then store them at 4 °C.

[0056] Example 2

[0057] Strain identification.

[0058] 1. Strain morphological characteristics

[0059] Strain BG-34 is a Gram-negative bacterium, coccobacillus, without spores and non-motile.

[0060] On the LB solid plate medium, the colonies of the strain are yellow, round, convex on the surface, smooth, semi-transparent, and with neat edges.

[0061] 2. 16S rDNA identification

[0062] Use a DNA extraction kit (Sangon) to extract bacterial DNA according to the extraction steps, and then perform PCR amplification of the target fragment. The PCR reaction conditions are: pre-denaturation at 94 °C for 5 min, and then 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 1.5 min, and finally extension at 72 °C for 5 min. The PCR amplification product is sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The strain is identified by 16S rDNA, and its result is compared for homology through the Blast program. The strain has a 99% homology with Bordetella sp. strain BAB-6428. The base sequence of the 16S rDNA of the strain is shown in SEQ ID No.1.

[0063] As Figure 1 shown, the strain BordetellaNeighbor-joining tree of sp. BG-34 based on 16S rDNA sequences and its closest 16S rDNA match in GenBank.

[0064] SEQ ID No.1 is:

[0065]

[0066] Example 3

[0067] Determination of the growth curve.

[0068] The bacterial suspension was inoculated into LB medium at an inoculation amount of 1%, and cultured at 30 °C and 160 rpm. Samples were taken every 4 h for the first 48 h to measure the OD 600 value, and three replicates were made. At the same time, a blank control was made.

[0069] As Figure 2 shown, the strain Bordetella sp. BG-34 was in the adjustment period from 0 to 12 h, entered the logarithmic growth phase at 12 h, and entered the stationary growth phase after 40 h.

[0070] Example 4

[0071] 1. Determination of the degradation curve and degradation ability:

[0072] The degradation strain bacterial suspension was inoculated into an inorganic salt medium (pH 7.0) containing 100 mg / L atrazine at an inoculation amount of 1%, and cultured with shaking at 30 °C and 160 rpm. The initial sampling time interval was 12 h, and the sampling time was adjusted subsequently according to the measured atrazine concentration. The concentration of atrazine was detected by high performance liquid chromatography (HPLC).

[0073] Atrazine detection method: The chromatographic conditions for HPLC detection were: C 18 chromatographic column, the mobile phase was methanol: aqueous solution with a volume ratio of 70:30, the flow rate was 1.0 mL / min, the wavelength was 220 nm, the injection volume was 10 mL, and the column temperature was room temperature.

[0074] As Figure 3 shown, the strain Bordetella sp. BG-34 could reach a degradation rate of 90% for atrazine within 36 h.

[0075] 2. Degradation effect of the strain:

[0076] The degradation effect of atrazine was detected by liquid chromatography. In a 250 mL inorganic salt liquid medium reaction system with a cell concentration of 8.0×10 7 cfu / ml (at an inoculation amount of 1%), after culturing at 30 °C in a constant temperature shaker at 160 rpm for 4 d, the strain BG-34 had the highest degradation efficiency for atrazine, reaching 93.8%, and the degradation rate of the blank control was 11.2%. This strain is a highly efficient degradation strain.

[0077] Table 1 shows the strain Bordetella sp.The degradation ability of BG-34 to atrazine is shown in Table 1. The degradation rate of strain Bordetella sp. BG-34 to atrazine can reach 93.8%.

[0078] Table 1 Degradation ability of strains to atrazine

[0079] Strain number BG-34 CK Inoculation concentration (CFU / ml) 8.0*107 0 Initial substrate concentration in the medium (mg / L) 100 100 Substrate concentration in the system on the 5th day (mg / L) 6.2 88.8 Degradation rate (%) 93.8 11.2

[0080] Example 5

[0081] Effect of different temperatures on degradation:

[0082] The bacterial suspension with OD = 1.0 was transferred to the atrazine inorganic salt medium with an initial concentration of 100 mg / L at a pH value of 7.0 and a shaker speed of 160 rpm according to an inoculation amount of 1%, and the culture time was 48 h. The degradation rate of atrazine at different temperatures was investigated, and the detection method was the same as that in Example 4.

[0083] As Figure 4 shown, the degradation ability of strain Bordetella sp. BG-34 to atrazine is different under different temperature conditions. The degradation ability of the strain is the strongest in the range of 30~35 °C and can reach 90%. Below 30 °C or above 35 °C, the degradation ability decreases. Therefore, the optimal growth temperature range of the strain is 30~35 °C.

[0084] Example 6

[0085] Effect of different pH values on degradation:

[0086] The bacterial suspension with OD = 1.0 was transferred to the atrazine inorganic salt medium with an initial concentration of 100 mg / L at 30 °C and a shaker speed of 160 rpm according to an inoculation amount of 1%, and the culture time was 48 h. The degradation rate of atrazine at different pH values was investigated, and the detection method was the same as that in Example 4.

[0087] As Figure 5 shown, the degradation ability of strain Bordetella sp. BG-34 to atrazine is different under different pH conditions. The degradation ability of the strain is the strongest at a pH value of 7.0 and reaches 91%. Therefore, the optimal growth pH value of the strain is 7.0.

[0088] Example 7

[0089] Effect of different rotation speeds on degradation:

[0090] The bacterial suspension with OD = 1.0 was transferred to the atrazine inorganic salt medium with an initial concentration of 100 mg / L at 30 °C and pH 7.0 according to an inoculation amount of 1%, and the culture time was 48 h. The degradation rate of atrazine under different shaker speeds was investigated, and the detection method was the same as that in Example 4.

[0091] As Figure 6 shown, the degradation ability of strain Bordetella sp. BG-34 to atrazine is different under different shaker speed conditions. The degradation ability of the strain is the strongest at 150 - 180 rpm, reaching 93%. When the speed is lower than 150 rpm or higher than 180 rpm, the degradation ability decreases. The optimal shaker speed for the growth of the strain is 160 rpm.

[0092] Example 8

[0093] Effect of different initial concentrations on degradation: The bacterial suspension with OD = 1.0 was transferred to the atrazine inorganic salt medium with different initial concentrations at 30 °C, pH 7.0, and a shaker speed of 160 rpm according to an inoculation amount of 1%, and the culture time was 48 h. The degradation rate of atrazine under different initial concentrations was investigated, and the detection method was the same as that in Example 4.

[0094] As Figure 7 shown, the degradation rate of strain Bordetella sp. BG-34 is different under different initial concentrations of atrazine. When the initial concentration is 50 mg / L and 100 mg / L, the atrazine degradation rate can reach more than 90%. However, when the initial concentration of the strain is 200 mg / L and 500 mg / L, the degradation rate can reach more than 60%, indicating that the strain has strong tolerance.

[0095] All of the above samples were made in triplicate, and each experiment had a blank medium as a control.

[0096] This application is the first to isolate the highly efficient degradation strain Bordetella sp. BG-34 of the genus Bordetella from soil samples. However, so far, the degradation research on atrazine by the genus Bordetella ( Bordetella sp.) is still blank. This application studied the degradation conditions of atrazine for the isolated strain Bordetella sp. BG-34, filling the blank in the degradation research of this genus on atrazine and having high reference value for subsequent research. The strain of this application can be used for the remediation of environments contaminated by atrazine such as wastewater or soil. The strain ( Bordetella sp. BG-34) was isolated from the soil of farmland corn fields in the Gongzhuling demonstration area. The strain ( Bordetella sp.BG-34) can grow using atrazine as the sole carbon and nitrogen source and has the ability to efficiently degrade atrazine. The atrazine degradation rate can reach 93.8% after 4 days. The strain Bordetella sp. BG-34 has an optimal growth temperature of 30 - 35 °C, an optimal growth pH of 7.0, and an optimal growth shaker speed of 160 rpm. Moreover, this strain can tolerate high concentrations of atrazine (1500 mg / L).

Claims

1. An atrazine-degrading bacterium, characterized in that: Bordetella strain BG-34, classified and named as Bordetella sp. , was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on November 09, 2022, with the deposit number CGMCC No. 26091.

2. The atrazine-degrading bacterium according to claim 1, characterized in that: The base sequence of the 16S rDNA of the strain is as shown in SEQ ID NO.

1.

3. The atrazine-degrading bacterium according to claim 1, characterized in that: The morphological identification of the strain is as follows: Gram-negative bacteria, coccobacilli, without spores, and non-motile.

4. The atrazine-degrading bacterium according to claim 1, characterized in that: The colonies of the strain on the LB solid plate medium are yellow, semi-transparent, round, convex on the surface, smooth, and with regular edges.

5. A strain of atrazine-degrading bacteria according to claim 1, characterized in that: The strain can grow with atrazine as the sole carbon source or nitrogen source.

6. The atrazine-degrading bacterium according to claim 5, characterized in that: The tolerance of the strain to atrazine reaches 1500 mg / L.

7. Use of an atrazine-degrading bacterium, characterized in that: The strain described in any one of claims 1-6 is applied to the degradation of atrazine.

8. Use of an atrazine-degrading bacterium according to claim 7, characterized in that: The strain is applied to the preparation of a microbial agent for degrading atrazine.

9. Use of an atrazine-degrading bacterium according to any one of claims 7 or 8, characterized in that: The temperature of the degradation is 30-35 °C, and the pH value is 6-8.

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