Energetic material NTO degrading bacterium and application thereof

Abstract

The invention belongs to the technical field of environmental pollution bioremediation, and particularly relates to an energetic material NTO degrading bacterium and application thereof. The NTO degrading bacterium is a cellulosimicrobium cellulans strain which is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the NTO degrading bacterium is CGMCC No.28714. The NTO degrading bacterium has the advantages that the NTO degrading bacterium can be used for degrading the NTO, and the NTO degrading bacterium can be used for degrading the NTO. The strain supplements microbial resources of the genus, and reserves and provides strain resources for in-situ remediation of energetic material polluted environment. The NTO degrading bacteria can effectively degrade NTO and can completely degrade 1 mmol / L of NTO within 72 hours, so that the NTO in China can be produced and used in an environment-friendly and non-toxic manner. When the degrading bacterium is added into an NTO polluted water body or soil environment, NTO can be efficiently degraded, and the degrading bacterium has a wide bioremediation application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental pollution bioremediation, and specifically relates to an energetic material NTO-degrading bacterium and application thereof. Background Art

[0002] 3-nitro-1,2,4-triazol-5-one (NTO) is a leading material for a new generation of insensitive explosives with great application prospects. NTO is widely used in the defense industry due to its extremely low impact sensitivity, good thermal stability, excellent resistance to X-rays and ultraviolet radiation, and easy synthesis. It requires large amounts of storage and continuous production tests. It has been used in weapons and equipment abroad. The U.S. Army has approved NTO as one of the main components of insensitive ammunition. It has also been widely used in propellants and insensitive melt-cast explosives in China.

[0003] Toxicological data show that NTO is irritating to rabbit skin. In the body, NTO will be decomposed into 3-amino-1,2,4-triazol-5-one (3-amino1, 2, 4-triazol-5-one, ATO) and uracil. NTO will not be reabsorbed after being filtered into the urine. It has been confirmed that NTO has multiple toxic effects on organisms and ecosystems, mainly testicular toxicity, embryotoxicity, teratogenicity, liver toxicity and potential genotoxicity to mammals. In addition, due to its high water solubility (12.8 g / L, 19°C), NTO has a high migration ability in the environment. It will become a source of public soil and water pollution with the migration and transformation of soil and water bodies, causing harm to birds, frogs and aquatic organisms, and causing neurobehavioral abnormalities in birds.

[0004] At present, there are few studies on the microbial degradation of NTO. Foreign studies mainly tend to focus on the overall components of insensitive munitions (IM), focusing on wastewater treatment in engineering applications, or completing degradation analysis in environmental soil. There is a lack of targeted research on NTO-degrading bacteria, and there are no reports on the microbial degradation of NTO in China. Some existing reports on microbial treatment technology for NTO abroad mainly focus on the biodegradation of NTO by microbial communities, and the strains that play a specific degradation role are still unclear. Therefore, it is urgent to solve the problems of environmental pollution and health damage effects caused by NTO during production, use and storage. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of NTO-degrading bacteria of energetic materials and reserve bacterial resources for in-situ repair of an environment polluted by energetic materials.

[0006] Another object of the present invention is to provide an application of energetic material NTO-degrading bacteria to provide technical support for the remediation of soil and water environmental pollution caused by NTO.

[0007] To this end, the technical solution provided by the present invention is as follows: A strain of NTO-degrading bacteria containing energetic materials, wherein the NTO-degrading bacteria are fibrogenic microbacteria cellulosimicrobium cellulans The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No. 28714. The energetic material NTO-degrading bacterial solution is obtained by expanding and culturing the fibrogenic microbacteria strain.

[0008] The specific process of expanded culture was as follows: a single colony of the fibrogenic microbacterium strain was picked and inoculated into NTO inorganic salt liquid culture medium, and cultured for 48 h in a constant temperature shaker at 37 °C and 120 rpm.

[0009] The invention discloses an application of energetic material NTO-degrading bacteria in preparing NTO-degrading products.

[0010] The invention discloses an application of an energetic material NTO-degrading bacterial liquid in preparing a product degrading NTO.

[0011] The beneficial effects of the present invention are: The present invention separates and obtains NTO-degrading bacteria from activated sludge collected from aeration tanks of sewage treatment plants of NTO production enterprises, which belongs to the classification level. cellulosimicrobium cellulans , supplementing the microbial resources of this genus, and providing bacterial resources for in-situ remediation of energetic material-contaminated environments. This strain can effectively degrade NTO, and can completely degrade 1mmol / L of NTO within 72h, thereby enabling my country's NTO to be produced and used in an environmentally friendly and non-toxic manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the NTO structural formula; Figure 2 This is the NTO degradation bacteria screening process of the present invention; Figure 3 This is the colony morphology of the NTO-degrading bacteria of the present invention; Figure 4 is a Gram staining diagram of the NTO-degrading bacteria of the present invention; Figure 5 This is a graph showing the agarose gel electrophoresis results of 16s rDNA PCR amplification of NTO-degrading bacteria of the present invention; Figure 6 is a 16s rDNA gene phylogenetic tree of the NTO-degrading bacteria of the present invention; Figure 7It is a schematic diagram of the growth of NTO-degrading bacteria of the present invention under 37°C; Figure 8 It is the standard curve of liquid phase detection of NTO; Fig. 9 The degradation effect of the NTO-degrading bacteria of the present invention on NTO is monitored within 72 hours. DETAILED DESCRIPTION

[0013] The following describes the implementation of the present invention through specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0014] The exemplary embodiments of the present invention are now described with reference to the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided to disclose the present invention in detail and completely and to fully convey the scope of the present invention to those skilled in the art. The terms used in the exemplary embodiments shown in the accompanying drawings are not intended to limit the present invention.

[0015] Unless otherwise specified, the terms (including technical terms) used herein have the commonly understood meanings to those skilled in the art. In addition, it is understood that the terms defined in commonly used dictionaries should be understood to have the same meanings as those in the context of the relevant fields, and should not be understood as idealized or overly formal meanings.

[0016] Example 1 The present invention provides a strain of NTO-degrading bacteria of energetic materials, wherein the NTO-degrading bacteria is a fibrogenic microbacterium cellulosimicrobium cellulans The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No. 28714. NTO is 3-nitro-1,2,4-triazol-5-one, with the structural formula Figure 1 shown.

[0017] Screening of NTO-degrading bacteria: Activated sludge was collected from the aeration tank of a sewage treatment plant of an NTO production enterprise. 100g of sludge was added to an inorganic salt culture medium with an NTO content of 13g / L, and enriched and cultured in a constant temperature shaker at 37°C and 120rpm. Every 10 days was a cycle, and the inorganic salt culture medium was replaced with a 1% transfer. After 5 generations of continuous enrichment, various wild bacteria gradually decreased under strict pressure conditions, and a relatively single bacterial solution that could utilize NTO was obtained. Figure 2 .

[0018] Example 2 This embodiment provides an energetic material NTO-degrading bacterial solution, which is obtained by expanding and culturing a fibrogenic microbacterium strain.

[0019] The specific process of expanded culture was as follows: a single colony of the fibrogenic microbacterium strain was picked and inoculated into NTO inorganic salt liquid culture medium, and cultured for 48 h in a constant temperature shaker at 37 °C and 120 rpm.

[0020] Purification and preservation of degrading bacteria: Based on the screening of degradation bacteria in Example 1, the bacterial solution in the fifth generation culture bottle was streaked and inoculated on the NTO inorganic salt solid culture medium plate for purification. After constant temperature culture at 37°C for 24 hours, a single colony was picked and inoculated into the NTO inorganic salt liquid culture medium, and continued to be cultured in a constant temperature shaker at 37°C and 120 rpm. This was repeated, and a purified degradation bacteria was obtained according to the colony morphology, named CzL-03. The purified bacterial solution was centrifuged, collected, added to 20% glycerol LB culture medium for suspension, and stored in a -80°C refrigerator.

[0021] Among them, NTO inorganic salt solid culture medium (g / L): liquid NTO inorganic salt culture medium plus 2% agar powder was sterilized at high temperature at 121℃ for 25min.

[0022] NTO inorganic salt liquid culture medium (g / L): KH 2 PO 4 1.5 g, Na 2 HPO 4 •12H 2 O 3.8g, water 1000 mL, high temperature sterilization at 121℃ for 25 min, then add 10mL of high temperature sterilized 1g / L CaCl 2 •2H 2 O, 10 mL of filter-sterilized 5 g / L MgSO 4 •7H 2 O, 0.2g / L MnSO 4 •4H 2 O and 0.5g / L FeSO 4 •7H 2 O mixture, 10 mL of filter-sterilized 0.1 mol / L NTO solution.

[0023] Preparation of 0.1 mol / L NTO solution: Accurately weigh 0.13 g of NTO and dissolve it in 10 mL of sterile water, and filter and sterilize. NTO (purity > 99.0%, Xi'an Modern Chemistry Research Institute) Strain identification 1. Morphological observation and Gram staining Observe the colony morphology of LB solid medium plate after 12 h of culture. Figure 3 The colony morphology of CzL-03 is regular, the colonies are small, dark yellow, convex in the middle, moist and smooth surface, opaque, and the edges are neat and round.

[0024] The single colony was observed under a Gram staining microscope after smearing. CzL-03 bacteria were spherical, 1-2 μm, and Gram-positive bacteria without capsules and spores. Figure 4 .

[0025] 2. 16s rDNA identification and phylogenetic tree analysis The total DNA of the degrading bacteria genomic DNA was extracted using a kit and PCR amplified using 16S rDNA universal primers (27F: AGAGTTTGATC TGGCTCAG; 1492R: GGTTACCTTGTTACGACTT). The PCR reaction system was 20 μL: 2×GC Mix 10 μL, universal primers 1 μL each (10 mmol / L), template DNA 1 μL, and sterile deionized water 7 μL.

[0026] Amplification procedure: 95 °C pre-denaturation for 5 min, 95 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C primer extension for 30 s, 35 cycles, 72 °C extension for 5 min. Finally, the PCR purified product was entrusted to Beijing Novogene Technology Co., Ltd. for sequencing, and the sequencing results were compared with the NCBI database.

[0027] The 16s rDNA sequencing result of CzL-03 was 1398 bp, and the agarose electrophoresis result was shown in Figure 5 The nucleotide sequence of its 16s rDNA was consistent with that of the cellulosic aquatic microbacterium in GenBank ( cellulosimicrobium aquaticle ) 16s rDNA gene sequence homology is 99.36%, and the phylogenetic tree was constructed using MEGA11.0 software, see Figure 6 , combined with the morphological characteristics of the strain, it was determined that the strain was fibrogenic fibromicrobacterium ( cellulosimicrobium cellulans ). It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 23, 2023, with the deposit number CGMCC No.28714. The address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, and the classification name is fibrosing fiber microbacteria ( cellulosimicrobium cellulans ).

[0028] The 16s rDNA sequence of CzL-03 bacteria is as follows: 3. Determination of growth curve The preserved strain was streaked and inoculated into LB solid medium and cultured at 37°C. A single colony was inoculated into LB liquid medium. The medium without inoculated colonies was used as blank control. The culture was cultured in a constant temperature shaker at 37°C and 120 rpm. Samples were taken at intervals and OD was detected by UV spectrophotometer. 600 The results showed that the fibroblast-forming microbacteria CzL-03 entered the exponential growth phase from the 24th hour and entered the stable phase after 48 hours. Figure 7 .

[0029] Among them, LB liquid culture medium (g / L): NaCl 10g, Tryptone 10g, Yeast Extract 5g, add water to make up to 1000mL, and sterilize at high temperature at 121℃ for 25min. LB solid medium (g / L): Liquid LB medium plus 2% agar powder and sterilized at 121°C for 25 min.

[0030] Example 3 This embodiment provides an application of energetic material NTO-degrading bacteria in the preparation of NTO-degrading products.

[0031] Determination of degradation curve NTO was prepared into solutions with different concentration gradients, and a standard curve was drawn using high performance liquid chromatography detection technology. Figure 8 The strain CzL-03 was inoculated into an inorganic salt medium containing 1mmol / L NTO, and samples were taken regularly to determine the NTO content. The change in NTO content in the inorganic salt medium without inoculation was used as a control. The trend of NTO concentration change was calculated based on the standard curve. The results showed that CzL-03 could completely degrade 1mmol / L NTO within 72h. Fig. 9 During the growth process, strain CzL-03 will use NTO in the culture medium. The OD of the bacterial solution obtained after 48 hours is 600 The value is 1.4.

[0032] Adding this degrading bacteria to NTO-contaminated water or soil environment can efficiently degrade NTO and has broad application prospects in bioremediation.

[0033] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.

Claims

1. A strain of energetic material NTO-degrading bacteria, characterized in that: The NTO-degrading bacteria are fibrogenic microbacteria cellulosimicrobium cellulans The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number CGMCC No. 28714.

2. Energetic material NTO degrading bacterial liquid, characterized by: The fiber-forming microbacteria strain is obtained by expanding and culturing the fiber-forming microbacteria strain described in claim 1.

3. The energetic material NTO-degrading bacterial solution according to claim 2, characterized in that: The specific process of expanded culture was as follows: a single colony of the fibrogenic microbacterium strain was picked and inoculated into NTO inorganic salt liquid culture medium, and cultured for 48 h in a constant temperature shaker at 37 °C and 120 rpm.

4. Use of the energetic material NTO-degrading bacteria according to claim 1 in the preparation of NTO-degrading products.

5. Use of the energetic material NTO-degrading bacterial solution according to claim 2 in the preparation of NTO-degrading products.

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