Nitroglycerin degrading bacterium and application thereof
By screening and identifying Pseudomonas stutzeri WN1, the problems of limited species and poor tolerance of nitroglycerin-degrading bacteria were solved, efficient and economical nitroglycerin wastewater treatment was achieved, and the degradation efficiency and environmental friendliness were significantly improved.
Patent Information
- Application Number
- CN202511055120.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-30
- Publication Date
- 2025-10-14
AI Technical Summary
In the existing technology, the types of nitroglycerin-degrading bacteria are limited, and their tolerance to high concentrations of nitroglycerin is poor. Traditional treatment methods are costly and prone to secondary pollution.
A strain of Pseudomonas stutzeri WN1 was screened and identified, with the preservation number CGMCC No. 33673. It can efficiently degrade nitroglycerin in a high-concentration environment without the need for exogenous carbon and nitrogen sources, and directly uses nitroglycerin as the only carbon and nitrogen source.
It achieves efficient degradation of nitroglycerin with a degradation rate of 98%, and can tolerate high concentrations of nitroglycerin of 500 mg/L. It significantly shortens the pollutant removal cycle, reduces treatment costs, and has both environmental and economic benefits.
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Figure CN120775744A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of environmental microorganisms and relates to a nitroglycerin-degrading bacterium and an application thereof. Background Art
[0002] Nitroglycerin (C3H5N3O9) is a nitrate compound widely used in industries such as explosives and pharmaceuticals. Its chemical properties are stable and it is easily discharged into the environment through wastewater, posing a serious threat to ecosystems and human health. Traditional physical and chemical treatment methods for nitroglycerin-containing wastewater (such as activated carbon adsorption and chemical oxidation) have problems such as high cost and the tendency to cause secondary pollution (Dang Yunfei, Wei Zhixian, Yue Pan, et al. Research progress in nitroglycerin wastewater treatment [J]. Fine Chemical Intermediates, 2018, 48(06): 6-11+14. DOI: 10.19342 / j.cnki.issn.1009-9212.2018.06.002.). Biodegradation has attracted much attention due to its environmental friendliness. However, the reported nitroglycerin-degrading bacteria are limited in species and have poor tolerance to high concentrations of nitroglycerin (Yang Shishan, Zhang Wei, Wang Jigui. Research Progress on Microbial Degradation of Nitroglycerin and Nitrocellulose [J]. Chemical Industry and Engineering Progress, 2011, 30(08):1854-1857. DOI:10.16085 / j.issn.1000-6613.2011.08.030.). Therefore, screening for efficient and tolerant nitroglycerin-degrading strains is of great significance for the treatment of industrial wastewater containing nitroglycerin. Summary of the Invention
[0003] In order to develop a nitroglycerin-degrading strain with high degradation efficiency and strong tolerance for industrial wastewater treatment, the present invention screened a strain WN1 capable of efficiently degrading nitroglycerin from activated sludge. The strain is a Gram-negative aerobic bacillus. After morphological and molecular biological identification, it was determined that the strain is Pseudomonas stutzeri ( Stutzerimonas stutzeri ) and deposited with the General Microbiology Center of the China Culture Collection Administration under the deposit number CGMCC No. 33673, with a deposit date of February 27, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. This strain can achieve a 98% degradation rate of 300 mg / L of nitroglycerin within three days and is tolerant to high concentrations of nitroglycerin (no significant inhibition below 500 mg / L), making it suitable for use in wastewater treatment applications with high levels of nitroglycerin contamination. Furthermore, this strain requires no external carbon and nitrogen sources and can directly utilize nitroglycerin as its sole carbon and nitrogen source, reducing treatment costs and providing an innovative solution for the biological treatment of industrial wastewater containing nitroglycerin.
[0004] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions: The first object of the present invention is to provide a Pseudomonas stutzeri ( Stutzerimonas stutzeri )WN1, the deposit number is CGMCC No.33673.
[0005] The second object of the present invention is to provide the use of the above-mentioned Pseudomonas stutzeri WN1 in degrading industrial wastewater containing nitroglycerin.
[0006] The third object of the present invention is to provide the use of the above-mentioned Pseudomonas stutzeri WN1 in the preparation of a microbial preparation for degrading industrial wastewater containing nitroglycerin.
[0007] The fourth object of the present invention is to provide a microbial preparation containing the above-mentioned Pseudomonas stutzeri WN1.
[0008] A fifth object of the present invention is to provide the use of the above-mentioned microbial preparation in degrading industrial wastewater containing nitroglycerin.
[0009] A sixth object of the present invention is to provide use of the Pseudomonas stutzeri WN1 or the microbial preparation in preparing an industrial wastewater treatment agent, wherein the industrial wastewater contains nitroglycerin.
[0010] A seventh object of the present invention is to provide a method for degrading nitroglycerin in industrial wastewater, wherein the method comprises adding the above-mentioned Pseudomonas stutzeri WN1 into industrial wastewater containing nitroglycerin for treatment.
[0011] In one embodiment of the present invention, the Pseudomonas stutzeri WN1 is a bacterial liquid of Pseudomonas stutzeri WN1 cultured to the logarithmic growth phase.
[0012] In one embodiment of the present invention, the treatment temperature is 20-37°C.
[0013] In one embodiment of the present invention, the treatment is carried out at 150-250 r / min.
[0014] Beneficial effects of the present invention: The Pseudomonas stutzeri WN1 screened by the present invention has the following advantages: (1) High-efficiency degradation performance: In a 300 mg / L nitroglycerin system, the 3-day degradation rate of strain WN1 reached 98%, which was significantly better than the degradation efficiency of existing reported strains and significantly shortened the pollutant removal cycle.
[0015] (2) Strong tolerance: The strain still maintains metabolic activity in a high concentration of 500 mg / L nitroglycerin environment, breaking through the tolerance limit of traditional strains to high pollution loads and providing a new solution for the treatment of industrial high-concentration wastewater.
[0016] (3) Metabolic uniqueness: The strain does not require additional carbon and nitrogen sources and directly uses nitroglycerin as the only carbon and nitrogen source, which reduces processing costs and has both environmental benefits and economic value. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 is the colony morphology of Pseudomonas stutzeri WN1; Figure 2 This is the phylogenetic tree analysis result of Pseudomonas stutzeri WN1; Figure 3 This is the growth curve of Pseudomonas stutzeri WN1 at a concentration of 300 mg / L nitroglycerin; Figure 4 This is the degradation curve of Pseudomonas stutzeri WN1 under 300 mg / L nitroglycerin conditions. DETAILED DESCRIPTION
[0018] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings. It should be noted that the embodiments mentioned below are only applicable to explaining the present invention, but are not intended to limit the scope of the present invention. The embodiments mentioned below are only a part of the embodiments of the present invention and not all of the embodiments. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve them. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention. In this field, if other technical personnel have not made creative work, the embodiments they obtain are all protected by the present invention.
[0019] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, culture media, and instruments used are conventional in the art, and can be obtained commercially by those skilled in the art unless otherwise specified. The molecular biology experimental procedures involved in the present invention are conventional in the art or can be performed according to the product instructions of the corresponding reagents unless otherwise specified.
[0020] The culture medium and its composition of the present invention are as follows: Enrichment medium: KH2PO4 0.9 g / L, K2HPO4 2.1 g / L, glycerol 0.55 g / L, MgSO4·7H2O 0.25 g / L, citric acid 0.58 g / L, nitroglycerin 100 mg / L, trace elements 1 mL / L, pH 7.2, and 15 g / L of agar were added to the solid culture medium.
[0021] Degradation medium: KH2PO4 0.9 g / L, K2HPO4 2.1 g / L, MgSO4·7H2O 0.25 g / L, nitroglycerin 100-500 mg / L, trace elements 1 mL / L, pH 7.2.
[0022] The trace element components are boric acid 0.57 g / L, FeCl3·6H2O 0.24 g / L, CoCl2·6H2O 0.04 g / L, CuSO4·5H2O 0.06 g / L, MnSO4·4H2O 0.3 g / L, ZnSO4·7H2O 0.31 g / L, and Na2MoO4·2H2O 0.03 g / L. The product was sterilized with high-pressure steam at 121°C for 20 min before use.
[0023] The stock concentration of nitroglycerin was 1.4 g / L.
[0024] Example 1: Screening of Pseudomonas stutzeri WN1 (1) Enrichment culture of degrading bacteria Using activated sludge from the 7416 Plant of the Fourth Research Institute of the China Aerospace Science and Technology Corporation as an inoculum, 10 g of sludge sample was added to an enrichment culture. After incubation at 35°C and 180 rpm for 5 days, a 5% inoculum was transferred to fresh enrichment culture. This process was repeated for six generations of continuous enrichment and acclimation to obtain a nitroglycerin-degrading enrichment culture. During this period, 1 mL of the bacterial culture was regularly sampled, sterilized by filtration through a 0.22 μm filter, and nitroglycerin concentration was determined by liquid chromatography. The resulting enrichment culture can consume approximately 80% of 100 mg / L of nitroglycerin within 5 days.
[0025] (2) Isolation and purification of degradation bacteria Take 1 mL of the above enriched culture and dilute it serially using the 10-fold dilution method. Select three gradients (e.g. 10 -5 , 10 -6 , 10 -7 Evenly spread 1 mL of each dilution onto solid enrichment medium plates containing 100 mg / L nitroglycerin, with three replicates for each gradient. Incubate the dilution plates upright for 30 minutes, then invert them in a 35°C incubator. Observe colony growth daily.
[0026] Use an inoculating loop to pick up single colonies of different morphologies and streak them on a new culture plate. Pick up the single colony at the end of the streak and streak it again. Burn the inoculating loop each time you streak. The source of the bacteria for the last streak comes from the end of the previous streak. Repeat this process several times until you obtain nitroglycerin-degrading bacteria with good growth and stable passage.
[0027] (3) Acclimation of degradation bacteria To improve the degradation and tolerance of nitroglycerin by the degrading bacteria, an acclimation experiment was conducted. The glycerol and citric acid components of the enrichment medium were removed, and nitroglycerin was added as the sole carbon source. The strain was inoculated into a degradation medium with an initial nitroglycerin concentration of 100 mg / L. Cultures were shaken at 35°C and 180 rpm for 48 hours before transfer. The nitroglycerin concentration was increased to 200, 300, and 500 mg / L, and the strain was acclimated three times in each concentration gradient.
[0028] Example 2: Identification of Pseudomonas stutzeri WN1 (1) Morphological identification The acclimated strain WN1 was plated on a solid enrichment medium containing 100 mg / L nitroglycerin and incubated in a 35°C incubator for 5 days. The colonies were observed for morphology, texture, color, size, edges, and surface conditions. The colonies of strain WN1 were characterized by yellow, cauliflower-like color, irregular edges, and a dry texture. Figure 1 ).
[0029] (2) Molecular biological identification Well-growing strains were selected and purified from single colonies to be inoculated into 10 mL of enrichment medium. The culture was shaken for 5 days. Genomic DNA from strain WN1 was extracted using a bacterial genomic DNA extraction kit (spin column format) according to the kit instructions. The extracted genomic DNA was amplified by PCR using universal primers 27F (SEQ ID NO. 2) and 1541R (SEQ ID NO. 3). The amplification system (50 μL total volume) consisted of 45 μL ddH2O, 1 μL template DNA, 1 μL 2.5 mM dNTPs, 1 μL of each primer, and 1 μL Taq DNA polymerase. PCR amplification was performed using a pre-denaturation step at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 90 s, followed by extension at 72°C for 10 min. The PCR products were detected by 1% agarose gel electrophoresis (120 V, 100 mA, electrophoresis for 20 min) and purified. The 16S rRNA sequence of strain WN1 obtained by sequencing is shown in SEQ ID NO. 1. The obtained gene sequence was compared with known sequences in GenBank by BLAST to find the most similar strain sequence. Homology analysis was performed, and a phylogenetic tree was constructed using MEGA 7.0 software ( Figure 2 ), to determine the systematic taxonomic status of strain WN1. The results showed that the strain was consistent with the Stutzerimonas stutzeri The similarity of strain CIFRI.BTL.S-24 (PP780384.1) was the highest, reaching 100%, and it was preliminarily confirmed that the strain was Pseudomonas stutzeri ( Stutzerimonas stutzeri ), and named Stutzerimonas stutzeri WN1.
[0030] The present invention deposits strain WN1, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC No. 33673 and a deposit date of February 27, 2025. The depository address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.
[0031] SEQ ID NO.1: SEQ ID NO.2: 5'-AGAGTTTTGATCCTGGCTCA-3'; SEQ ID NO. 3: 5'-AAGGAGGTGATCCAGCCGCA-3'.
[0032] Example 3: Application of Pseudomonas stutzeri WN1 in the degradation of nitroglycerin (1) Growth determination of strain WN1 The bacterial suspension of strain WN1 was inoculated into degradation medium with nitroglycerin concentrations of 100, 200, 300 and 500 mg / L at a 5% inoculum volume, and cultured at 35°C and 180 rpm for 5 days. Samples were taken every 12 hours to measure the OD value. 600 Value, with time as the horizontal axis, OD 600 The value is the vertical axis, and the growth curve is drawn. The result is as follows Figure 3 As shown in the figure, strain WN1 grew best at a concentration of 300 mg / L nitroglycerin. It entered the logarithmic growth phase 12 hours after inoculation, and the maximum absorbance value reached 0.9, indicating that strain WN1 had good tolerance to 300 mg / L nitroglycerin.
[0033] (2) Determination of nitroglycerin degradation rate by strain WN1 The bacterial suspension of strain WN1 was inoculated into degradation medium with nitroglycerin concentrations of 100, 200, 300, and 500 mg / L at a 5% inoculum volume. The culture was shaken at 35°C and 180 rpm for 5 days. During this period, samples were taken every 12 hours to measure the nitroglycerin concentration. The degradation curve was plotted with time as the horizontal axis and nitroglycerin concentration as the vertical axis. The results are shown in Figure 2. Figure 4 As shown in the data, when the nitroglycerin concentration was 300 mg / L, the degradation rate of nitroglycerin by strain WN1 reached 98% within 3 days, which was significantly better than the performance of strains in similar studies.
[0034] (3) Study on the degradation mechanism of degrading bacteria To further clarify the metabolic pathway of strain WN1 to nitroglycerin, four groups of experiments were designed, respectively, control group, glycerol group, ammonium sulfate group, blank group, wherein the culture medium of the control group is the degradation culture medium added with 300 mg / L nitroglycerin; the glycerol group does not add nitroglycerin in the degradation culture medium, and 0.5 g / L glycerol is added as a carbon source, and no external nitrogen source is contained; the ammonium sulfate group does not add nitroglycerin in the degradation culture medium, and 0.3 g / L ammonium sulfate is added as a nitrogen source, and no external carbon source is contained; the blank group removes the nitroglycerin component in the degradation culture medium, and no carbon source and nitrogen source are added. The bacterial liquid of strain WN1 cultured to the logarithmic phase is centrifuged at a speed of 10000 r / min for 5 min, the supernatant is discarded, and the bacterial liquid is washed 2-3 times with sterile water to remove the components of the culture medium containing nitroglycerin, and finally the bacterial liquid is resuspended with sterile water and inoculated into the above experimental groups, and cultured at 35℃, 180 r / min for 96 h, and the OD 600 value is measured every 12 h. The results show that the OD 600 of the control group can reach 0.92 after 72 h of culture, and stable growth is achieved; while the strains in the glycerol group, the ammonium sulfate group and the blank group do not grow. It is confirmed that WN1 cannot maintain metabolism by using single glycerol or ammonium sulfate, and the growth is completely inhibited when lacking carbon source and nitrogen source. This shows that nitroglycerin can be used as the only carbon source and nitrogen source for the growth and metabolism of WN1 strain, and provides a metabolic basis for its high-efficiency repair ability in the oligotrophic nitroglycerin-polluted environment.
[0035] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore, the protection scope of the present application should be defined by the claims.
Claims
1. A Pseudomonas stutzeri ( Stutzerimonas stutzeri ) WN1, characterized in that, The deposit number is CGMCC No.33673.
2. Use of the Pseudomonas stutzeri WN1 according to claim 1 in degrading industrial wastewater containing nitroglycerin.
3. Use of the Pseudomonas stutzeri WN1 according to claim 1 in the preparation of a microbial preparation for degrading industrial wastewater containing nitroglycerin. A microbial preparation containing the Pseudomonas stutzeri WN1 according to claim 1 .
5. Use of the microbial preparation according to claim 4 in degrading industrial wastewater containing nitroglycerin.
6. Use of the Pseudomonas stutzeri WN1 according to claim 1 or the microbial preparation according to claim 4 in the preparation of an industrial wastewater treatment agent, characterized in that: The industrial wastewater contains nitroglycerin.
7. A method for degrading nitroglycerin in industrial wastewater, characterized in that: The method comprises adding the Pseudomonas stutzeri WN1 described in claim 1 into industrial wastewater containing nitroglycerin for treatment.
8. The use according to claim 7, characterized in that The Pseudomonas stutzeri WN1 is a bacterial liquid of Pseudomonas stutzeri WN1 cultured to the logarithmic growth phase.
9. The use according to claim 7, characterized in that The treatment temperature is 20-37°C.
10. The use according to claim 7, characterized in that The treatment is carried out under the condition of 150-250 r / min.