Rhodococcus sp. And application thereof in biodegradation of octogen

Abstract

The invention discloses rhodococcus and application thereof in biodegradation of octogen, and belongs to the technical field of microorganisms. The invention aims to provide a strain of rhodococcus capable of degrading oversaturated high-concentration octogen. The invention provides a strain of Rhodococcus sp. LH-2, the name of the Rhodococcus sp. LH-2 is Rhodococcus sp. LH-2, the Rhodococcus sp. LH-2 is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation date is July 15, 2024, and the preservation number is CGMCC No.31308. The invention also provides a method for preparing the Rhodococcus sp. LH-2. The Rhodococcus sp.LH-2 used in the invention can degrade saturated high-concentration octogen, which indicates that the Rhodococcus sp.LH-2 has a certain application prospect in the resource utilization of octogen wastewater.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Rhodococcus and application thereof in biodegradation of octogenes. Background Art

[0002] Octogen (HMX) has an eight-membered ring nitramine structure and is the best explosive with the best overall performance today because of its good explosive performance. However, improper operation during the production and use of Octogen causes serious pollution to the environment around the factory, resulting in changes in the structure of soil microbial communities and inhibition of soil metabolic processes; and Octogen has significant toxicity and strong irritation to the eyes. It also has certain toxicity and mutagenic effects on the central nervous system of mammals. When Octogen is injected intravenously into animals, it causes circulatory system weakness and central nervous system disorders; the direct symptoms for humans are cardiovascular depression or atrophy, hyperfunction and convulsions; in addition, the solubility of Octogen in water is 5 mg·L -1 Due to its low n-octanol-water partition coefficient (logkow=0.16), octogen dissolved in water is not easily adsorbed by soil or sediment, but is transferred to groundwater with soil pore water, thus affecting the ecological balance of the water environment. Therefore, it is particularly important to find a reasonable method to treat octogen waste liquid.

[0003] Common treatment methods for removing octogen include activated carbon adsorption, iron-dependent consumption, catalytic conversion, electrolysis, incineration and oxidative extraction, membrane separation, flocculation and other physical and chemical methods. However, these traditional treatment technologies are not cost-effective, consume a lot of energy, and produce harmful end products such as non-reusable catalysts, wastewater and ash as residual waste, and the treated pollutants or hazardous substances need to be further treated. Many researchers have studied the microbial degradation of explosives earlier. Biodegradation is a treatment method that uses the metabolism of organisms to transform and stabilize waste octogen pollutants to make them harmless. Because the process of treating waste octogen materials has the advantages of low energy consumption, no pollution, can be used in common with existing biochemical pools, and low operating costs, it is an environmentally friendly and cost-effective method, so biodegradation has become a more promising treatment method.

[0004] At present, there are many strains used for microbial degradation of octogen wastewater, such as Bacillus toyonensis and Plasmodium flavidum strains. However, Bacillus toyonensis only has a good degradation effect on octogen at an initial concentration of 6 mg / L (Meda, A., Sangwan, P. & Bala, K. Optimization of process parameters for degradation of HMX with Bacillus toyonensis using response surface methodology. Int. J. Environ. Sci. Technol. 17, 4601–4610 (2020)); Plasmodium flavidum strains can only degrade octogen under aerobic conditions, with an initial concentration of only 6 mg / L, and a degradation period of 20 days (Nagar, S., Shaw, AK, Anand, S. et al. Aerobic biodegradation of HMX by Planomicrobium flavidum. 3Biotech 8, 455 (2018)). Summary of the invention

[0005] The purpose of the invention is to provide a rhodococcus capable of degrading supersaturated high-concentration octogene.

[0006] The present invention provides a strain of Rhodococcus sp. LH-2, the name of the Rhodococcus sp. is Rhodococcus LH-2, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit date of July 15, 2024 and a deposit number of CGMCC No.31308.

[0007] The present invention provides a microbial preparation containing the above-mentioned Rhodococcus.

[0008] The present invention provides an application of the above-mentioned Rhodococcus or the above-mentioned microbial preparation in resisting Octogen.

[0009] The present invention provides an application of the above-mentioned Rhodococcus or the above-mentioned microbial preparation in degrading octochrome.

[0010] The present invention provides a method for degrading octogene, which comprises fermenting the above-mentioned Rhodococcus at 37° C. for at least 84 hours.

[0011] It is further defined that the nitrogen source of the fermentation medium is octogen.

[0012] It is further defined that the carbon source of the fermentation medium is glucose.

[0013] It was further defined that the maximum concentration of Octogen was 28.68 mg / L.

[0014] It is further defined that the added amount of Rhodococcus is 3%-10%.

[0015] The present invention provides the use of the above-mentioned Rhodococcus or the above-mentioned microbial preparation in the preparation of biological cleaning agents, degradation agents or soil repair agents.

[0016] Beneficial effects: Rhodococcus can degrade octogen under aerobic / anaerobic conditions, and tolerates high concentrations of octogen, and can degrade saturated high-concentration octogen, making it more suitable for existing sewage treatment systems. The present invention fermented and separated a high-concentration octogen-tolerant and degrading strain Rhodococcus sp.LH-2 from the seed-keeping bacteria of Rhodococcus sp.LMD. Compared with the disclosed degradation strains, the Rhodococcus sp.LH-2 provided by the present invention can tolerate supersaturated high-concentration octogen wastewater (28.68 mg / L, i.e., 0.0968 mmol / L) through experiments and grow and reproduce in an inorganic salt culture medium with octogen as the only nitrogen source. Under the conditions of temperature of 37°C, pH of about 7 and rotation speed of 220r / min, this strain can degrade octoquinone in the supersaturated state in the culture medium by 78.85% within a period of 84 hours, and can even degrade it to 89.85% after 132 hours. The degradation cycle is about 84 hours. The degradation rate will increase in the later period but the increase is not large. Therefore, in a cycle of 84 hours, it can basically degrade 80%. The degradation rate is relatively fast, which is better than that reported in the literature.

[0017] Compared with other disclosed octoquinone-degrading strains, the Rhodococcus sp. LH-2 provided by the present invention has its own metabolic characteristics and can degrade saturated high-concentration octoquinone, while no Rhodococcus strain that can degrade such a high concentration has been found in literature reports.

[0018] [Biological Deposit Information]: A strain of Rhodococcus sp., the name of which is Rhodococcus sp. LH-2, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is July 15, 2024, and the deposit number is: CGMCC No.31308. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the result of the isolation and identification of Rhodococcus sp.LH-2;

[0020] Figure 2This is the colony picture of Rhodococcus sp.LH-2;

[0021] Figure 3 This is the growth curve of Rhodococcus sp. LH-2 in Octogen solution, where the horizontal axis is time and the vertical axis is biomass. DETAILED DESCRIPTION

[0022] Example 1. Method for isolating and identifying Rhodococcus

[0023] Fermentation medium: 0.31g potassium dihydrogen phosphate, 0.99g potassium dihydrogen phosphate, 146uL glycerol, 0.18g glucose, 0.294g sodium citrate, 200mL RO water, 200uL trace elements, pH adjusted to 7.2, autoclaved and cooled, then added the sole nitrogen source Octogen.

[0024] LB liquid medium: 1 g sodium chloride, 1 g tryptone, 0.5 g yeast extract, 100 mL RO water.

[0025] LB solid medium: 2 g sodium chloride, 2 g tryptone, 1 yeast extract, 3 g agar powder, 100 mL RO water.

[0026] The pH of the above three culture media is about 7. They were sterilized by high pressure at 121°C for 30 min before inoculation.

[0027] Screening, separation and purification of degrading bacteria: Rhodococcus sp. LH-2 was inoculated at a 5% inoculation rate, the inoculated bacterial liquid was centrifuged, the supernatant was discarded and the bacteria were inoculated into 200 mL fermentation medium containing 24 mg / L octoquinone as the only nitrogen source, and the culture was placed in a 37°C constant temperature shaker at 220 r / min. After the fermentation was completed, the fermented Rhodococcus was obtained, which can tolerate and degrade supersaturated high-concentration octoquinone solution and is stored in a -80°C refrigerator.

[0028] The test results showed that the strain was a Gram-positive bacillus that did not form spores. Figure 2 This is the colony formed by the strain in LB solid culture medium. It can be seen that the colony is round, arched, orange-yellow, opaque, with a smooth and moist surface and complete edges.

[0029] The physiological and biochemical characteristics of this strain are: Gram-positive, polymorphic bacteria, may form rudimentary mycelium, immediately break into irregular cells, generally light orange-yellow, pink, orange or red, with colorless variants.

[0030] Identification of degrading bacteria: The present invention uses 16S rDNA to perform homology analysis on the gene sequence of the screened strains, and compares the sequences through the NCBI (National Center for Biotechnology Information) database to obtain a chart.

[0031] result:

[0032] The results of molecular biological identification of the 16S rDNA gene of the strain (sequence https: / / www.ncbi.nlm.nih.gov / nuccore / MK351311.1?from=1&to=1471) showed 100% homology with Rhodococcus ruber strain R1chromosome. The results of isolation and identification of the strain are shown in Figure 1 The obtained Rhodococcus was named Rhodococcus sp.LH-2.

[0033]

[0034] Example 2. A method for degrading octoquinone

[0035] Octogen degradation test:

[0036] The seed-keeping bacteria of Rhodococcus sp. LH-2 obtained in Example 1 was inoculated at a rate of 5%, the inoculated bacterial liquid was centrifuged, the supernatant was discarded and the bacteria were retained, and the culture medium was inoculated into 200 mL of fermentation medium containing 24 mg / L octogen as the sole nitrogen source, and the culture was placed in a constant temperature shaker at 37°C and cultured at 220 r / min. The control group used potassium nitrite as the sole nitrogen source, and the OD value was measured by an ultraviolet spectrophotometer every 12 hours to observe the changes in the bacterial liquid, and the fermentation liquid was taken before and after inoculation to determine the concentration of octogen before and after degradation.

[0037] The results are as follows Figure 3 As shown in the figure, the Rhodococcus containing 24 mg / L octochrome enters the logarithmic growth phase after 24 hours of culture. With the continuous consumption of nutrients in the culture medium, the growth rate of the strain slows down, the activity gradually decreases, and the overall growth effect is general, indicating that octochrome has a certain stress effect on Rhodococcus. The degradation rate of octochrome in the solution is shown in Table 1. When octochrome is the only nitrogen source in experimental group 1, the concentration of octochrome drops from 28.67584 mg / L to 6.06376 mg / L in 84 hours, and the degradation rate reaches 78.85%; when octochrome is the only nitrogen source in experimental group 2, the fermentation time is extended, and the concentration of octochrome drops from the initial 19.463615 mg / L to 1.975208 mg / L in 132 hours, and the degradation rate reaches 89.85%.

[0038] Degradation rate formula: (C-C0)×100% / C (C: initial octogenin concentration; C0: octogenin concentration at the end of fermentation).

[0039] Table 1 Liquid phase and degradation results of Rhodococcus sp.LH-2 using octoquinone as the only nitrogen source

[0040]

Claims

1. A strain of Rhodococcus sp. LH-2, characterized in that: The name of the Rhodococcus is Rhodococcus LH-2, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration. The deposit date is July 15, 2024, and the deposit number is: CGMCC No.31308.

2. A microbial preparation containing the Rhodococcus according to claim 1.

3. Use of the Rhodococcus described in claim 1 or the microbial preparation described in claim 2 in resisting Octogen.

4. Use of the Rhodococcus described in claim 1 or the microbial preparation described in claim 2 in degrading octochrome.

5. A method for degrading octogen, characterized in that: The Rhodococcus bacteria according to claim 1 are fermented at 37° C. for at least 84 hours.

6. The method according to claim 5, characterized in that The nitrogen source of the fermentation medium was octogen.

7. The method according to claim 5, characterized in that The carbon source of the fermentation medium was glucose.

8. The method according to claim 5, characterized in that The highest concentration of Octogen was 28.68 mg / L.

9. The method according to claim 5, characterized in that The addition amount of Rhodococcus is 3%-10%.

10. Use of the Rhodococcus according to claim 1 or the microbial preparation according to claim 2 in the preparation of a biological cleaning agent, a degradation agent or a soil remediation agent.

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