Chryseobacterium P6 for degrading unsymmetrical dimethylhydrazine and degradation method thereof

Abstract

The invention provides Chryseobacterium P6 for degrading unsymmetrical dimethylhydrazine and a degradation method of the Chryseobacterium P6, the strain is obtained by separating from activated sludge which is domesticated by the unsymmetrical dimethylhydrazine for a long time, and the strain is identified as Bacteroidetes door and Chryseobacterium by 16SrDNA (Deoxyribose Nucleic Acid) gene sequence analysis. After being amplified and cultured for 12 hours by an LB culture medium, the strain can show remarkable degradation activity on unsymmetrical dimethylhydrazine under the conditions that the pH is 7.0 and the temperature is 32 DEG C, and the degradation rate within 48 hours can reach 95% or above. The method is suitable for in-situ remediation of industrial wastewater, polluted soil and waste without additionally adjusting the pH value or adding an oxidizing agent; rapid propagation of the strain can be realized by using a conventional culture medium (such as an LB culture medium), complex equipment or energy input is not needed in the degradation process, and the unit treatment cost is reduced by more than 60% compared with that of a chemical oxidation method; no heavy metal catalyst, high-concentration oxidant or adsorbent is used in the whole process, and secondary pollution risks of bromate, regenerated waste liquid and the like are completely eradicated.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a Chlorella P6 strain for degrading unsymmetrical dimethylhydrazine and its degradation method. Background Technology

[0002] Unsymmetrical dimethylhydrazine (UDMH) is a colorless, transparent liquid with a strong ammonia odor and a boiling point of 63.5°C. It is miscible with water and most organic solvents (such as ethanol and ether). Its high-energy N / N bonds and methyl groups in its molecular structure give it strong reducing properties, making it readily ignited with oxidizers (such as nitrogen tetroxide). It is widely used as a liquid rocket propellant. However, UDMH's high volatility (vapor pressure of 16.4 kPa at 20°C) and mobility cause it to rapidly diffuse into soil, groundwater, and the atmosphere after leakage. Furthermore, in acidic or oxidizing environments, it readily generates toxic derivatives such as nitrosodimethylamine and formaldehyde, exacerbating environmental risks.

[0003] Unsymmetrical dimethylhydrazine (UDMH) is classified as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC), meaning it can cause acute and chronic harm to humans through skin contact, inhalation, or ingestion. Acute exposure can lead to hemolytic anemia, liver and kidney damage, and central nervous system depression. Long-term low-dose exposure is significantly associated with the development of liver and lung cancer. Its metabolic intermediate, nitrosodimethylamine, is a potent genotoxic substance that can persist in the environment for extended periods and accumulate through the food chain, threatening biosafety. Around space launch sites, UDMH contamination has led to the destruction of soil microbial communities, vegetation degradation, and groundwater deterioration, becoming a key factor limiting regional ecological restoration.

[0004] Space launch sites frequently face contamination from unsymmetrical dimethylhydrazine (UDMH) due to fuel loading, testing, and accidental leaks. UDMH often coexists with nitrogen tetroxide and hydrazine derivatives, forming complex mixed pollutants that are easily triggered by traditional chemical oxidation methods. Areas with high launch activity require rapid decontamination to ensure subsequent operations, but the adsorbed form of UDMH in the soil (Koc = 120–180) is difficult to completely remove through physical washing.

[0005] While existing physicochemical treatment technologies (such as ozone oxidation and activated carbon adsorption) can reduce unsymmetrical dimethylhydrazine (UDMH) concentrations in the short term, they suffer from high energy consumption, secondary pollution (such as bromate formation), and the inability to eradicate nitrosodimethylamine. Therefore, developing a green remediation technology that can adapt to extreme environments and completely mineralize UDMH and its toxic derivatives has become an urgent need in the aerospace environmental protection field. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] This invention proposes a Chlorella P6 strain for degrading unsymmetrical dimethylhydrazine (UDMH) and its degradation method, providing a simple, low-cost, and convenient microbial strain suitable for the biodegradation of UDMH for the remediation of rocket propellant contaminated sites.

[0008] (II) Technical Solution

[0009] To address the aforementioned technical problems, this invention proposes Chryseobacterium sp. P6, which belongs to the phylum Bacteroidetes and the genus Chryseobacterium. It was deposited on April 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34173, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its 16S rDNA gene sequence is shown in SEQ ID NO:1.

[0010] Furthermore, this invention also proposes a method for degrading unsymmetrical dimethylhydrazine using the aforementioned Chryseobacterium sp. P6, the method comprising the following steps:

[0011] S1. Chryseobacterium sp. P6 was inoculated onto slant culture medium and cultured to obtain slant culture;

[0012] S2. Inoculate the slant culture into an Erlenmeyer flask containing LB medium and culture to obtain a bacterial suspension;

[0013] S3. Inoculate the bacterial suspension into an Erlenmeyer flask containing a degradation solution of unsymmetrical dimethylhydrazine and culture it to degrade the unsymmetrical dimethylhydrazine.

[0014] Furthermore, in step S1, Chryseobacterium sp. P6 is inoculated into slant culture medium at a temperature of 37°C for 24 hours.

[0015] Further, in step S1, the slant culture medium includes: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 15 g / L agar powder.

[0016] Further, in step S2, the slant culture is inoculated into 50 mL of LB medium and cultured at 32 °C and 140 r / min for 12 h.

[0017] Furthermore, in step S2, the LB medium comprises: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0018] Further, in step S3, the bacterial suspension is inoculated into the degradation solution of unsymmetrical dimethylhydrazine at an inoculum of 3%, and degraded at 32°C and 140 r / min for 48 h.

[0019] Further, in step S3, the degradation solution of unsymmetrical dimethylhydrazine includes: glucose 0.1 g / L, Na2HPO4 2.8 g / L, KH2PO4 1.0 g / L, (NH4)2SO4 0.5 g / L, MgCl2 0.053 g / L, CaCl2 0.05 g / L, which are aseptically added after autoclaving and cooling.

[0020] Furthermore, the pH of the slant culture medium, LB medium, and the degradation solution of unsymmetrical dimethylhydrazine were all 7.0.

[0021] (III) Beneficial Effects

[0022] This invention proposes a Chryseobacterium P6 strain for degrading unsymmetrical dimethylhydrazine (UDMH) and its degradation method. This strain was isolated from activated sludge that had been acclimated to UDMH for a long period and identified by 16S rDNA gene sequence analysis as belonging to the phylum Bacteroidetes and the genus Chryseobacterium. After 12 hours of expansion culture in LB medium, the strain exhibited significant degradation activity against UDMH at pH 7.0 and a temperature of 32°C, achieving a degradation rate of over 95% within 48 hours. This invention requires no additional pH adjustment or oxidant addition, making it suitable for in-situ remediation of industrial wastewater, contaminated soil, and waste. Rapid expansion of the strain can be achieved using conventional culture media (such as LB medium), and the degradation process requires no complex equipment or energy input, reducing the unit treatment cost by more than 60% compared to chemical oxidation methods. The entire process involves no heavy metal catalysts, high-concentration oxidants, or adsorbents, eliminating the risk of secondary pollution such as bromate and regenerated wastewater.

[0023] The strains of this invention are suitable for the bioremediation of industrial wastewater, contaminated soil and waste containing unsymmetrical dimethylhydrazine. They have advantages such as simple operation, low cost and no secondary pollution, and can provide new microbial resources and technical solutions for the treatment of rocket fuel pollution. Attached Figure Description

[0024] Figure 1 This is a morphological diagram of the Chlorella P6 degrading strain of the present invention;

[0025] Figure 2 Gram staining image of the Chlorella P6 degrading strain of the present invention;

[0026] Figure 3 This is a phylogenetic tree of the *Aureobacterium* P6 degrading strain of the present invention. Detailed Implementation

[0027] To make the objectives, contents, and advantages of the present invention clearer, the specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples.

[0028] This invention proposes a Chryseobacterium sp. P6 strain, which belongs to the phylum Bacteroidetes and the genus Chryseobacterium. It was deposited on April 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34173, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its 16S rDNA gene sequence is shown in SEQ ID NO:1.

[0029] (1) Isolation and identification of the strain: A highly efficient degrading strain was isolated and purified from activated sludge acclimated to unsymmetrical dimethylhydrazine (UDMH) through enrichment culture, serial dilution, and streak plating. 16S rDNA gene sequence analysis (as shown in SEQ ID NO: 1) identified it as belonging to the phylum Bacteroidetes and the genus Chryseobacterium, and named it Chryseobacterium sp. P6, with accession number CGMCC No. 34173. This strain can efficiently degrade UDMH.

[0030] (2) Culture and degradation characteristics of the strain: After the strain was cultured in LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) at 32℃ and 140 r / min for 12 h, a highly active bacterial suspension was formed. When the suspension was added to a degradation system containing 50 mg / L unsymmetrical dimethylhydrazine (pH 7.0, 32℃, 140 r / min) at an inoculum of 3%, the degradation rate of unsymmetrical dimethylhydrazine reached more than 90% within 48 h.

[0031] (3) Degradation process conditions: The bacterial strain was inoculated into slant culture medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar powder, pH 7.0) and cultured at 37℃ for 24 h; the slant culture was then inoculated into LB liquid medium and cultured at 32℃ and 140 r / min for 12 h with shaking to obtain a bacterial suspension with OD600≈1.5; the bacterial suspension was added at 3% inoculation to a degradation solution containing unsymmetrical dimethylhydrazine (0.1 g / L glucose, 2.8 g / L Na2HPO4, 1.0 g / L KH2PO4, 0.5 g / L (NH4)2SO4, 0.053 g / L MgCl2, 0.05 g / L CaCl2, 50 mg / L unsymmetrical dimethylhydrazine, pH 7.0), and reacted at 32℃ and 140 r / min for 48 h to achieve efficient degradation of unsymmetrical dimethylhydrazine.

[0032] In this embodiment, the specific steps for screening unsymmetrical dimethylhydrazine degrading bacteria are as follows:

[0033] The sludge suspension was mixed by shaking in sterile phosphate-buffered saline (PBS, pH 7.0) (200 rpm, 30 min), and after standing, the supernatant was used as the bacterial source. Ultrapure water was sterilized at 121°C for 30 min and cooled to obtain sterile water. The bacterial source solution was then serially diluted with sterile water in 10-fold increments to a final concentration of 10. -6 Concentration. Take 10 -4 10 -5 10 -6 100 μL of each diluent was evenly spread onto LB solid isolation medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar powder, pH 7.0) and incubated upside down at 37°C for 24 h. Colonies with significantly different morphologies were picked and repeatedly streaked three times on LB solid medium containing unsymmetrical dimethylhydrazine (UDMH, 50 mg / L) to obtain a single, pure UDMH-degrading strain. This strain was then inoculated onto slant agar and incubated at 37°C for 24 h. The UDMH-degrading strain was named *Chlorella vulgaris* P6. The morphology of *Chlorella vulgaris* P6 is shown in the figure below. Figure 1 As shown.

[0034] In this embodiment, the specific steps for identifying unsymmetrical dimethylhydrazine degrading bacteria are as follows:

[0035] Fresh cultures of the purified strains were collected, and single colonies were picked up using a sterile inoculation loop and evenly spread onto a clean glass slide to form a bacterial film approximately 1 cm in diameter. After air drying, the film was heat-fixed by rapidly passing it through the outer flame of an alcohol lamp three times. Crystal violet staining solution was added to cover the bacterial film and stained for 1 min; then gently rinsed with deionized water until no residual staining remained. Lugol's iodine solution (1.0 g iodine, 2.0 g potassium iodide, 300 mL deionized water) was added and incubated for 1 min to enhance the binding of the dye to the cell wall. The Lugol's iodine solution was rinsed with deionized water. The slide was tilted, and 95% ethanol was added to decolorize the film for 20–30 s (until the ethanol flowed out colorlessly). Safranin staining solution was added for counterstaining for 1 min, rinsed with deionized water, and blotted dry with absorbent paper before microscopic examination. Under an optical microscope (oil immersion, 1000×), *Chlorella vulgaris* P6 appeared red, indicating it was a Gram-negative bacterium. Gram staining of *Chlorella vulgaris* P6 is shown below. Figure 2 As shown.

[0036] Figure 3 As shown.

[0037] In this embodiment, the specific steps for testing the degradation performance of unsymmetrical dimethylhydrazine-degrading bacteria are as follows:

[0038] The slant culture was inoculated into 50 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and cultured at 32 °C with shaking at 140 rpm for 12 h to obtain a bacterial suspension with OD600 = 1.5 ± 0.1. A degradation solution containing unsymmetrical dimethylhydrazine was prepared, consisting of: 0.1 g / L glucose, 2.8 g / L Na₂HPO₄, 1.0 g / L KH₂PO₄, 0.5 g / L (NH₄)₂SO₄, 0.053 g / L MgCl₂, and 0.05 g / L CaCl₂. The solution was autoclaved (115 °C, 20 min), cooled, and then under aseptic conditions, unsymmetrical dimethylhydrazine was added to a final concentration of 50 mg / L (pH 7.0). The bacterial suspension was added to the degradation solution at an inoculum of 3%, and cultured under continuous shaking at 32℃ and 140r / min for 72h. Samples were taken every 12h to detect the concentration of unsymmetrical dimethylhydrazine.

[0039] The concentration of unsymmetrical dimethylhydrazine (UDMH) was determined by high-performance liquid chromatography (HPLC). The chromatographic conditions were: C18 reversed-phase column (4.6 mm × 250 mm, 5 μm); flow rate 1.0 mL / min; mobile phase: potassium dihydrogen phosphate buffer solution-water (85:15, v / v). The potassium dihydrogen phosphate buffer solution was prepared as follows: 0.68 g of potassium dihydrogen phosphate was weighed, dissolved in water, and diluted to 500 mL. The solution was mixed thoroughly, transferred to a reagent bottle, and stored at 4 °C protected from light.

[0040] Chlorella P6 exhibits highly efficient degradation capabilities for unsymmetrical dimethylhydrazine, with a degradation rate of 71.8% in 24 hours and 96.43% in 48 hours, and poses no risk of secondary pollution, thus meeting the rapid remediation needs of aerospace fuel-contaminated sites.

[0041] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Chryseobacterium sp. P6, characterized by, The *Chryseobacterium* sp. P6 belongs to the phylum *Bacteroidetes* and the genus *Chryseobacterium*. It was deposited on April 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34173. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The 16S rDNA gene sequence is shown in SEQ ID NO:

1.

2. A method for degrading unsymmetrical dimethylhydrazine using *Chryseobacterium sp.* P6 as described in claim 1, characterized in that, The method includes the following steps: S1. Chryseobacterium sp. P6 was inoculated onto slant culture medium and cultured to obtain slant culture; S2. Inoculate the slant culture into an Erlenmeyer flask containing LB medium and culture to obtain a bacterial suspension; S3. Inoculate the bacterial suspension into an Erlenmeyer flask containing a degradation solution of unsymmetrical dimethylhydrazine and culture it to degrade the unsymmetrical dimethylhydrazine.

3. The method for degrading unsymmetrical dimethylhydrazine as described in claim 2, characterized in that, In step S1, Chryseobacterium sp. P6 was inoculated into slant culture medium at a temperature of 37°C for 24 hours.

4. The method for degrading unsymmetrical dimethylhydrazine as described in claim 2, characterized in that, In step S1, the slant culture medium includes: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 15 g / L agar powder.

5. The method for degrading unsymmetrical dimethylhydrazine as described in claim 2, characterized in that, In step S2, the slant culture was inoculated into 50 mL of LB medium and cultured at 32 °C and 140 r / min for 12 h.

6. The method for degrading unsymmetrical dimethylhydrazine as described in claim 2, characterized in that, In step S2, the LB medium consists of: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

7. The method for degrading unsymmetrical dimethylhydrazine as described in claim 2, characterized in that, In step S3, the bacterial suspension was inoculated into the degradation solution of unsymmetrical dimethylhydrazine at an inoculation rate of 3%, and degraded at 32°C and 140 r / min for 48 h.

8. The method for degrading unsymmetrical dimethylhydrazine as described in claim 2, characterized in that, In step S3, the degradation solution of unsymmetrical dimethylhydrazine includes: glucose 0.1 g / L, Na2HPO4 2.8 g / L, KH2PO4 1.0 g / L, (NH4)2SO4 0.5 g / L, MgCl2 0.053 g / L, CaCl2 0.05 g / L, which are aseptically added after autoclaving and cooling.

9. The method for degrading unsymmetrical dimethylhydrazine as described in claim 2, characterized in that, The pH of the slant culture medium, LB medium, and unsymmetrical dimethylhydrazine degradation solution was 7.0.

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