A bacterial strain for degrading methaqualone and application thereof

Abstract

The application belongs to the technical field of environmental microbiology and is applied to the biodegradation of residual methaqualone in the environment, and particularly relates to a methaqualone-degrading strain and application thereof. Sphingobium sp . S-6 belongs to the sphingomonas genus and has been preserved in the China Center for Type Culture Collection on April 2, 2025, with a preservation number of CCTCC NO: M2025674. The strain has good degradation effects on methaqualone at a temperature of 16-30 DEG C and a pH of 4-8, has strong environmental adaptability, provides a high-efficiency degradation strain resource for the elimination and control of residual methaqualone in the environment, and has important theoretical and application values.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology. Specifically, this invention relates to a 2,4-dichlorodegrading strain and its application. Background Technology

[0002] 2,4-D (2,4-D) is a hormone-type herbicide with some systemic and selective properties. It boasts advantages such as low cost, high efficacy, and a broad weed control spectrum, leading to its widespread application in agricultural production. However, its stable benzene ring structure results in slow natural degradation in soil, making it a difficult-to-adsorb organic pollutant. In recent years, due to its large-scale and frequent use, 2,4-D has been frequently detected in both soil and water bodies. Residual 2,4-D in the soil readily diffuses into terrestrial runoff, seriously endangering ecological security and agricultural product quality safety. Therefore, eliminating residual 2,4-D in the environment has become an urgent problem to solve. Currently, 2,4-D pollution remediation mainly includes physical remediation, chemical remediation, and bioremediation. Physical and chemical remediation are limited in practical application due to their potential for secondary pollution and high remediation costs. In contrast, bioremediation technology is considered an environmentally friendly remediation method due to its low cost, thoroughness, and low environmental risk. With increasing emphasis on food and ecological security, bioremediation has become one of the important strategies for achieving sustainable development in modern agriculture. Summary of the Invention

[0003] The purpose of this invention is to provide a 2,4-D degrading strain and its application. This strain not only has a strong substrate degradation ability, but also a strong environmental adaptability, providing a highly efficient degrading strain resource for the elimination and control of residual 2,4-D in the environment.

[0004] In one aspect, the present invention provides a bacterial strain that degrades 2,4-D. Sphingobium sp . S-6 belongs to the genus *Sphingomonas* (… Sphingobium sp . The sample was isolated from a soil sample and is now deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on April 2, 2025, with accession number CCTCCNO: M2025674.

[0005] The strain described in this invention has been identified as *Sphingomonas* genus (…). Sphingobium sp . Gram-negative, strictly aerobic, cultured on LB solid medium for 3 days, colonies are yellow, round, smooth, and have neat edges. The 16S rRNA gene sequence is shown in SEQ ID NO.1.

[0006] In one aspect of the present invention, the strain described hereinSphingobium sp . When supplied with an aerobic inorganic salt medium at an optimal temperature of 25°C and an optimal initial pH of 5.0, strain S-6 was able to completely degrade 0.05 mM dimethyltetrachloride within 4 h.

[0007] strains of the present invention Sphingobium sp . S-6 was inoculated into an inorganic salt culture medium, and its degradation of 2,4-D was investigated. At initial concentrations of 0.025, 0.05, and 0.1 mM, 100%, 100%, and 50% of 2,4-D were removed within 12 h, respectively. This result demonstrates the effectiveness of the present invention. Sphingobium sp. S-6 can efficiently remove dimethyltetrachlorine.

[0008] The optimal degradation temperature and pH for strain S-6 of this invention are 25℃ and 5.0, respectively. Within a temperature range of 16-37℃ and a pH range of 3-8, strain S-6 exhibits the ability to degrade 2,4-D. These results demonstrate that strain S-6 possesses excellent environmental adaptability and good application potential.

[0009] The inorganic salt culture medium (MSM) formula of the present invention is as follows: NaCl 1 g, NH4Cl 1 g, K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, add ultrapure water to make up to 1 L, and adjust the pH to 7.0.

[0010] In one aspect, the present invention provides the strain said inventive. Sphingobium sp . Application of S-6 in the preparation of microbial agents. The microbial agents of this invention are liquid formulations, lyophilized powder formulations, and granular formulations. Preferably, the liquid formulation is a suspension concentrate. The liquid formulations and lyophilized powder formulations contain... Sphingobium sp . Fermented product of S-6.

[0011] In one aspect, the present invention provides a microbial agent containing a bacterial strain. Sphingobium sp . S-6.

[0012] In one aspect, the present invention provides a bacterial strain. Sphingobium sp . Application of S-6 and its microbial agents in the degradation of residual 2,4-D in soil and water. Beneficial effects

[0013] (1) The present invention isolates a strain capable of efficiently degrading 2,4-D from soil samples. Sphingobium sp .S-6. Under optimal conditions, this strain can completely degrade and remove 0.05 mM dimethyltetrachloride within 12 h.

[0014] (2) The present invention Sphingobium sp . S-6 exhibits the ability to degrade 2,3-Tetrachloroethylene at temperatures ranging from 16 to 30°C and pH values ​​ranging from 3 to 8. This strain demonstrates excellent environmental adaptability and promising application potential.

[0015] (3) The strain disclosed in this invention Sphingobium sp . S-6, isolated from soil samples, can adapt well to harsh natural environmental conditions. Compared with physicochemical methods, it significantly reduces costs and produces no toxic byproducts. It has important application value for the bio-enhanced remediation of 2,4-D in water and soil environments. Attached Figure Description

[0016] Figure 1 The strain of the present invention Sphingobium sp . Colony morphology diagram of S-6.

[0017] Figure 2 The strain of the present invention Sphingobium sp . Phylogenetic tree of S-6.

[0018] Figure 3 The strain of the present invention Sphingobium sp . Degradation characteristics of S-6 under different inoculation conditions.

[0019] Figure 4 The strain of the present invention Sphingobium sp . Degradation characteristics of S-6 under different temperature conditions.

[0020] Figure 5 The strain of the present invention Sphingobium sp . Degradation characteristics of S-6 under different pH conditions.

[0021] Figure 6 The strain of the present invention Sphingobium sp . Degradation characteristics of S-6 for different concentrations of dimethyltetrachloride.

[0022] Figure 7 The strain of the present invention Sphingobium sp . Liquid chromatograms of S-6 degradation of different substrates.

[0023] Figure 8The strain of the present invention Sphingobium sp . S-6 against 20 mg kg -1 Research on the remediation of soil contaminated with 2,4-D.

[0024] Figure 9 The strain of the present invention Sphingobium sp . S-6 against 40 mg kg -1 Research on the remediation of soil contaminated with 2,4-D.

[0025] In this invention, " Sphingobium sp . "S-6", "strain S-6", and "Sphingomonas S-6" all refer to the strain with accession number CCTCC NO: M2025674 in this invention. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the technical solutions described in this invention are conventional methods in the art, and the reagents or materials described, unless otherwise specified, are conventional reagents derived from commercial sources.

[0027] Example 1 Strain S-6 ( Sphingobium sp . S-6) Separation and Identification

[0028] 1.1 Isolation of MCPA-degrading strain S-6

[0029] Take 3 g of soil sample and add it to 100 mL of basic salt medium (MSM). Add dimethyl tetrachloride to a final concentration of 0.1 mM and incubate at 30℃ and 180 rpm for 5 days. Then, transfer it to fresh sterile MSM with a final concentration of 0.1 mM dimethyl tetrachloride at an inoculum of 4% (v / v). Repeat this process three times. Detect the concentration of dimethyl tetrachloride in the fourth-generation enrichment solution using high performance liquid chromatography (HPLC). The enrichment solution with degradation effect was diluted and spread onto the surface of LB solid medium and incubated at 30℃ for 3 days. Single colonies were picked and transferred to LB test tubes and cultured to the exponential phase. The obtained bacterial culture was then inoculated into MSM containing 0.1 mM dimethyltetrachloride and cultured at 30℃ and 180 rpm for 2 days. 500 μL of culture medium sample was taken, an equal volume of methanol was added, and the sample was thoroughly shaken and mixed. The sample was then filtered through a 0.22 μm organic filter membrane, and the concentration of dimethyltetrachloride in the sample was detected by HPLC. If the concentration of dimethyltetrachloride decreased and new products were produced, it was inferred that the strain had degradation ability.

[0030] The formulation of 1 L LB liquid culture medium in this invention is as follows: 5 g NaCl, 5 g yeast extract, 10 g peptone, and ultrapure water is added to bring the volume to 1 L, and the pH is adjusted to 7.0. The LB solid culture medium is based on the LB liquid culture medium with the addition of 1.8% (g / v) agar powder.

[0031] The chromatographic conditions for HPLC detection of dimethyltetrachloride concentration in this invention are as follows: the column is a Syncronis C18 (ThermoFisher Scientific) reversed-phase column (size: 250 mm × 4.6 mm × 5 μm); the mobile phase is acetonitrile: 0.5% phosphoric acid water (80:20 (v:v)); the flow rate is 0.8 mL·min -1 The detection wavelength was 230 nm; the column temperature was 40°C; and the injection volume was 5 μL.

[0032] A 2,4-D (dimethyltetrachloroethyl) degrading strain, named S-6, was successfully isolated using enrichment culture and dilution plating separation methods. This strain can completely degrade 0.05 mM 2,4-D within 12 hours.

[0033] 1.2 Identification and biological characteristics of MCPA-degrading strain S-6

[0034] Strain S-6 was cultured on solid LB medium for 3 days. Colonies were round, yellow, opaque, with neat edges and smooth surfaces. Using fresh bacterial culture of strain S-6 as a template, 16S rRNA gene sequence was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The amplified product was purified and ligated into the vector pCE2TA / Blunt-Zero. Sequencing yielded a gene sequence of approximately 1450 bp (SEQ ID NO. 1). This sequence was compared and analyzed in the EzBioCloud database (https: / / www.ezbiocloud.net). The results showed that strain S-6 was similar to... Sphingobium xenophagum NBRC 107872 T The similarity was the highest, at 99.93%, combined with the colony morphology of strain S-6 ( Figure 1 Physiological and biochemical characteristics and phylogenetic tree of 16S rRNA gene ( Figure 2 It was identified as belonging to the genus *Sphingosomalmonella*. Sphingobium The strain S-6 was deposited as a patent strain at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on April 2, 2025, with accession number CCTCC NO: M2025674.

[0035] Example 2 Strain S-6 ( Sphingobium Test of the ability of sp. S-6 to degrade dimethyltetrachlorine

[0036] Sphingobium Preparation of seed liquid (s. S-6)

[0037] Select a single colony of strain S-6 and culture it in an LB tube until the exponential phase. Take 2 mL of strain S-6 culture and transfer it to 100 mL of LB liquid medium. Then, culture the culture at 30℃ and 150 rpm until the exponential phase. Centrifuge at 8000 rpm to collect the cells. Wash the cells three times with MSM liquid medium and resuspend them in 10 mL of MSM to obtain the seed culture of strain S-6.

[0038] 2.2 Degradation characteristics of strain S-6 under different inoculum amounts

[0039] Seed culture of strain S-6 was inoculated into 20 mL of MSM at different inoculation amounts (0.1, 0.5, 1.0, 1.5, and 2.0) with a final concentration of 0.05 mM 2,4-D. Each treatment was cultured at 30 °C and 150 rpm using a shaker. Every 12 h, 500 μL of culture medium was collected, an equal volume of methanol was added, and the mixture was thoroughly mixed. The samples were then filtered through a 0.22 μm organic filter membrane, and the concentration of 2,4-D in the samples was determined by HPLC. The change in 2,4-D concentration over time was plotted on the x-axis, representing the degradation characteristics of strain S-6 under different inoculation conditions. The results are as follows: Figure 3 As shown, when the inoculum concentration is in the range of 0.5-2.0, strain S-6 can completely degrade 2,4-D within 12 hours. When the initial concentration is OD... 600 At a concentration of 2.0, it degrades dimethyltetrachloro at the fastest rate, reaching 100% degradation rate in 4 hours.

[0040] 2.3 Degradation characteristics of strain S-6 under different temperature conditions

[0041] The seed culture of strain S-6 was inoculated into 20 mL of MSM (pH 7.0) with a final concentration of 0.05 mM dimethyltetrachloro, and the OD was adjusted. 600 The concentration of dimethyltetrachlorochloride (DMC) was 0.5, and each treatment was cultured at different temperatures (16, 25, 30, 37, and 42°C) using a shaker at 150 rpm. Every 2 hours, 500 μL of culture medium was collected, an equal volume of methanol was added, and the mixture was thoroughly mixed. The samples were then filtered through a 0.22 μm organic filter membrane, and the concentration of DMC in the samples was determined by HPLC. A curve showing the change in DMC concentration over time was plotted with time as the abscissa, representing the degradation characteristics of strain S-6 under different temperature conditions. The results are as follows: Figure 4As shown, strain S-6 exhibits good degradation ability for 2,000 mM 2,000 chlorine at temperatures ranging from 16 to 30°C, with the optimal degradation temperature being 25°C. It can completely remove 0.05 mM 2,000 chlorine within 12 hours.

[0042] 2.4 Degradation characteristics of strain S-6 under different pH conditions

[0043] Seed culture of strain S-6 was inoculated into 20 mL of MSM with different initial pH values ​​(pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) at a final concentration of 0.05 mM dimethyltetrachlorochloride, and the OD was adjusted. 600 The concentration of dimethyltetrachlorochloride (DMC) was 0.5, and each treatment was incubated at 30℃ with shaking at 150 rpm. Every 2 h, 500 μL of culture medium was taken, an equal volume of methanol was added, and the mixture was thoroughly mixed. The samples were then filtered through a 0.22 μm organic filter membrane, and the concentration of DMC in the samples was determined by HPLC. A curve showing the change in DMC concentration over time was plotted with time as the abscissa, representing the degradation characteristics of strain S-6 under different pH conditions. The results are as follows: Figure 5 As shown, strain S-6 has the ability to degrade 2,000 mM 2,000 chlorine at pH 3-8, with the optimal degradation pH being 5.0. It can completely remove 0.05 mM 2,000 chlorine in 4 hours.

[0044] 2.5 Degradation characteristics of strain S-6 for different concentrations of 2,4-D

[0045] Seed culture of strain S-6 was inoculated into 20 mL of MSM, and OD was adjusted. 600 The concentration of dimethyltetrachloride (DMC) was 0.5. For each treatment, final concentrations of 0.025 mM, 0.05 mM, 0.1 mM, 0.15 mM, 0.2 mM, and 0.25 mM were added. Each treatment was incubated at 30℃ with shaking at 150 rpm. Every 2 h, 500 μL of culture medium was collected, an equal volume of methanol was added, and the mixture was thoroughly mixed. The sample was then filtered through a 0.22 μm organic filter membrane, and the concentration of DMC in the sample was determined by HPLC. A curve showing the change in DMC concentration over time was plotted with time as the abscissa, representing the degradation curve of DMC by strain S-6. The results are as follows: Figure 6 As shown, strain S-6 has the ability to degrade 0.025-0.15 mM dimethyltetrachloride, and it can completely degrade 0.025-0.05 mM dimethyltetrachloride within 12 h.

[0046] 2.6 Ability of strain S-6 to degrade different substrates

[0047] Seed culture of strain S-6 was inoculated into 20 mL of MSM, and OD was adjusted. 600The concentration of each substrate was 0.05 mM. Standard working solutions of MCPA, sodium MCPA-Na, 2,4-dichlorophenoxyacetic acid (2,4-D), NAP, and DCP were added to the culture medium. An equal volume of sterile water was added to the blank control to replace the bacterial culture. All other conditions were the same as the treatment groups. The culture medium was adjusted to the optimal pH for bacterial degradation, ensuring a final concentration of 0.05 mM for each substrate in all cultures. The cultures were incubated at 25 °C and 150 rpm. Sampling was performed periodically to determine the residual concentration of each compound. Each experiment was conducted in triplicate. The HPLC detection methods for MCPA, MCPA-Na, 2,4-D, NAP, and DCP were the same as for MCPA. The HPLC detection method for dimethyltetrachloride was as follows: Column: ZORBAX Eclipse XDB-C18 (4.6 × 250 mm; 5 µm); Mobile phase: acetonitrile: 0.5% phosphoric acid water (80:20 (v:v)); Flow rate: 0.8 mL·min -1 The detection wavelength was 230 nm; the column temperature was 40°C; and the injection volume was 5 μL.

[0048] The degradation capabilities of strain S-6 for different phenoxycarboxylic acid herbicides are shown in Table 1. Within 48 h, strain S-6 achieved degradation rates of 100%, 100%, 100%, 83.3%, and 43.1% for 2,4-methyltetrachlorophenoxyacetic acid, dichlorvos, and 2,4-D propionic acid, respectively. The liquid chromatography results are shown in Table 1. Figure 7 As shown: Table 1. Degradation substrate profile of strain S-6 ;

[0049] Example 3 Preparation of liquid inoculum of strain S-6

[0050] Sphingosomalids spp. preserved at low temperature Sphingobium sp . Strain S-6 was inoculated onto solid culture medium and cultured at 30 ℃ for 48 h. A single colony was picked and placed in a 3 mL seed culture medium test tube, and cultured at 30 ℃ and 150 rpm for 24 h to obtain the inoculum. Activated bacterial culture was inoculated into the seed culture medium at a 1% inoculation rate and cultured at 30 ℃ and 150 rpm for 24 h to obtain the seed culture. According to OD... 600 Seed culture was inoculated into the fermentation medium at an inoculum size of 0.5, with a liquid volume of 100 mL / L. The temperature was 21 ℃, the rotation speed was 160 rpm, and the incubation time was 24 h to obtain the fermentation broth, namely Sphingosine Monoclonal liquid inoculum.

[0051] The seed culture medium in the above step consists of: 5 g / L yeast extract, 10 g / L peptone, and 10 g / L NaCl. The fermentation culture medium in the above step consists of: 10 g / L yeast extract, 3% maltose, 1 g / L NaCl, 0.2 g / L MgSO4, 1.5 g / L K2HPO4, 0.5 g / L KH2PO4, 1.0 g / L NH4Cl, and pH 7.0.

[0052] Example 4: Degradation of 2,4-D by strain S-6 in soil

[0053] Soil samples uncontaminated with 2,4-D were selected as test samples. The soil samples were air-dried, ground, and passed through a 20-mesh sieve. A certain amount of 2,4-D was dissolved in methanol and evenly mixed into 1 kg of the air-dried soil sample, bringing the final concentration of 2,4-D in the soil to 20 mg / kg. -1 and 40 mg kg -1 Next, the S-6 degrading agent prepared in Example 2 was inoculated into 1 kg of air-dried soil sample to achieve an initial inoculum size of 6.0 × 10⁻⁶. 6 CFU / kg was incubated in a constant temperature incubator at 25℃. Identical soil without inoculation with inoculant S-6 was used as a control. Both the control and treatment were performed in triplicate. Soil moisture content was maintained at approximately 60% during the incubation period. After 5 days of incubation, soil samples were taken and extracted three times consecutively with dichloromethane. The organic phases were combined, and after complete volatilization, methanol was added to dissolve the organic phase. The residual amount of dimethyltetrachloromethane in the extracted samples was then detected using high-performance liquid chromatography (HPLC). Results are as follows: Figure 8 , Figure 9 As shown, strain Sphingobium sp. S-6 showed efficacy against 20 mg kg of soil at 12 and 18 days. -1 and 40 mg kg -1 The degradation rate of 2,4-dimethyltetrachloro reached 100%.

[0054] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A bacterial strain that degrades DDT Sphingobium sp . S-6, characterized in that, The strain belongs to the genus Sphingomonas and is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. The deposit date is April 2, 2025, and the accession number is CCTCC NO: M2025674.

2. The strain according to claim 1 Sphingobium sp . S-6, characterized in that, The Sphingobium sp . The 16S rRNA gene sequence of S-6 is shown as SEQ ID NO.

1.

3. A strain according to claim 1 Sphingobium sp . The use of S-6 in the degradation of Dicamba, characterized in that, The strain exhibits the ability to degrade 0.025-0.15 mM dimethyltetrachloride at temperatures ranging from 16 to 30°C and pH values ​​ranging from 3 to 8.

4. The application according to claim 3, characterized in that, The strain is used to degrade 2,4-D in soil or water.

5. A strain according to claim 1 Sphingobium sp . Application of S-6 in the preparation of 2,4-D degrading agent.

6. A bacterial agent for degrading 2,4-D, characterized in that, The bacterial agent contains the strain described in claim 1. Sphingobium sp . S-6.

7. The microbial agent according to claim 6, characterized in that, The bacterial agent is a liquid bacterial agent, powder, or granule preparation.

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