PAEs degrading bacterium trichoderma strain CSP1 derived from tea garden soil and application of PAEs degrading bacterium trichoderma strain CSP1

Abstract

The invention provides a PAEs degrading bacterium trichoderma strain CSP1 derived from tea garden soil and application of the PAEs degrading bacterium trichoderma strain CSP1. According to the invention, a trichoderma fungus CSP1 is separated from tea garden soil, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.41624. According to the bacterial strain, DBP and DEHP serve as unique carbon sources at the same time, degradation conditions of the bacterial strain are optimized through a response surface design method, it is found that the bacterial strain has a good degradation effect on main PAEs (DBP and DEHP) in tea garden soil, an application technology of the bacterial strain is given, and the bacterial strain has important application value for relieving PAEs pollution of the tea garden soil.

Description

Technical Field

[0001] The invention belongs to the field of tea garden soil pollutant remediation, and specifically relates to a PAEs-degrading fungus Trichoderma strain CSP1 originating from tea garden soil and an application thereof. Background Art

[0002] Phthalate esters (PAEs) are a class of polymer material additives with excellent plasticizing and adhesion properties. They can be used as plasticizers in plastic products. PAEs are not chemically bonded to plastic polymers and can easily migrate into the environment through evaporation, leaching and abrasion. They have been detected in air, water, soil and sediments and are ubiquitous pollutants in the environment. Soil is the main reservoir of various pollutants. PAEs can exist in soil for a long time, and the half-life is usually several months or longer. PAEs pollution to varying degrees has been found in agricultural soils such as greenhouses, vegetable fields, rice fields, and cotton fields, which may be caused by pesticides, plastic films, fertilizers, and atmospheric deposition. Studies have shown that PAEs in soil can be absorbed and accumulated by crops, thus posing risks to humans through the food chain. Therefore, how to effectively alleviate PAEs pollution in soil is an urgent problem that needs to be solved and has great social and ecological significance.

[0003] As one of the most popular non-alcoholic beverages in the world, tea has received widespread attention for its health benefits. Organic pollutants, heavy metals, pesticide residues, etc. in tea are important factors affecting the quality and safety of tea, and have become an urgent concern. In recent years, domestic and foreign researchers have detected PAEs from tea leaves of different types with a probability of up to 100%. Studies have shown that PAEs in tea leaves and tea garden soil are mainly DBP (dibutyl phthalate) and DEHP (di(2-ethylhexyl) phthalate), accounting for more than 70%. At present, microbial degradation has become an effective method for removing PAEs pollution in soil with its advantages of high efficiency, low cost, low energy consumption, and no secondary pollution. So far, more than 80 strains of degrading bacteria have been reported and studied, most of which are bacteria. However, no research and reports on the application of PAEs-degrading bacteria in tea garden soil have been found. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide a PAEs-degrading fungus Trichoderma strain CSP1 derived from tea garden soil and applications thereof.

[0005] The technical solution adopted by the present invention is:

[0006] The present invention provides a PAEs-degrading fungus Trichoderma sp. CSP1 derived from tea garden soil, which is isolated from tea garden soil. The strain is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 41624 and a deposit date of November 11, 2024.

[0007] The 16S rDNA sequence of the strain is shown as SEQ ID No.1.

[0008] The present invention also provides the use of the Trichoderma strain CSP1 in treating PAEs pollution in tea garden soil. Further, the PAEs are dibutyl phthalate (DBP) and di(2-ethyl)hexyl phthalate (DEHP).

[0009] According to a preferred embodiment of the present invention, the application is: culturing the Trichoderma strain CSP1, and preparing the cultured Trichoderma strain CSP1 into bacterial powder, adding sterilized water to the bacterial powder and mixing it to obtain a CSP1 bacterial agent, and pouring the CSP1 bacterial agent into the soil at the roots of tea trees, and utilizing the Trichoderma strain CSP1 therein to degrade PAEs in the tea garden soil.

[0010] The present invention also provides a CSP1 bacterial agent for treating PAEs pollution in tea garden soil, wherein the CSP1 bacterial agent contains the above-mentioned Trichoderma strain CSP1; the concentration of the Trichoderma strain CSP1 in the CSP1 bacterial agent is 3×10 7 spores / mL.

[0011] According to a preferred embodiment of the present invention, the CSP1 bacterial agent is prepared by the following method: the Trichoderma strain CSP1 is inoculated into an MSM inorganic liquid culture medium containing DBP and DEHP, and cultured in a shaking dark environment at a certain temperature, a certain pH and a certain PAEs mixture concentration, wherein a mixture of dibutyl phthalate (DBP) and di(2-ethyl)hexyl phthalate (DEHP) is used as the sole carbon source in the culture medium;

[0012] The cultured Trichoderma strain CSP1 was prepared into bacterial powder, added into sterilized water and mixed evenly to obtain the CSP1 bacterial agent.

[0013] Preferably, the temperature is 30° C., the total concentration of DBP and DEHP in the culture medium is 10 mg / kg, and the pH is 5.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention isolates a strain of Trichoderma fungus CSP1 from tea garden soil, and the strain mainly uses a mixture of DBP and DEHP, which has the highest proportion of PAEs in tea leaves and tea garden soil, as its sole carbon source. In order to further understand the degradation characteristics of the strain, a response surface optimization analysis method is used to perform multiple regression fitting on the basis of a single factor experiment under the action of three factors: different temperatures, different initial concentrations of DBP and DEHP mixtures, and different pH values, to confirm the optimal degradation conditions. The strain is very suitable for the acidic soil remediation required for tea tree planting, and can also effectively reduce the accumulation of PAEs in tea garden soil, thereby ensuring the quality and safety of tea. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the phylogenetic tree of strain CSP1;

[0017] Figure 2 This is the colony morphology of strain CSP1 on MSM solid medium;

[0018] Figure 3 The relationship between culture temperature and PAEs degradation rate was analyzed by univariate analysis;

[0019] Figure 4 The relationship between the initial concentration of DBP and DEHP mixture and the degradation rate of PAEs was analyzed by single factor;

[0020] Figure 5 To analyze the relationship between medium pH and PAEs degradation rate by single factor;

[0021] Figure 6 It is the three-dimensional response surface diagram of the interaction between culture temperature and initial concentration;

[0022] Figure 7 is the three-dimensional response surface diagram of the interaction between culture temperature and pH;

[0023] Figure 8 It is a three-dimensional response surface diagram of the interaction between pH and initial concentration.

[0024] Fig. 9 This is the degradation effect of strain CSP1 on DBP and DEHP in tea garden soil. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0026] Example 1 Isolation and identification of strain CSP1

[0027] Take 5g tea garden soil and place it in a sterilized glass test tube, add 50mL sterile water, and incubate at 30℃ and 180r·min. -1Oscillate in the dark for 20 min, let stand for 30 min, take 5 mL of supernatant, add 45 mL of MSM liquid medium containing DBP and DEHP respectively, and perform gradient acclimatization three times. 30℃, 150 r·min -1 The cells were incubated in dark for 7 days, and the concentrations of DBP and DEHP mixtures were 5, 10, and 20 mg kg -1 Take the cultured bacterial suspension for 10 2 , 10 3 , 10 4 Then 100 μL of bacterial solution was diluted to 10 mg kg -1 DBP and DEHP were spread on MSM solid plates, cultured at 30°C for 7 days, and further streaked to obtain single colonies. The MSM liquid culture medium contained Na2HPO4 2440.0mg / L, KH2PO4 1520mg / L, (NH4)2SO4500.0mg / L, Mg2SO4 98.0mg / L, CaCl2 37.8mg / L, EDTA-2Na 5.0mg / L, FeSO4·7H2O 2.0mg / L, ZnSO4·7H2O 0.1mg / L, MnCl2·4H2O 0.03mg / L, H3BO3 0.3mg / L, CuCl2·2H2O 0.01mg / L, CoCl2·6H2O 0.2mg / L, NiCl2·6H2O 0.02mg / L, Na2MoO4·2H2O 0.03mg / L, biotin 0.02mg / L, vitamin B1, B2, B3, B5, B6, B9, B12, K1, K3 (0.05, 0.05, 0.05, 0.1, 0.02, 0.001, 0.22, 1.0mg / L respectively), p-aminobenzoic acid 0.22mg / L, lipoic acid 0.05mg / L; add 20g / L agar into solid culture medium.

[0028] After enrichment and culture of the above single colonies, PCR amplification was performed using universal primers ITS1 and ITS4 for fungal species identification, and the amplified products were sequenced to construct a phylogenetic tree ( Figure 1 ). Based on the 16S rDNA sequence (shown in SEQ ID No.1) and the Blast alignment of the related sequences in NCBI, the strain was identified as Trichoderma sp.CSP1. Figure 2 As shown, the conidiophores have a long main axis, the aerial hyphae are white, the colony surface is velvety, the hyphae are compact, and the basal hyphae are green.

[0029] SEQ ID No.1:

[0030] .

[0031] Therefore, the strain was named Trichoderma sp.CSP1 and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.41624 and the deposit date: November 11, 2024.

[0032] Example 2 Optimization of PAEs degradation conditions by strain CSP1 based on response surface methodology

[0033] The purified cultured CSP1 was selected and inoculated into the PDB culture medium for 3 days under shaking and dark conditions. The OD value at 660nm was measured by spectrophotometer and diluted with sterile water to control the consistency of bacterial solution concentration. 5 mL of the adjusted bacterial solution was added to the MSM liquid culture medium with different treatments, and the same amount of sterile water was used as the control. 180 r·min -1The culture was kept in a dark and shaken state for 7 days. The residual amounts of DBP and DEHP in the culture medium were determined by gas chromatography-mass spectrometry (GC-MS). The specific method steps of GC-MS refer to the Chinese invention patent (patent number ZL202210059785.4). The specific treatment is as follows. Different culture temperatures (20°C, 25°C, 30°C, 35°C), different initial concentrations of PAEs (1 mg / kg, 5 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, 100 mg / kg, in different initial concentration groups, the PAEs included were DBP and DEHP, and the volume ratio of DBP and DEHP was 1:1) and different pH (3, 4, 5, 6, 7) were set, and a single factor experiment was conducted, and each experiment was repeated 3 times. Based on the single factor experiment, temperature (A), initial concentration (B) and pH (C) were used as independent variables in the response surface experiment, and the degradation rate of PAEs was used as the response value. Box-Behnken optimization was performed using Design-ExertV8.0.6, and the optimal degradation conditions of CSP1 were determined through variance analysis and response contour map analysis.

[0034] As the culture temperature increased, the degradation rate of PAEs in the culture medium gradually increased. At 30°C, the degradation rate of PAEs reached the highest level, exceeding 80% ( Figure 3 ). Within the range of 20℃-35℃, the degradation rate of PAEs is above 50%, and the degradation rate of DBP at 30℃ can reach above 90%. The results show that the strain has good degradation ability within the normal temperature range, especially for the degradation of DBP. At different initial concentrations (mixture of DBP and DEHP), the degradation rate of PAEs showed a trend of first increasing and then decreasing. At 10mg / kg, the degradation rate of PAEs reached the highest ( Figure 4 ). In the pH range of 3.0-7.0, the degradation rate of PAEs is above 50%, especially at pH 5.0, where the degradation rate is the highest ( Figure 5 ), indicating that the strain has a good degradation ability under acidic conditions, which is consistent with the requirement that tea trees need acidic conditions to grow.

[0035] On the basis of single factor experiment, temperature (A), initial concentration (B), and pH (C) were selected as independent variables according to the central composite design principle of Box-Behnken, and PAEs degradation rate was selected as the response value. The degradation conditions were optimized, and the effects of various factors and their interactions on PAEs degradation rate were investigated. The variance analysis of the regression model is shown in Table 1.

[0036] Table 1. Significance analysis of regression coefficients and variance analysis of models

[0037]

[0038] The model P<0.05, the lack of fit term P>0.05, it can be seen that the model regression is significant, the lack of fit term is not significant, indicating that there is no lack of fit factor, the model is statistically significant, and other factors have little effect on the experiment. The model's corrected determination coefficient R 2 adj =0.9692, indicating that the model has only 3.08% variation and is highly accurate. 2 =0.9846, predicted R 2 =0.9419, R 2 With R 2 adj The difference is <0.1, R 2 adj and predict R 2 The difference is <0.2, and the signal-to-noise ratio Adeq Precision=23.5359>4. The above parameters fully demonstrate that the model is significant, the degree of fitting is high, the experimental prediction error is small, the data repeatability is good, and it can better reflect the influence of the three factors on the degradation rate of PAEs. Factor A 2 , B 2 and C 2 All of them were extremely significant (P<0.01). According to the F value, the order of the influence of the three factors on the degradation rate of PAEs is: initial concentration (B)> pH (C)> temperature (A). 3D response diagram of the interaction of temperature, initial concentration and pH on the degradation rate of PAEs ( Figure 6-Figure 8 ), it can be seen that the interaction between temperature and initial concentration is the largest, followed by the interaction between temperature and pH, and the interaction between initial concentration and pH is the least obvious.

[0039] According to the results of response surface analysis, the optimal conditions for the strain to degrade PAEs were temperature 30°C, initial concentration of DBP and DEHP mixture (DBP and DEHP volume ratio 1:1) 10 mg / kg, and pH 5.0.

[0040] Example 3 Application of strain CSP1 in PAEs contamination of tea garden soil

[0041] Preparation of CSP1 inoculant: Weigh 450g of fine rice bran and 450g of rice husk into a high temperature and heat sterilization bag, add 18g of sucrose, 9g of ammonium sulfate, 0.9g of potassium dihydrogen phosphate, 0.45g of calcium chloride, 0.45g of magnesium sulfate and 1.35L of purified water, and mix thoroughly. Autoclave at 121°C for 30min. After cooling to room temperature, add 100mL of bacterial suspension (pick colonies from the plate and inoculate in 50mL of MSM liquid medium containing 10mg / kg PAEs, amplify at 180r / min and 30°C for 2 days, and then use the hemocytometer to calculate the colony content of the bacterial solution to 10 7), ferment in a sterilized bag at 30℃ for 3-4 days, mix well after mycelium grows, and ferment quickly. Stir every 3 days to mix. After 10 days, wash the spores on the substrate with sterilized water, dry at room temperature and collect to obtain bacterial powder. Take 1g of bacterial powder and add 500mL of sterilized water and mix well to obtain CSP1 bacterial agent (3×10 7 spores / mL).

[0042] In a normal 4-year-old tea garden, the tea tree variety was Zhongcha 108. 500 mL of the CSP1 inoculant prepared above was poured into the soil at the root of each tea tree, and 500 mL of the inoculant was poured into the root every 10 days. After 30 days, the tea garden soil was collected and the PAEs content in the tea garden soil was determined by GC-MS. The results showed that the degradation rates of DBP and DEHP in the tea garden soil by strain CSP1 were 37.95% and 29.93%, respectively ( Fig. 9 ), indicating that strain CSP1 has a good ability to degrade PAEs in soil and has application potential in the field of tea garden soil remediation.

[0043] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A PAEs-degrading fungus, Trichoderma strain CSP1, derived from tea garden soil, characterized in that: This strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCCNo.41624 and the deposit date: November 11, 2024.

2. Use of the Trichoderma strain CSP1 according to claim 1 in treating PAEs pollution in tea garden soil.

3. The use according to claim 2, characterized in that: The PAEs are dibutyl phthalate (DBP) and di(2-ethyl)hexyl phthalate (DEHP).

4. The use according to claim 2, characterized in that: The application is: culturing the Trichoderma strain CSP1 described in claim 1, and preparing the cultured Trichoderma strain CSP1 into bacterial powder, adding sterilized water to the bacterial powder and mixing it to obtain a CSP1 bacterial agent, pouring the CSP1 bacterial agent into the soil at the root of the tea tree, and using the Trichoderma strain CSP1 therein to degrade PAEs in the tea garden soil.

5. A CSP1 bacterial agent for treating PAEs pollution in tea garden soil, characterized in that: The CSP1 bacterial agent contains the Trichoderma strain CSP1 according to claim 1; the concentration of the Trichoderma strain CSP1 in the CSP1 bacterial agent is 3×10 7 spores / mL.

6. The CSP1 bacterial agent according to claim 5, characterized in that The CSP1 bacterial agent is prepared by the following method: the Trichoderma strain CSP1 described in claim 1 is inoculated into an MSM inorganic liquid culture medium containing DBP and DEHP, and cultured in a shaking dark environment at a certain temperature, a certain pH and a certain PAEs mixture concentration, wherein a mixture of dibutyl phthalate (DBP) and di(2-ethyl)hexyl phthalate (DEHP) is used as the sole carbon source in the culture medium; The cultured Trichoderma strain CSP1 was prepared into bacterial powder, added into sterilized water and mixed evenly to obtain the CSP1 bacterial agent.

7. The CSP1 bacterial agent according to claim 6, characterized in that The temperature is 20-35° C., the total concentration of DBP and DEHP in the culture medium is 10 mg / kg, and the pH is 3.0-7.

0.

8. The CSP1 bacterial agent according to claim 6, characterized in that The temperature was 30°C and the pH was 5.

Images