Bio-preparation of immobilized pseudomonas aeruginosa psl by silkworm excrement carrier and application thereof

Abstract

The application discloses a kind of silkworm excrement carrier fixed pseudomonas aeruginosa PS1 biological agent and application thereof.Pseudomonas aeruginosa PS1 has strong degradation ability to PAEs, the degradation rate of DBP reaches 97.5%, the degradation rate of DEHP reaches 60.2%, and can use catechol, BA, PCA, PA, MBP, benzoic acid monobutyl ester, 4,5-dihydroxy phthalic acid, DEP, DMP and DOP and other various PAEs as substrate.The silkworm excrement is used as the carrier of fixed pseudomonas aeruginosa PS1, and the biological agent for repairing PAEs contaminated environment is prepared, is used to significantly improve soil pH, increase soil organic matter content and / or increase soil nitrate nitrogen and / or ammonium nitrogen content, increase the abundance of phylum Proteobacteria and / or phylum Synergistetes, and / or reduce the abundance of actinomycetales and / or Pseudonocardiales, so as to repair the environment contaminated by PAEs.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a biological agent for immobilizing Pseudomonas aeruginosa PS1 on silkworm excrement and its application. Background Technology

[0002] Phthalate esters (PAEs) are plasticizers widely used in the plastics, rubber, paint, pesticide, and cosmetic industries. Because PAEs exist in a free state in plastics and other products, they readily migrate into the environment, resulting in their widespread presence in the atmosphere, water bodies, soil, and organisms. PAEs are endocrine disruptors, exhibiting bioaccumulation and strong toxicity. Therefore, effectively controlling and eliminating PAE pollution in the environment has become a pressing issue.

[0003] Physical and chemical remediation can effectively remove some organic pollutants from soil, but they can severely damage soil ecology and physicochemical quality. Furthermore, the high cost of these two technologies limits their large-scale application in agricultural soils contaminated with organic pollutants. Phytoremediation is an environmentally friendly, low-cost, and sustainable remediation method. However, there are few plant species and small biomass that hyperaccumulate organic pollutants, resulting in significant limitations due to phytotoxicity and long remediation cycles.

[0004] Given the advantages of microorganisms, especially bacteria, in pollutant degradation and environmental remediation, including complete pollutant degradation, low treatment costs, high safety, and minimal environmental impact, bacterial-driven biodegradation is considered the most promising remediation strategy. Among the isolated degrading bacteria, some strains can use phthalic acid (PAEs) as their sole carbon and energy source for growth, while others can only convert PAEs into certain degradation intermediates, such as phthalic acid (PA) or benzoic acid (BA). Furthermore, various environmental factors also influence the ability of bacteria to degrade PAEs. However, most isolated PAE-degrading bacteria exhibit limited substrate degradation capabilities and poor resistance to abiotic stress. Therefore, it remains necessary to isolate, screen, or artificially cultivate new, high-performance, and superior degrading bacteria from naturally polluted environments to enrich the resources of highly efficient PAE-degrading strains and enhance the bioremediation potential of PAE-contaminated environments.

[0005] Considering that free strains are easily lost in practical applications and have difficulty competing with native soil microorganisms, developing suitable immobilization technologies is also key to ensuring that functional microorganisms can efficiently degrade pollutants. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned deficiencies of the prior art and to provide a biological agent for immobilizing Pseudomonas aeruginosa PS1 on silkworm excrement carrier and its application.

[0007] The first objective of this invention is to provide a Pseudomonas aeruginosa PS1.

[0008] A second object of the present invention is to provide the application of the aforementioned Pseudomonas aeruginosa PS1 in the remediation of environments contaminated with phthalates.

[0009] A third objective of this invention is to provide a biological agent for environmental remediation.

[0010] A fourth object of the present invention is to provide the application of the said biological agent in the remediation of environments contaminated with phthalates.

[0011] A fifth objective of this invention is to provide a method for remediating environments contaminated with phthalates.

[0012] To achieve the above objectives, the present invention is implemented through the following solution:

[0013] A strain of Pseudomonas aeruginosa, PS1, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 27, 2021, with accession number GDMCC No: 62009.

[0014] The application of Pseudomonas aeruginosa PS1 in the remediation of environments contaminated with phthalates.

[0015] Preferably, the phthalate comprises one or more of dibutyl phthalate, di(2-ethylhexyl) phthalate, catechol, benzoic acid, protocatechuic acid, phthalic acid, monobutyl phthalate, monobutyl benzoate, 4,5-dihydroxyphthalic acid, diethyl phthalate, dimethyl phthalate, and dioctyl phthalate.

[0016] Preferably, the remediation of the phthalate-contaminated environment involves degrading the phthalates in the phthalate-contaminated environment, restoring the physicochemical properties of the phthalate-contaminated environment, and / or restoring the microbial community structure of the phthalate-contaminated environment.

[0017] More preferably, the restoration of the physicochemical properties of the phthalate-contaminated environment involves increasing soil pH, increasing soil organic matter content, and / or increasing soil nitrate nitrogen and / or ammonium nitrogen content; the restoration of the microbial community structure of the phthalate-contaminated environment involves increasing the abundance of Proteobacteria and / or Digestorhynchus, and / or decreasing the abundance of Actinomycetes and / or Pseudonocardiales.

[0018] An environmental remediation biological agent, characterized in that the biological agent contains the aforementioned Pseudomonas aeruginosa PS1.

[0019] Preferably, the biological agent also contains silkworm excrement.

[0020] The application of the described biological agent in the remediation of environments contaminated with phthalates.

[0021] Preferably, the phthalate comprises one or more of dibutyl phthalate, di(2-ethylhexyl) phthalate, catechol, benzoic acid, protocatechuic acid, phthalic acid, monobutyl phthalate, monobutyl benzoate, 4,5-dihydroxyphthalic acid, diethyl phthalate, dimethyl phthalate, and dioctyl phthalate.

[0022] Preferably, the remediation of the phthalate-contaminated environment involves degrading the phthalates in the phthalate-contaminated environment, restoring the physicochemical properties of the phthalate-contaminated environment, and / or restoring the microbial community structure of the phthalate-contaminated environment.

[0023] More preferably, the restoration of the physicochemical properties of the phthalate-contaminated environment involves increasing soil pH, increasing soil organic matter content, and / or increasing soil nitrate nitrogen and / or ammonium nitrogen content; the restoration of the microbial community structure of the phthalate-contaminated environment involves increasing the abundance of Proteobacteria and / or Digestorhynchus, and / or decreasing the abundance of Actinomycetes and / or Pseudonocardiales.

[0024] Preferably, the preparation method of the biological agent is as follows: pick up the Pseudomonas aeruginosa PS1 colony, inoculate it into the culture medium, incubate for 23-25 ​​h, remove the supernatant, wash the Pseudomonas aeruginosa PS1 cells, centrifuge, resuspend the Pseudomonas aeruginosa PS1 cells in 4-6 mL of physiological saline, and mix with 19-21 g of silkworm excrement.

[0025] More preferably, the preparation method of the biological agent is as follows: pick the Pseudomonas aeruginosa PS1 colony, inoculate it into the culture medium, incubate for 24 h, remove the supernatant, wash the Pseudomonas aeruginosa PS1 cells, centrifuge, resuspend the Pseudomonas aeruginosa PS1 cells in 5 mL of physiological saline, and mix with 20 g of silkworm excrement.

[0026] A method for remediating an environment contaminated with phthalates, using the aforementioned Pseudomonas aeruginosa PS1 or the aforementioned biological agent.

[0027] Preferably, the phthalate comprises one or more of dibutyl phthalate, di(2-ethylhexyl) phthalate, catechol, benzoic acid, protocatechuic acid, phthalic acid, monobutyl phthalate, monobutyl benzoate, 4,5-dihydroxyphthalic acid, diethyl phthalate, dimethyl phthalate, and dioctyl phthalate.

[0028] Preferably, the remediation of the phthalate-contaminated environment involves degrading the phthalates in the phthalate-contaminated environment, restoring the physicochemical properties of the phthalate-contaminated environment, and / or restoring the microbial community structure of the phthalate-contaminated environment.

[0029] More preferably, the restoration of the physicochemical properties of the phthalate-contaminated environment is to increase soil pH, increase soil organic matter content, and / or increase soil nitrate nitrogen and / or ammonium nitrogen content; the restoration of the microbial community structure of the phthalate-contaminated environment is to increase the abundance of Proteobacteria and / or Digestorhynchus, and / or decrease the abundance of Actinomycetes and / or Pseudonocardiales.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention discloses a biological agent for immobilizing Pseudomonas aeruginosa PS1 on silkworm excrement and its application. Pseudomonas aeruginosa PS1 exhibits strong degradation ability for phthalates (PAEs), achieving a degradation rate of 97.5% for dibutyl phthalate (DBP) and 60.2% for di(2-ethylhexyl) phthalate (DEHP). Furthermore, it can utilize various PAEs as substrates, including catechol, benzoic acid (BA), protocatechuic acid (PCA), phthalic acid (PA), monobutyl phthalate (MBP), monobutyl benzoate, 4,5-dihydroxyphthalic acid, diethyl phthalate (DEP), dimethyl phthalate (DMP), and dioctyl phthalate (DOP). A biological agent for remediating PAE-contaminated environments was prepared using silkworm excrement as a carrier for immobilizing Pseudomonas aeruginosa PS1. This agent is used to significantly increase soil pH, increase soil organic matter content and / or increase soil nitrate nitrogen and / or ammonium nitrogen content, increase the abundance of Proteobacteria and / or Digestorhynchites, and / or decrease the abundance of Actinomycetes and / or Pseudonocardiales, thereby remediating PAE-contaminated environments. Attached Figure Description

[0032] Figure 1 The images show the colony morphology and scanning electron microscope (SEM) images of PS1 bacteria grown on LB solid medium, where a represents the colony morphology characteristics and b represents the SEM image (×10000).

[0033] Figure 2 Phylogenetic analysis of strain PS1.

[0034] Figure 3 The degradation rate of DBP by strain PS1 under different single-factor conditions is shown in Figure 1, where a represents different pH values, b represents different temperature values, and c represents different inoculum amounts.

[0035] Figure 4 To optimize the optimal degradation conditions using the response surface methodology, a represents the response surface analysis of PS1 bacteria to DBP degradation rate in relation to temperature and pH; b represents the contour lines of PS1 bacteria to DBP degradation rate in relation to temperature and pH.

[0036] Figure 5 The curve shows the relationship between the growth and DBP degradation of strain PS1.

[0037] Figure 6 The degradation curves of DBP by strain PS1 are shown at different initial concentrations of DBP.

[0038] Figure 7 To infer the biochemical pathway of DBP degradation by strain PS1.

[0039] Figure 8 The adsorption kinetics curves of DBP on soil and silkworm excrement are shown.

[0040] Figure 9 Infrared spectra of silkworm excrement and vegetable garden soil before and after adsorption of DBP.

[0041] Figure 10 The images are scanning electron microscope (SEM) images of silkworm excrement with bacteria. Image a is a scanning electron microscope image of silkworm excrement before bacteria fixation; image b is a scanning electron microscope image of silkworm excrement after bacteria fixation at 1kx magnification; image c is a scanning electron microscope image of silkworm excrement after bacteria fixation at 5kx magnification; and image d is a scanning electron microscope image of silkworm excrement after bacteria fixation at 10kx magnification.

[0042] Figure 11 The concentration of PS1, a degrading bacterium, in silkworm excrement degrading agent and sterilized soil within 30 days.

[0043] Figure 12 The aboveground and underground biomass (fresh weight) of plants in each treatment group is represented by the biomass of the aboveground and underground parts.

[0044] Figure 13 Figure 1 shows the physicochemical properties of soils in each treatment. Figure a represents soil pH; b represents soil organic matter content; c represents soil nitrate nitrogen content; and d represents soil ammonium nitrogen content.

[0045] Figure 14 Venn diagram of bacterial-specific and shared OTUs in soils from different treatments.

[0046] Figure 15 Principal coordinate analysis of soil bacterial communities for each treatment.

[0047] Figure 16 At the phylum level, the relative abundance distribution of bacterial community species in soils of each treatment is shown.

[0048] Figure 17 To determine the significantly different bacterial species in soils under different treatments (compared to the control) using linear discriminant analysis (LDAScore>4.5, P<0.05), where a represents group M, b represents group SE, and c represents group SEM.

[0049] Figure 18 To determine the significantly different bacterial species in soil treated with SE and SEM, a linear discriminant analysis was performed (LDA Score > 4, P < 0.05).

[0050] Figure 19 The relative abundance of Pseudomonas spp. in soils of each treatment is given.

[0051] Figure 20 RDA analysis of environmental indicators and major bacterial communities. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0053] Environmental samples: Sludge samples used to isolate PAE-degrading bacteria were taken from a wastewater treatment plant in Guangzhou.

[0054] Chemical reagents: All inorganic reagents used in the laboratory were of analytical grade and purchased from Tianjin Damao Reagent Co., Ltd.; PAEs-type organic reagents (analytical grade), including DBP, DMP, DEP, DEHP, and DOP, were purchased from Aladdin (Shanghai) Chemical Co., Ltd.; chromatographic reagents were purchased from Sigma-Aldrich (USA); and soil ammonium nitrogen and nitrate nitrogen detection kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0055] Culture medium: (1) Liquid basic inorganic salt culture medium (MSM, g / L): potassium hydrogen phosphate (5.8), potassium dihydrogen phosphate (4.5), ammonium sulfate (2.0), magnesium chloride (0.16), calcium chloride (0.02), sodium molybdate (0.0024), ferric chloride (0.0018), manganese chloride (0.0015), double distilled water 1000mL, pH 7.0.

[0056] (2) LB medium (g / L): peptone (10), sodium chloride (5), yeast extract (5), double distilled water 1000mL, pH 7.0, mainly used for the expansion culture of the strain.

[0057] Carrier material: Silkworm excrement was provided by Guangdong Plant Dragon Biotechnology Co., Ltd. The silkworm excrement was sterilized twice by high-pressure steam (121℃, 30min) and then dried for later use.

[0058] The soil used in the experiment was collected from a vegetable garden on a farm in Guangzhou. The soil used in the adsorption process was sterilized (sterilized at 121℃ for 1 hour).

[0059] Experimental Instruments: Constant temperature incubator (LRH, Shanghai); Nucleic acid analyzer (Nanodrop, Thermo (USA)); Ultraviolet spectrophotometer (UV-2600, Shimadzu (Japan)); Electrophoresis apparatus (DYY-6C, Beijing Liuyi); Gel imaging system (Tanon 4100, Tanon); PCR instrument (PTC200, Bio-Rad (USA)); Ultrasonic cleaner (KQ-300TDB, Kunshan); Muffle furnace (SX2, Shanghai Boxun Instruments Co., Ltd.); Rotary evaporator (R-300, Buqi GmbH, Switzerland); Gas chromatography-mass spectrometry (QP2010Plus, Shimadzu (Japan)); Scanning electron microscope (Ulrta 55, ZEISS (Germany)); Electronic balance (SI-234, Denver Instruments (Germany)); LC-TOF-MS / MS (Trip TOF 5600, AB Sciex) MA (USA)); Clean bench (SW-CJ-1F, Suzhou); Autoclave (75KBS, Shanghai); High performance liquid chromatograph (1100, Agilent (USA)); FITR spectrometer (VERTEX70, Germany); Freeze dryer (LL3000, Thermo (USA)).

[0060] Example 1: Screening of degrading strains

[0061] I. Experimental Methods

[0062] (1) Pre-acclimatization of phthalic acid ester (PAE) degrading bacteria

[0063] Add dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) stock solutions (acetone as solvent) to 100 mL of sterilized MSM medium, achieving a final concentration of 50 mg / L for both DBP and DEHP. Add 5 g of activated sludge sample and incubate in a shaker (30℃, 150 rpm) in the dark for 7 days. Then, transfer (1% inoculum) to the same fresh inorganic salt medium, repeating this process 5 times. The PAE concentration in the medium is doubled with each transfer, ultimately reaching a concentration of 1600 mg / L. Finally, streak the culture onto solid LB agar plates to isolate the colonies, and store single colonies at 4℃. Pick a single colony and inoculate it into MSM medium containing a 200 mg / L DBP and DEHP mixture, incubating at 30℃ and 180 rpm for 3 days to assess its degradation effect. Single colonies were inoculated into LB agar slant in test tubes and stored at 4°C for later use.

[0064] (2) Preparation of standard solutions and construction of standard curves

[0065] A mixed standard of DBP and DEHP was dissolved in dichloromethane (chromatographic grade) and serially diluted to prepare a mixed solution of DBP and DEHP with concentrations of 0.1, 0.2, 0.5, 1.0, 2.0, 4.0 and 8.0 mg / L for DBP and the same concentration of DEHP for DEHP. This solution was used to plot a standard curve for quantitative analysis of PAEs.

[0066] (3) Extraction of PAEs

[0067] The specific extraction method for sample pretreatment is as follows: Add 20 mL of dichloromethane to a culture flask containing culture medium, and shake at 120 r / min for 10 min at room temperature (25℃) to ensure that the dichloromethane extractant is thoroughly mixed with the remaining PAEs in the culture flask. The culture medium is the culture medium from step (1) of Example 1, inoculated with a single colony and cultured in MSM medium for 3 days. Transfer to a separatory funnel, let stand for 10 min, collect the lower organic phase dichloromethane, and pour the upper aqueous phase back into the original culture flask. Repeat the extraction twice (adding 20 mL and 10 mL of dichloromethane respectively). Pass the final organic phase dichloromethane through a 15 cm column of anhydrous Na2SO4 (dried in a muffle furnace at 400 m℃) to dry the residual water. Collect the extract in a volumetric flask and dilute to 50 mL with dichloromethane to obtain the extract. After diluting the extract 50 times, take 1 mL, filter it, collect it in a sample vial, and determine its residue using gas chromatography-mass spectrometry (GC-MS).

[0068] (4) GC-MS determination

[0069] GC-MS detection conditions: column, DB-5ms; carrier gas: high-purity helium; injection port temperature: 280℃; autosampler injection; initial pressure: 33.6 kPa; flow rate: 1.0 mL / min; splitless injection; injection volume: 1.0 μL. Temperature program: initial temperature 60℃, hold for 1 min; increase to 280℃ at 60℃ / min, hold for 2 min; then increase to 290℃ at 10℃ / min, hold for 1 min. Qualitative and quantitative analysis by mass spectrometry: The mass spectrometer used an EI ion source at a temperature of 200℃; the target ion for DEHP was 149 m / z, and the reference ions were 167 and 57 m / z, with a retention time of 7.137 min; the target ion for DBP was 149 m / z, and the reference ions were 41 and 150 m / z, with a retention time of 5.393 min; DBP and DEHP were qualitatively analyzed by comparing the sample with the standard solution using the target ion, reference ion, and retention time, and the concentrations of DBP and DEHP in the sample were quantified using the plotted standard curve.

[0070] II. Experimental Results

[0071] Using a gradient pressure acclimatization method, a strain capable of growing on solid MSM medium with PAEs as the sole carbon source was screened and named PS1. After inoculating strain PS1 into MSM medium containing 200 mg / L DBP and DEHP for 3 days, its degradation ability was measured. The results showed that strain PS1 achieved a DBP degradation rate of 97.5% and a DEHP degradation rate of 60.2%. Due to the superior DBP degradation effect of strain PS1, DBP was selected as the degradation substrate for strain PS1 in subsequent experiments.

[0072] Example 2: Identification of degrading bacterial strain PS1

[0073] I. Experimental Methods

[0074] (1) Morphological identification of strains

[0075] Cell morphology: The colony characteristics of strain PS1 in Example 1 were observed. The size and shape of individual cells of strain PS1, as well as the morphological characteristics such as cell arrangement, were observed using a scanning electron microscope.

[0076] (2) Identification of physiological and biochemical characteristics

[0077] Refer to "Bergey's Manual of Determinative Bacteriology" (Holt R, Lawton J. The ecological consequences of shared natural enemies. Annual review of Ecology and Systematics, 1994, 25 (1): 495-520.).

[0078] (3) Homology analysis of 16S rDNA sequence of strain PS1

[0079] Genomic DNA was extracted from strain PS1 in Example 1. The 16S rDNA fragment of strain PS1 was amplified using the universal bacterial primers F27 (5'-AGAGTTTGATCMTGGCTCAGTAC-3') and R1492 (5'-GGYTACCTTGTTACGACTT-3'). The amplified product was purified and sequenced. The sequencing results were subjected to BLAST alignment analysis and a phylogenetic tree was constructed.

[0080] II. Experimental Results

[0081] Example 1: Colony and strain morphology of strain PS1 are shown in [reference needed]. Figure 1 ,in Figure 1 a represents the colony morphology characteristics. Figure 1b is a scanning electron microscope image (×10000). The colonies are pale yellow, irregularly shaped, and have rough, opaque edges. The scanning electron microscope shows that the cells are short rods, approximately 1.0–2.5 μm in length and 0.5 μm in width. The physiological and biochemical characteristics of strain PS1 are shown in Table 1.

[0082] Table 1. Physiological and biochemical characteristics of strain PS1

[0083]

[0084] Note: "+" indicates that it can be used or is positive, and "-" indicates that it can not be used or is negative.

[0085] The 16S rDNA gene nucleotide sequence of strain PS1 in Example 1 is SEQ ID NO: 1.

[0086] BLAST analysis showed that strain PS1 shared over 99% homology with strain Pseudomonas aeruginosa 50071 (accession number: NR_117678.1), and also exhibited high homology with other species in the same genus. A phylogenetic tree of the 16S rRNA sequence of strain PS1 was constructed as follows: Figure 2 As shown. Based on all the above identification results, strain PS1 was identified as belonging to the genus *Pseudomonas aeruginosa*, and named *Pseudomonas aeruginosa* PS1. It was deposited on October 27, 2021, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No.: 62009), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0087] Example 3: Effect of degradation conditions on DBP degradation by strain PS1

[0088] I. Experimental Design

[0089] 1. Preparation of PS1 bacterial suspension

[0090] Single colonies of strain PS1 from Example 1 were picked and cultured overnight in LB medium. After centrifugation to remove the supernatant, the culture was washed three times with sterile PBS and resuspended to obtain the seed suspension. For degradation assays, unless otherwise specified, OD values ​​were used. 600nm The bacterial suspension was prepared at a concentration of 0.6 for subsequent degradation experiments.

[0091] 2. Effects of single factors on the degradation of dibutyl phthalate (DBP) by strain PS1

[0092] (1) Effect of temperature on the degradation ability of strain PS1

[0093] Add DBP to an Erlenmeyer flask containing 50 mL of sterile MSM medium to achieve a concentration of 200 mg / L. Inoculate the bacterial suspension from step 1 with an inoculation rate of 0.6 (OD200). 600nm Then, the Erlenmeyer flasks were placed in shakers at 20℃, 25℃, 30℃, 35℃ and 40℃ respectively, and the shaking speed of each shaker was kept at 180 r / min. After 3 days of cultivation, the degradation rate of DBP in the bacterial solution was finally measured.

[0094] (2) Effect of pH on the degradation of DBP by degrading strains

[0095] Adjust the pH of the prepared MSM medium to 5, 6, 7, 8, and 9 using a pH meter. Divide the medium into 250 mL flasks (50 mL each) and autoclave. Add DBP to the sterilized medium to a concentration of 200 mg / L. Inoculate the bacterial suspension from step 1 with an inoculum size of 0.6 (OD200). 600nm After being placed in a shaker and continuously shaken for 3 days (30℃, 180r / min), the degradation rate of DBP by strain PS1 was measured.

[0096] (3) Effect of inoculum quantity on DBP degradation by the strain

[0097] The bacterial suspension from step 1 was inoculated at inoculation rates of 0.4, 0.6, 0.8, 1.0, and 1.2 (OD200). 600nm The culture medium was inoculated into a 250 mL culture flask containing 50 mL of sterile MSM medium (pH 7.0). DBP was added simultaneously as a growth substrate to a final concentration of 200 mg / L. After culturing in a shaker (30℃, 180 r / min) for 3 days, the degradation rate of DBP by strain 2G was determined.

[0098] All the above experiments used the non-inoculated treatment as a control, and each treatment was set up in 3 replicates.

[0099] 3. Optimization of degradation conditions using response surface methodology

[0100] After initially determining the range of DBP degradation ability of strain PS1 with varying factors through single-factor experiments, response surface methodology was used to determine the influence of three factors on the DBP degradation ability of strain PS1. pH (X1), temperature (X2), and inoculum size (X3) were used as independent variables, with -1, 0, and 1 representing the three levels of the independent variables. The degradation rate of 200 mg / L DBP by strain PS1 on day 3 was used as the response value (Y). The experimental results were optimized using response surface methodology to determine the optimal combination of conditions for DBP degradation by strain PS1. The level codes for each factor are shown in Table 2. The final regression model was derived: Y... i =b0+Σb i X i +Σb ijX i X j +Σb ii X i 2 , where Y i To predict the degradation rate of DBP, the reliability of the regression model was evaluated through model significance and variance analysis. Finally, the degrading strain PS1 was cultured under the theoretical values ​​of the optimal degradation conditions, and the degradation rate was used as the evaluation index to verify the reliability of the model conclusions.

[0101] Table 2. Levels and codes of experimental factors based on Box-Behnken design.

[0102]

[0103] II. Experimental Results

[0104] The effects of different single factors on the ability of strain PS1 to degrade DBP, such as Figure 3 As shown, at different temperatures ( Figure 3 b) and pH ( Figure 3 a) significantly affected the ability of strain PS1 to degrade DBP, while the inoculum quantity had no significant effect on the ability of strain PS1 to degrade DBP. Figure 3 c) The degradation rate of the strain was highest at pH 7, reaching 97.6%, and its optimal pH range was 6–8. The optimal temperature for strain PS1 was 30℃, at which strain PS1 exhibited the highest degradation rate of DBP, reaching 97.4%, and its optimal temperature range was 25–35℃.

[0105] The degradation condition design and response values ​​of strain PS1 were obtained using Box-Behnken design (see Table 3).

[0106] The equation for the DBP degradation rate (Y) was obtained by fitting the data:

[0107]

[0108] The parameters are shown in Table 4.

[0109] Statistical analysis results show its coefficient of determination (R²) 2 The coefficient of variation (CV) was 0.9533, indicating a high correlation between the measured and predicted values, making it suitable for theoretical prediction of DBP degradation by strain PS1. The CV was 2.1%, indicating a high good fit of the model equation. Analysis of variance showed that the model was highly significant (P < 0.0001). Analysis of variance of the regression equations showed that the linear effects of pH (X1) and temperature (X2) on the DBP degradation rate of strain PS1 were extremely significant (P < 0.01), with the quadratic coefficient X1 being particularly significant. 2 and X2 2The linear effect on the DBP degradation rate of strain PS1 was extremely significant (P<0.01).

[0110] Table 3 Box-Behnken Design Experiments and Results

[0111]

[0112] Note: In the table, X1 represents the pH value; X2 represents the temperature value; and X3 represents the inoculum quantity value. The results are the mean of three replicates ± standard error. Data in the same column with the same letter indicate no significant difference at the 5% level.

[0113] Table 4. Model and Analysis of Variance

[0114]

[0115] Note: R 2 =0.9533 (corrected R) 2 =0.9155), coefficient of variation (CV) = 2.1%; df refers to degrees of freedom; SS refers to mean square; P<0.05 indicates that the factor under investigation has a significant effect; P<0.01 indicates that it has a highly significant effect.

[0116] Use Design-Expert 8.0 software to draw response surfaces, such as... Figure 4 . Figure 4 a is the surface curve analyzing the response of PS1 bacteria to DBP degradation rate in response to temperature and pH; Figure 4 b represents the contour lines of DBP degradation rate by PS1 bacteria against temperature and pH. Based on the equation for DBP degradation rate (Y) obtained above, the inoculum quantity is fixed at 0.9 (OD). 600nm The effects of temperature and pH on the degradation rate of DBP by strain PS1 were analyzed. Three-dimensional response surfaces and contour plots were generated, and the optimal degradation conditions for DBP by strain PS1 were determined: temperature 28.7℃, pH 7.2, and inoculum size 0.9 (OD200). 600nm At this point, the model predicted a DEHP degradation rate of 97.6%. Repeated verification experiments were conducted to test the accuracy of the predictions under the obtained optimal degradation conditions. The theoretically optimal degradation conditions were selected to culture the degrading strain PS1, with other culture conditions identical to those in Example 3. After 3 days of treatment, the degradation rate of 200 mg / L DBP reached 98.1%, which was not significantly different from the theoretically predicted maximum degradation rate, indicating that the model fits the actual situation and the optimized conditions are usable.

[0117] The optimal pH for DBP degradation by strain PS1 is 7.2, indicating that the bacteria exhibit stronger degradation activity under neutral and slightly alkaline conditions. Within a temperature range of 25–40℃, strain PS1 achieves a DBP degradation rate exceeding 85%; at the optimal temperature of 28.7℃, the degradation rate reaches as high as 98.1%. In summary, strain PS1 demonstrates a wide pH and temperature adaptability range in DBP degradation, thus making it more widely applicable for the remediation of PAE-contaminated environments.

[0118] Example 4: Study on the degradation characteristics of DBP by strain PS1

[0119] I. Experimental Methods

[0120] (1) Determination of the relationship between the growth of strain PS1 and the degradation of dibutyl phthalate (DBP):

[0121] Inoculate 50 mL of sterilized MSM medium with the OD from step 1 of Example 3. 600nm A bacterial suspension with a concentration of 0.6 was prepared, and DBP working stock solution was added to achieve a final mass concentration of 200 mg / L. The suspension was then cultured in a constant temperature incubator with shaking (180 rpm) under the optimal conditions confirmed in Example 3 (pH = 7.2, 28.7 °C). Samples were taken at regular intervals at 4, 8, 12, 24, 36, and 48 hours of culture. The residual DBP content was determined by GC-MS (under the same detection conditions as in Example 1), and the OD of the bacterial suspension samples was measured using a UV spectrophotometer. 600nm The values ​​were set up with no inoculation as a control, and each treatment was repeated in 3 replicates. Finally, DBP degradation curves and bacterial growth curves were plotted.

[0122] (2) Degradation kinetics of strain PS1 at different initial concentrations of DBP:

[0123] Different amounts of DBP working stock solution were added to 50 mL of sterile culture medium to achieve final concentrations of 100, 200, 400, 800, and 1000 mg / L. The culture was then incubated under the optimal conditions (pH = 7.2, 28.7 °C) confirmed in Example 3 (180 rpm) with shaking. Samples were taken at 0, 12, 24, 36, 48, and 72 h, and the residual DBP in each bacterial culture sample was determined by GC-MS (detection conditions were the same as in Example 1). Degradation kinetic curves of different DBP concentrations were plotted. Three replicates were set up for each treatment, with no inoculation as a control.

[0124] II. Experimental Results

[0125] like Figure 5As shown, under the optimal conditions confirmed in Example 3 (pH = 7.2, 28.7℃), the strain underwent an adaptation period of 0–4 hours; a logarithmic growth phase of 4–8 hours; and reached a stationary phase after 8 hours. During the adaptation period, DBP was hardly degraded, with a low degradation rate; however, during the logarithmic growth phase, the rate of DBP degradation gradually increased, reaching its maximum at the early stage of stationary growth, after which the rate of degradation slowed down, reaching a maximum of 95.3% at 48 hours.

[0126] When strain PS1 of the present invention degrades DBP under the optimal conditions (pH = 7.2, 28.7℃) confirmed in Example 3, its growth curve shows that the environmental adaptation process of the strain is extremely short, and it reaches the growth stationary phase within 8 hours of degradation. Furthermore, the subsequent growth activity of the strain remains stable over a long period of time, indicating that the acidic metabolic intermediates of strain PS1 do not accumulate over a long period of time during the degradation of DBP, thus not causing changes in the pH value of the bacterial solution.

[0127] The degradation kinetics curves of strain PS1 for different initial concentrations (100–1000 mg / L) of DBP are shown in the figure. Figure 6 The experimental results showed that at high concentrations (800–1000 mg / L), the strain did not exhibit significant lag in DBP degradation, indicating that it could tolerate even higher concentrations of DBP. Within 72 hours, the degradation rate reached 80.95%–90.83%. At low concentrations (50–800 mg / L), degradation was almost complete within 72 hours. Overall, the degradation rate of DBP by strain PS1 decreased with increasing initial DBP concentration, indicating that high concentrations of DBP had a certain inhibitory effect on the growth of strain PS1.

[0128] The residual DBP levels at different initial concentrations and corresponding time points were fitted using a first-order degradation kinetic model. The first-order degradation kinetic model was C0. t =C0×e -kt In the formula, C t Let Ct be the residual concentration of DBP in the fermentation broth at time t (g / L), C0 be the initial concentration of DBP, and k be the degradation rate constant (tt). -1 Degradation half-life The fitting results are shown in Table 5. The correlation coefficient R is obtained from the fitting at each initial DBP concentration. 2The values ​​all exceeding 0.95 indicate that the degradation process of strain PS1 conforms to a first-order fitting model under different initial DBP concentrations. The degradation half-life of strain PS1 for DBP increases with increasing initial DBP concentration. At an initial DBP concentration of 100 mg / L, the degradation half-life is 7.31 h, increasing to 29.50 h as the initial DBP concentration increases to 1000 mg / L. Strain PS1 exhibits a wide range of initial DBP concentrations for degradation, a short degradation half-life, and excellent DBP tolerance and degradation efficiency.

[0129] Table 5. Degradation kinetics of strain PS1 at different initial concentrations of DBP

[0130]

[0131] Example 5: Determination of the broad-spectrum substrate activity of strain PS1

[0132] I. Experimental Methods

[0133] Five common PAEs (phthalates and their derivatives) were selected: dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), di(2-ethylhexyl) phthalate (DEHP), and dioctyl phthalate (DOP). Potential biodegradation intermediates of DBP, including monobutyl phthalate (MBP), phthalic acid (PA), protocatechuic acid (PCA), and 4,5-dihydroxyphthalic acid, as well as common aromatic compounds such as monobutyl benzoate, benzoic acid, and catechol, were used as test substrates. These substrates were added to sterile basal MSM medium to a final concentration of 200 mg / L, and then inoculated with fresh OD from step 1 of Example 3. 600nm A bacterial suspension with a concentration of 0.6 was cultured and the OD value of the fermentation broth was measured. 600nm The method was the same as in Example 4, determining whether strain PS1 could utilize these substrates for growth. A culture medium without bacterial suspension but with the same concentration of substrate served as a control, and each treatment was repeated in triplicate.

[0134] II. Experimental Results

[0135] Table 6 shows the utilization of various substrates by strain PS1. All substrates could be utilized by strain PS1, but the utilization varied considerably. Substrates with short side chains, such as DEP, DMP, DBP, MBP, PA, and PCA, were readily utilized by PS1, resulting in faster strain growth. Conversely, substrates with longer side chains (DEHP, DOP) were less readily utilized by the degrading bacteria, leading to slower strain growth. The steric hindrance created by PAEs compounds with long side chains inhibited further hydrolysis of PAEs compounds by the degrading bacteria.

[0136] Table 6. Substrate utilization of strain PS1

[0137]

[0138] Note: (++) indicates vigorous growth; (+) indicates that growth is possible.

[0139] Example 6: Determination and Analysis of Degradation Intermediates of Dibutyl Phthalate (DBP) by Strain PS1

[0140] I. Experimental Methods

[0141] Inoculate the bacterial suspension into 50 mL of sterilized MSM medium and add DBP working stock solution to make the final DBP concentration 200 mg / L. Place it under optimal conditions and shake to culture. Take samples when the degrading bacteria PS1 grows to the mid-log phase (6 h) and stationary phase (12 h), respectively, filter through a 0.22 μm membrane, and wait for instrumental analysis.

[0142] The analysis of DBP degradation products was performed using high performance liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS / MS). The chromatographic and mass spectrometric conditions are described in the literature (Zhao H, Hu R, Chen X, et al. Biodegradation pathway of di-(2-ethylhexyl)phthalate by a novel Rhodococcus pyridinivorans XB and its bioaugmentation for remediation of DEHPcontaminated soil. Science of the Total Environment, 2018, 640: 1121-1131).

[0143] II. Experimental Results

[0144] EESI primary and secondary mass spectrometry data of DBP and its degradation products were obtained using LC-TOF-MS / MS in full scan mode. Data for the inferred structure and degradation products obtained from the full scan are shown in Table 7.

[0145] Table 7. Identification of DBP degradation products by strain PS1 using LC-TOF-MS / MS

[0146]

[0147] Based on the DBP degradation intermediates identified by LC-TOF-MS / MS, the biochemical pathway of DBP degradation by strain PS1 was deduced. Figure 7First, DBP is hydrolyzed to produce monobutyl phthalate (MBP), which is then further hydrolyzed to produce phthalic acid (PA). The metabolism of PA varies significantly among strains of different genera. In the DBP degradation process of strain PS1 of this invention, PA is oxidized by phthalic acid 4,5-dioxygenase to produce 4,5-dihydroxybenzoic acid, which is then ring-opened by PCA oxidation and metabolized through the tricarboxylic acid cycle. Furthermore, butyl benzoate was also detected, which is produced by the decarboxylation of MBP, followed by hydrolysis to produce benzoic acid (BA), and then oxidized to catechol before entering the tricarboxylic acid cycle. This step is a unique degradation pathway of strain PS1. Strain PS1 of this invention possesses this unique degradation metabolic pathway, exhibiting a higher degradation capacity during DBP metabolism. The specific DBP mineralization pathway is as follows:

[0148] Pathway I: DBP→MBP→PA→4,5-dihydroxybenzoic acid→protocatechuic acid→tricarboxylic acid cycle.

[0149] Pathway II: DBP → MBP → Butyl benzoate → Benzoic acid → Catechol → Tricarboxylic acid cycle.

[0150] Example 7: Comparison of adsorption characteristics of phthalates (PAEs) in silkworm excrement and soil

[0151] I. Experimental Methods

[0152] 1. Determination of adsorption equilibrium time

[0153] The adsorption characteristics of PAEs on silkworm excrement and soil (garden soil) were studied using the OECD equilibrium method: A background solution containing 0.1 mol / L CaCl2 and 200 mg / L NaN3 was used, and DBP stock solution was added to achieve a final concentration of 12 mg / L to obtain the experimental dibutyl phthalate (DBP) solution. 0.5 g of silkworm excrement and soil were weighed and placed in Erlenmeyer flasks containing 50 mL of the experimental DBP solution. Each treatment was repeated in triplicate, and the flasks were placed at 25°C with constant temperature shaking (180 r / min). Samples were taken at 0.5, 1, 2, 3, 6, 18, 24, 30, and 36 h, centrifuged at 8000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm microporous membrane. The residual DBP concentration was analyzed by HPLC to determine the equilibrium time for DBP adsorption in silkworm excrement and soil.

[0154] 2. Adsorption kinetics analysis of PAEs in silkworm excrement and soil

[0155] The treatment groups for silkworm excrement and soil (garden soil) were set up in the same manner as in step 1. A control group containing the same DBP concentration but without any adsorbent was also included. All samples were placed in a shaker at 25°C with constant temperature shaking (180 r / min). For soil samples, 1 mL samples were taken at 0.5, 1, 2, 3, 6, and 18 h. For silkworm excrement samples, 1 mL samples were taken at 5, 10, 15, 30, 60, 90, 120, 150, and 180 min. Samples from the control group and the silkworm excrement and soil treatment groups were taken at the same time points. All samples were filtered through a 0.22 μm microporous membrane, and their residual concentrations were determined by HPLC for adsorption kinetic analysis.

[0156] 3. Calculation method

[0157] The formula for calculating the adsorption capacity of DBP by silkworm excrement is as follows:

[0158]

[0159] In the formula, Q t Ct represents the adsorption amount at time t, in mg / g; C0 represents the initial concentration of DBP in the solution, in mg / L; Ct represents the initial concentration of DBP in the solution. t V is the concentration of DBP in the solution at time t, in mg / L; V is the volume of the solution, in L; W is the mass of the adsorbent, in g.

[0160] (1) The pseudo-first-order dynamics expression is:

[0161] Q t =Q e (1-exp(-k1t))

[0162] In the formula: Q e Q represents the adsorption capacity at equilibrium, in mg / g; t t represents the amount of adsorption at time t, in mg / g; k1 represents the rate constant.

[0163] (2) The pseudo-second-order dynamic equation is expressed as follows:

[0164]

[0165] The constants in the formula have the same meaning as those in the pseudo-first-order dynamics expression.

[0166] 4. High-performance liquid chromatography determination of DBP in the supernatant

[0167] The determination method is as follows: A liquid chromatography system equipped with a diode array detector (1100, Agilent, USA) was used. The chromatographic column was Hypersil ODS (250 mm × 4 mm, 5 μm). The mobile phase was methanol and ultrapure water (V / V = 80 / 20). The flow rate was 1 mL / min, the column temperature was 25 °C, the detection wavelength was 242 nm, and the injection volume was 10 μL. The DBP retention time was 4.8 min. Quantification was performed using the external standard method. The standard deviation of parallel samples for each treatment was less than 10%, and the sample spike recovery rate was 85%–92%.

[0168] 5. Fourier transform infrared spectroscopy analysis

[0169] After the adsorption experiment was completed, the DBP adsorption equilibrium solution was placed in a centrifuge and centrifuged at 2000 r / min for 10 min. The supernatant was discarded, and the precipitate and the corresponding unadsorbed DBP soil and silkworm excrement samples were freeze-dried for 24 h. A small amount of powder sample was taken and mixed with anhydrous potassium bromide at a ratio of 1.5:300 (mass ratio) and ground evenly. The scanning wavenumber range was set to 500–4000 cm⁻¹. -1 The scanning resolution is 4cm. -1 Scan on the machine.

[0170] II. Experimental Results

[0171] 1. Adsorption equilibrium curve

[0172] The adsorption process of DBP by both silkworm excrement and garden soil exhibits three stages: rapid, slow, and equilibrium adsorption stages. Figure 8 The rapid adsorption phase of DBP by silkworm excrement was 0–1 h, and the slow adsorption phase was 1–6 h. After 6 h, the adsorption of DBP by silkworm excrement reached equilibrium. The rapid adsorption phase of DBP by garden soil was 0–3 h, and the slow adsorption phase was 3–24 h. After 24 h, the adsorption of DBP by garden soil reached equilibrium. Therefore, in subsequent experiments, 6 h was taken as the adsorption equilibrium time of DBP by silkworm excrement, and 24 h was taken as the adsorption equilibrium time of DBP by garden soil.

[0173] 2. Adsorption kinetics model

[0174] The fitting results (Table 8) show that the first-order (R) 2 =0.99) and the second-order dynamic model (R 2Both the first-order kinetic model (=0.99) and the second-order kinetic model (=0.99) can fit the adsorption process of DBP by silkworm excrement well. The adsorption processes of DBP by silkworm excrement and garden soil are more consistent with the first-order kinetic model. The first-order kinetic model assumes that the adsorption process is controlled by the physical adsorption mechanism; the second-order kinetic model is based on the assumption that the adsorption rate is controlled by the chemical adsorption mechanism. Therefore, the adsorption processes of DBP by silkworm excrement and garden soil do not involve the chemical adsorption mechanism and are more inclined to physical adsorption. In addition, the equilibrium adsorption capacity of DBP by silkworm excrement calculated by the first-order kinetic model is 1.75 mg / g, which is higher than that of garden soil under the same conditions (0.95 mg / g), indicating that under the same environmental conditions, silkworm excrement has a stronger adsorption capacity for DBP than garden soil.

[0175] Table 8. Kinetic parameters of DBP adsorption in silkworm excrement and vegetable garden soil

[0176]

[0177] 3. Analysis of the adsorption mechanism of DBP by silkworm excrement and vegetable garden soil

[0178] from Figure 9 It can be seen from the soil in the vegetable garden that it is 3419cm deep. -1 1636cm -1 1052cm -1 836cm -1 There is a distinct absorption peak at 2924 cm⁻¹, and this absorption peak is significantly enhanced after DBP adsorption; in addition to the above peaks, silkworm excrement also exhibits a peak at 2924 cm⁻¹. -1 2851cm -1 The absorption peaks were also very obvious, and all absorption peaks were significantly enhanced after DBP absorption. During the adsorption of DBP by garden soil, hydrogen bonds and π-π bonds were formed with DBP; while during the adsorption of DBP by silkworm excrement, in addition to forming hydrogen bonds and π-π bonds, hydrophobic bonds were also formed. This is one of the reasons why the adsorption capacity of silkworm excrement for DBP is greater than that of garden soil.

[0179] In addition, silkworm excrement is at 3425cm -1 1500cm -1 1100cm -1 All samples exhibited absorption peaks, which were subsequently confirmed to be the stretching vibration peaks of the -OH group of the carboxyl group, the stretching vibration peak of the NH bond, and the stretching vibration peaks of the CO bond in aliphatic ethers and alcohols. Carriers with functional groups such as carboxyl and amide groups are conducive to the adhesion and proliferation of microbial cells, making them more suitable for immobilized microorganisms. No new absorption peaks appeared in the infrared spectra of silkworm excrement and garden soil before and after DBP adsorption, and the positions of existing absorption peaks did not change significantly, indicating that the adsorption of DBP by silkworm excrement and garden soil is mainly physical adsorption, consistent with the results of absorption kinetic simulations.

[0180] Example 8: Characteristics of the silkworm excrement-degrading bacteria preparation

[0181] I. Experimental Methods

[0182] 1. Preparation of silkworm excrement-degrading bacteria preparation

[0183] A single colony of strain PS1 from Example 1 was picked and inoculated into 50 mL of LB medium. After incubation for 24 h, the supernatant was removed, and the bacterial cells were washed with water. After centrifugation again, the bacterial cells were resuspended in 5 mL of physiological saline. 20 g of silkworm excrement was weighed and added to a sterilized tissue culture flask. The resuspended bacterial cells were poured in, mixed well, and set aside to obtain the silkworm excrement-degrading bacterial preparation.

[0184] 2. Scanning electron microscopy observation of the morphology of silkworm excrement-degrading bacterial agent

[0185] A small amount of the silkworm excrement-degrading bacteria preparation from step 1 of Example 8 was taken and used to prepare an electron microscopy scanning sample. Unsolidified silkworm excrement was used as a control. The morphological changes and bacterial distribution of the silkworm excrement before and after solidification were observed. The preparation method is as follows:

[0186] Take 1g of sample, rinse twice with physiological saline, add 1mL of 2.5% glutaraldehyde fixative and mix thoroughly. Let stand for 4h (extend the time as much as possible or overnight) for fixation. After washing three times with physiological saline, perform ethanol gradient dehydration, that is, add 30%, 50%, 70%, 80% and 90% ethanol once each, and 100% ethanol twice. After standing for 15min each time, centrifuge to remove the supernatant (after dehydration with 70% ethanol, let stand overnight, and continue gradient dehydration the next day). Send the sample to dry and then observe it under a scanning electron microscope.

[0187] 3. Determination of the concentration of degrading bacteria in silkworm excrement-degrading bacteria preparation

[0188] On days 0, 1, 7, 14, and 30 after the silkworm excrement-degrading bacteria preparation prepared in step 1 of Example 8 was completed, 1 g of the silkworm excrement-degrading bacteria preparation was taken and placed in 99 mL of physiological saline, and shaken at 25°C and 200 r / min for 30 min; the shaken solution was serially diluted, and 100 μL of each solution was taken. 6 10 7 The solution was diluted 10 times, and the plate coating method was used. Sterile soil inoculated with the same concentration of degrading bacteria was set as the treatment control group. Each treatment was repeated three times, and the concentration of degrading bacteria (CFU) in the carrier was measured.

[0189] II. Experimental Results

[0190] 1. Solidified morphology of degrading bacteria in silkworm excrement

[0191] Before solidification ( Figure 10 a) The surface of silkworm excrement is not smooth, with irregular wrinkles and gaps; after solidification ( Figure 10(b, c, d) The surface morphology of silkworm excrement showed no obvious changes, while the degrading bacteria were trapped in the folds and crevices of the silkworm excrement surface; Under high magnification electron microscopy (5k× and 10k×), the morphology of strain PS1 on the surface of silkworm excrement was clearly visible, without any obvious changes and with abundant content, indicating that the silkworm excrement has a significant ability to fix bacteria.

[0192] 2. Concentration of degrading bacteria in silkworm excrement

[0193] The concentration of degrading bacteria in silkworm excrement carriers was measured over time using the plate coating method to assess the adaptation status of degrading bacteria PS1 in the carrier. Figure 11 Without external nutrient supply, the concentration of degrading bacteria in silkworm excrement remained at 10 on day 7. 9 The concentration of CFU / mL was above 10, decreasing slightly on days 14 and 30. On day 30, the concentration of degrading bacteria remained at 10. 8 The concentration of degrading bacteria in sterile garden soil changed significantly over time, reaching 10 CFU / mL on day 30. 5 At around CFU / mL, the concentration of degrading bacteria decreased significantly. This indicates that, compared to sterile garden soil, silkworm excrement can significantly prolong the survival period of degrading bacteria, which is beneficial for its practical application.

[0194] Example 9: Study on the actual remediation effect and related mechanisms of silkworm excrement-degrading bacteria preparation

[0195] I. Experimental Methods

[0196] 1. Greenhouse pot experiment design

[0197] Treatment of contaminated soil: A mixed phthalic acid ester (PAE) solution containing 25 g / L dibutyl phthalate (DBP) and 25 g / L di(2-ethylhexyl) phthalate (DEHP) was prepared using acetone as a solvent. The DBP solution was added to soil particles with a diameter of 1 mm and stirred thoroughly. The contaminated soil (1 mm particle size) was then mixed with uncontaminated soil (2 mm particle size) at a mass ratio of 1:9 to obtain 50 kg of contaminated soil with a DBP and DEHP concentration of 50 mg / kg. The contaminated soil was then allowed to age naturally in the dark for 7 days before use, and the PAE concentration in the aged soil was measured.

[0198] The pot experiment consisted of four treatment groups: (1) contaminated soil + Chinese cabbage (CK); (2) contaminated soil + Chinese cabbage + strain PS1 from Example 1 (M); (3) contaminated soil + Chinese cabbage + silkworm excrement (SE); and (4) contaminated soil + Chinese cabbage + silkworm excrement - silkworm excrement from Example 8 - degrading bacteria preparation (MSE). Each treatment group had three replicates, with 3 kg of contaminated soil used in each replicate. The amount of silkworm excrement added in group (3) was 1% (dry weight). The amount of silkworm excrement in the silkworm excrement-degrading bacteria preparation in group (4) should be consistent with that in group (3), and the inoculation amount should be added according to the preparation ratio of the silkworm excrement-degrading bacteria preparation. The inoculation amount of strain PS1 in group (2) should be consistent with that in group (4).

[0199] Cultivation method for Chinese cabbage: The Chinese cabbage variety selected for the pot experiment was Huaguan, a high-accumulation variety of phthalate esters (PAEs). (Zhao H, Du H, Xiang L, et al. Variations in phthalate ester (PAE) accumulation and their formation mechanism in Chinese flowering cabbage (Brassica parachinensis L.) cultivars grown on PAE-contaminated soils. Environmental Pollution, 2015, 206:95-103.). Seeds were first disinfected by soaking in 2% sodium hypochlorite solution for 30 minutes, then rinsed with water and sown directly into pots. Approximately 20 seeds were sown per pot. Thinning began when seedlings had 3 leaves, and finally, 10 plants were transplanted per pot. During the vegetable growth period, tap water was used for irrigation, natural sunlight was provided, and pests and diseases were controlled manually.

[0200] 2. Measurement Indicators

[0201] (1) Collection of soil and plant samples: After 40 days of planting, the experiment was completed, and the aboveground parts and roots of the Chinese cabbage and the soil were harvested. The roots and aboveground parts of the Chinese cabbage were washed clean, dried, and weighed fresh. They were then freeze-dried for the extraction of PAEs from the plants. The top 2 cm of soil was removed, the soil sample was mixed, and a portion was placed in 10 mL sterile centrifuge tubes and stored at -80℃ for high-throughput sequencing of bacterial 16S rRNA genes. Another portion was freeze-dried for the extraction of residual PAEs from the soil sample, and the remaining soil was air-dried.

[0202] (2) Soil pH and organic matter content (g / kg): The relevant tests were conducted in accordance with the steps described in the national agricultural industry standard NY / T 1121.2-2006.

[0203] (3) Determination of nitrate nitrogen and ammonium nitrogen in soil (mg / kg): Determined according to the instructions of the soil nitrate nitrogen and ammonium nitrogen content detection kit.

[0204] (4) Determination of PAE content in potted plant samples

[0205] ① The preparation method of the standard curve is the same as step (2) in Example 1.

[0206] ② Extraction of PAEs from soil: Weigh 2g of lyophilized and pulverized soil sample into a 50mL polytetrafluoroethylene centrifuge tube, add 15mL of extraction buffer (dichloromethane:methanol = 7:3, V:V), sonicate for 10min, centrifuge at 8000rpm for 5min, and pour the supernatant into a chromatography column (35cm long × 1cm diameter glass column, packed with 4cm anhydrous sodium sulfate, 10cm silica gel, and 4cm neutral alumina from top to bottom, pre-rinsed with 20mL of extraction buffer before use) for purification, and collect in a pistol bottle. Repeat the above extraction steps twice. After the supernatant has passed through the column, elute the residual PAEs from the chromatography column with 10mL of extraction buffer. Then, concentrate the eluent in the pistol bottle to dryness using a rotary evaporator, make up to 2mL with methanol, filter through a membrane, and store at 4℃ for analysis.

[0207] ③ Extraction of PAEs from Chinese cabbage heart: Weigh 0.2g of freeze-dried and ground root and 1g of aerial part sample into a 50mL polytetrafluoroethylene centrifuge tube. The extraction method is the same as the above-mentioned extraction method for PAEs from soil. After extraction, dilute to 2mL with methanol and add 1mg / L benzyl benzoate. Filter through a 0.22μm filter membrane and store at 4℃ in the dark until analysis.

[0208] ④ The GC-MS analysis conditions for PAEs were the same as in Example 1.

[0209] (5) Soil microbial community 16S rDNA high-throughput sequencing: using Soil DNAKit (Lianchuan Biotechnology, Hangzhou) was used to extract soil genomic DNA, and its quality was assessed by electrophoresis, while DNA quantification was performed. PCR amplification of the hypervariable region V4-V5 of 16S rDNA was performed using specific primers (Shao P, Liang C, Rubert-Nason K, et al. Secondary successional forests undergo tightly-coupled changes in soil microbial community structure and soil organic matter. Soil Biology and Biochemistry, 2019, 128: 56-65.). The PCR reaction conditions were: 94℃ for 5 min; 35 cycles of 98℃ for 40 s, 54℃ for 30 s, and 72℃ for 45 s; a final 72℃ for 10 min; and storage at 4℃. PCR products were detected by 2% gel electrophoresis. Qualified PCR products were purified using AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified using Qubit (Invitrogen, USA). Amplicon pools were used for sequencing. The size and number of amplicon libraries were evaluated using an Agilent 2100 bioanalyzer and an Illumina (Kapa Biosciences, Woburn, MA) library quantification kit, respectively. The libraries were finally sequenced on a NovaSeq PE250 platform.

[0210] Sequencing data processing: After sequencing, clean data is obtained through assembly and other steps. DADA2 is used for noise reduction, and then an OUT-like table is constructed to obtain the final feature table for further analysis, such as diversity analysis. Bacterial alpha diversity is calculated using QIIME2. Beta diversity is calculated using QIIME2 and plotted using R packages. Blast is used for sequence alignment, and each representative sequence is annotated with the SILVA database. Other figures are implemented using R packages (v3.5.2).

[0211] II. Experimental Results

[0212] 1. Residues of PAEs in the soil and inside the Chinese cabbage heart

[0213] The residual amount of PAEs in the soil was determined by GC-MS (Table 9). The residual amount of DBP in the soil of the treatment groups with added silkworm excrement (SE) and silkworm excrement-degrading bacteria preparation (SEM) was not detected. There was no significant difference in the residual amount of DBP in the soil of the blank control group (CK) and the treatment group (M) with strain PS1 alone, and the DBP removal rate of both groups exceeded 85%. Compared with the CK group, the residual amount of DEHP in the soil of the M, SE and MSE groups decreased significantly and the DEHP removal rate increased significantly. Among them, the DEHP removal rate of the soil of the MSE group reached 71.66%, which was the most significant.

[0214] The results of the determination of PAE residues in Chinese cabbage (Table 10) showed that the M, SE and MSE groups all promoted the removal of PAEs in the soil and reduced the accumulation of PAEs in Chinese cabbage, but the effect was more obvious in the SE and MSE groups, and the effect of MSE was the best.

[0215] Table 9. Residues of PAEs in Soil

[0216]

[0217] Note: In the same column, if the mean (± error) is followed by the same letter, the difference is not significant (p>0.05), and ND means not detected.

[0218] Table 10. PAEs residues in Chinese cabbage heart

[0219]

[0220] Note: In the same column, if the mean (± error) is followed by the same letter, the difference is not significant (p>0.05), and ND means not detected.

[0221] 2. Biomass of Chinese cabbage

[0222] Analysis of biomass of aboveground and belowground parts of Chinese pistache under different soil treatments Figure 12 Compared with the CK group, the M, SE, and SEM groups all increased the biomass of both the aboveground and underground parts of Chinese cabbage. Among them, the SE and SEM groups had the most significant effects, and the difference between the two groups in terms of biomass was not obvious.

[0223] 3. Soil physicochemical properties

[0224] This invention measured the pH, organic matter content, ammonia nitrogen, and nitrate nitrogen content in soils of different treatment groups. The results showed that ( Figure 13 Compared to the CK group, the M, SE, and SEM groups all increased the soil pH. The SE and SEM groups changed the soil pH from slightly acidic (pH = 6.43) to slightly alkaline (pH = 7.50–7.58), and there was no significant difference in soil pH between these two treatment groups. Figure 13a); The M group did not significantly affect the organic matter content in the soil, but the SE and SEM groups significantly increased the organic matter content in the soil, and the SEM group had the most obvious effect. Figure 13 b); By performing a correlation analysis on the soil pH, organic matter content, and PAEs residues in the soil, it was found that there was a highly significant negative correlation between the soil pH value, organic matter content, and the residues of DBP and DEHP in the soil (Table 10). In this invention, the increase in the soil organic matter content promoted the biodegradation of PAEs in the soil, indicating that the increase in the soil organic matter content had a smaller impact on the process of soil particles adsorbing PAEs than on the process of soil microorganisms degrading PAEs.

[0225] PAEs can promote denitrification in the soil, inhibit nitrification in the soil, cause the loss of soil nitrogen nutrients, and is not conducive to crops absorbing nitrogen. Figure 13 c is the content of nitrate nitrogen in the soil; Figure 13 d is the content of ammonium nitrogen in the soil. In this invention, the M group significantly reduced the nitrate nitrogen content, the SE and SEM groups both significantly increased the nitrate nitrogen content, and there was no significant difference in the nitrate nitrogen content between the two treatment groups; the M group had no significant effect on the ammonium nitrogen content, the SE and SEM groups significantly increased the ammonium nitrogen content, and the SEM group had the most obvious effect. By performing a correlation analysis on the ammonia nitrogen and nitrate nitrogen contents in the soil and the above-ground and underground biomass of Brassica parachinensis (Table 11), it was found that there was a significant positive correlation between the ammonia nitrogen content in the soil and the biomass of Brassica parachinensis, and there was no significant correlation between the nitrate nitrogen content and the biomass of Brassica parachinensis. It shows that both silkworm feces and the silkworm feces - degrading bacteria preparation promote soil nitrogen fixation and nitrification, thereby increasing the contents of ammonia nitrogen and nitrate nitrogen in the soil, promoting Brassica parachinensis to absorb nitrogen elements in the soil, and increasing its above-ground and underground contents.

[0226] Table 11 Correlation analysis of PAEs residues in the soil, plant biomass, and soil physical and chemical properties

[0227]

[0228] Note: p < 0.01 is highly significant, 0.01 < p < 0.05 is significant, and p > 0.05 is not significant.

[0229] 4. Analysis of the characteristics of the soil bacterial community structure

[0230] (1) Analysis results of the bacterial Alpha diversity index in the soil

[0231] A total of 728,142 high-quality valid sequences were recovered using 16S rDNA high-throughput sequencing. These sequences were clustered into 26,340 OTUs with 97% sequence similarity. The soil bacterial α-diversity index in each treatment group was analyzed, as shown in Table 12. Compared to the CK group, the Chao 1 index and Shannon index of soil microorganisms in the M, SE, and SEM groups all decreased to varying degrees, but none were significant, indicating that the degrading bacteria PS1, silkworm excrement, and silkworm excrement-degrading bacteria preparation had little effect on the diversity and richness of bacteria in the soil.

[0232] Venn diagram of soil bacteria shared and unique to different treatment groups ( Figure 14 It can be seen that SEM shared 1804, 855, and 883 OTUs with the SE, M, and CK groups, respectively, accounting for 24.87%, 9.69%, and 10.62% of the total OTUs of the corresponding two treatment groups; SE shared 1033 and 1048 OTUs with the M and CK groups, respectively, accounting for 12.27% and 13.22% of the total OTUs of the corresponding two treatment groups; while M and CK shared 1698 OTUs, accounting for 21.51% of the total OTUs of the two treatment groups. Among the treatment groups, SEM and SE groups and CK and M groups shared a relatively large number of OTUs, but the sharing rate was only between 21% and 25%, which is relatively small, indicating that there are significant differences in the types of soil bacteria among the treatment groups.

[0233] Table 12 Alpha diversity index of bacteria in soils of different treatments

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[0236] Note: Values ​​(± error) with the same letter following them in the same column do not differ significantly (p>0.05).

[0237] (2) Analysis of PCoA in Soil Bacteria

[0238] Based on the Bray-Curtis distance matrix algorithm, PCoA analysis was used to perform cluster analysis on all samples. Figure 15 PCoA1 explained 67.12% of the variance in the dataset, while PCoA2 explained 13.16%. Different colored points belong to different sample groups, and the scale lines on the horizontal axis represent the distance between samples; the greater the distance between samples, the greater the difference in bacterial composition. The bacterial compositions between the CK and M groups, and between the SE and SEM groups, were relatively similar, but differed significantly from the other two groups. This indicates that silkworm excrement and silkworm excrement-degrading bacteria preparations caused significant differences in soil bacterial variations, while strain PS1 had little impact on soil bacterial community changes.

[0239] (3) Species analysis of bacterial communities in soil

[0240] Based on annotations and classifications of 26,340 OUT entries from relevant databases, a total of bacteria were identified, comprising 35 phyla, 121 classes, 283 orders, 510 families, and 1,027 genera. At the phylum level (… Figure 16 In all four treatment groups, Proteobacteria, Actinobacteria, Chloroflexi, Gemmatimonadetes, and Planctomycetes were the dominant soil bacteria, but there were certain differences in the relative abundance of soil bacteria among the groups. As shown in Table 13, the relative abundance of Proteobacteria in the soil of each treatment group differed significantly, and the order of abundance was SEM>SE>CK>M. Actinobacteria, Chloroflexi, and Acidobacteria had lower relative abundance in the SEM and SE treatment groups, and the difference between the two was not significant. Gemmatimonadetes had the lowest relative abundance in SEM and the highest relative abundance in M, and the difference between the two was significant. Bacteroidetes, Verrucomicrobia, and Firmicutes had higher relative abundance in the SE and SEM treatment groups, and the difference between the two was not significant. The results indicate that silkworm excrement and the silkworm excrement-degrading bacteria preparation increased the relative abundance of Proteobacteria, Bacteroidetes, Verrucomicrobia, and Firmicutes in the soil, while decreasing the relative abundance of Actinobacteria, Chloroflexi, Acidobacteria, and Gemmatimonadetes. Among these, the addition of the silkworm excrement-degrading bacteria preparation significantly increased the relative abundance of Proteobacteria compared to silkworm excrement; while the addition of degrading bacteria PS1 alone decreased the relative abundance of Proteobacteria and increased the relative abundance of Gemmatimonadetes in the soil.

[0241] Table 13. Relative abundance (%) of the top 10 bacteria at the phylum level in soil samples from each treatment.

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[0244] Note: The differences between mean (± error) values ​​with the same letter in the same row are not significant (p>0.05).

[0245] (4) LEfSe analysis

[0246] Based on the distribution of OTUs in each treatment group, significant differences in species among treatment groups were analyzed using Lefse software. The non-parametric factorial Kruskal-Wallis (KW) sum-rank test was used to detect species with significant abundance differences among different groups, and linear discriminant analysis (LDA) was performed. Microbial species with an LDA score > 4 were defined as biomarkers between different treatments.

[0247] Compared with the CK group: M group ( Figure 17 a) The dominant bacterial species in the interstitial group was Chloroflexi, with no other dominant bacterial species; SE group ( Figure 17 The dominant bacterial species in b) are Deltaproteobacteria (class), Sandaracinaceae_unclassified (genus), Myxococcales (order), Proteobacteria (phylum), Sandaracinaceae (family), Haliangiaceae (family), Haliangium (species), and Sandaracinaceae (species), all belonging to the phylum Proteobacteria; SEM group ( Figure 17 The dominant bacterial species in c) are Deltaproteobacteria (class), Proteobacteria (order), Xanthomonadaceae (family), Sandaracinaceae (genus), Myxococcales (order), Proteobacteria (genus), Proteobacteria (class), Proteobacteria (phylum), Sandaracinaceae (family), Proteobacteria (family), Sandaracinaceae (species), and Proteobacteria (species), all of which belong to the phylum Proteobacteria.

[0248] LDA analysis was performed on the dominant bacteria in the soil of the SE and SEM groups. The results are as follows: Figure 18The dominant bacterial species in Group SE are Nitrospirae (phylum), Nitrospira (class), Nitrospirales (order), Nitrospiraceae (family), Nitrospira (genus), Nitrospira (species), Actinobacteria (order), Pseudonocardiales (order), Actinobacteria (family), Pseudonocardiaceae (family), Actinobacteria (genus), Actinophytocola (genus), and Actinobacteria (species). Among these, Nitrospirae (phylum) and Nitrospira (class) are dominant. The bacteria in the SEM group, classified as *Nitrospira* (class), *Nitrospirales* (order), *Nitrospiraceae* (family), *Nitrospira* (genus), and *Nitrospira* (species), belong to the phylum *Nitrospirae*. *Actinobacteria* (order), *Actinobacteria* (family), *Actinobacteria* (genus), and *Actinobacteria* (species) belong to the order *Actinobacteria*. The dominant bacterial species in the SEM group are *Proteobacteria* (order), *Sandaracinaceae* (genus), *Proteobacteria* (genus), *Proteobacteria* (class), *Proteobacteria* (phylum), *Sandaracinaceae* (family), *Proteobacteria* (family), *Sandaracinaceae* (species), and *Proteobacteria* (species), all belonging to the phylum *Proteobacteria*.

[0249] In summary, group M did not significantly affect the structure of the indigenous microbial community in the soil; both groups SE and SEM increased the relative abundance of Proteobacteria in the soil; compared with group CK, both groups SE and SEM increased the abundance of Nitrospirae in the soil; while compared with group SE, group SEM decreased the relative abundance of Nitrospirae, Actinobacteria, and Pseudonocardiales, and increased the relative abundance of Proteobacteria. It is noteworthy that the genus Pseudomonas, to which strain PS1 belongs, did not become the dominant genus in the soil microbial community (relative abundance <0.2), possibly because the rhizosphere microbial ecological structure is determined by plant type (host genotype) and soil characteristics; however, the relative abundance of Pseudomonas in the soil of group SEM was much greater than in other groups. Figure 19 Furthermore, strain PS1 belongs to the phylum Proteobacteria. Therefore, the silkworm excrement-degrading bacteria preparation promoted the growth of Pseudomonas and Proteobacteria in the soil, increasing the relative abundance of these bacteria.

[0250] (5) RDA analysis of environmental indicators and major dominant bacteria

[0251] This invention uses RDA analysis to analyze soil pH, organic matter content, nitrate nitrogen, and ammonia nitrogen content with the relative abundance of dominant bacteria in each soil treatment group to reveal the relationships between them. The results show ( Figure 20The redundancy analysis model explained 85.07% of the variables, with Axis 1 and Axis 2 explaining 62.23% and 22.84%, respectively. Furthermore, the relative abundance of dominant bacteria in each soil treatment group was completely separated in the redundancy analysis model, indicating that these environmental indicators play an important role in explaining soil microbial community disturbance. Among the dominant bacterial species, the relative abundance of Proteobacteria and Nitrospirae bacteria was negatively correlated with the residual amount of PAEs in the soil, but positively correlated with Chinese cabbage biomass, soil pH, soil organic matter content, and soil ammonia nitrogen and nitrate nitrogen content; Actinobacteria and Pseudonocardiales bacteria showed the opposite trend. Correlation analysis revealed that soil organic matter content and pH were significantly negatively correlated with the residual amount of PAEs in the soil, while soil ammonia nitrogen content was significantly positively correlated with Chinese cabbage biomass. Therefore, Proteobacteria and Nitrospirae bacteria in the soil exhibit a positive correlation with soil pH, soil organic matter, and ammonia nitrogen levels, collectively promoting the degradation of PAEs in the soil and simultaneously promoting the growth of Chinese cabbage. Conversely, Actinobacteria and Pseudonocardiales bacteria show a negative correlation with soil pH, soil organic matter, and ammonia nitrogen levels, inhibiting the degradation of PAEs and the growth of Chinese cabbage. Nitrospira is a nitrite-oxidizing bacterium with nitrogen-fixing capabilities in the soil, promoting plant growth and providing a nitrogen source for degrading bacteria, thus promoting the degradation of PAEs.

[0252] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A type of Pseudomonas aeruginosa ( Pseudomonas aeruginosa PS1, characterized in that, The Pseudomonas aeruginosa ( Pseudomonas aeruginosa PS1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 27, 2021, with accession number GDMCC No: 62009.

2. The *Pseudomonas aeruginosa* as described in claim 1 (… Pseudomonas aeruginosa Application of PS1 in the remediation of environments contaminated with di(2-ethylhexyl) phthalate.

3. The application according to claim 2, characterized in that, The remediation of the environment contaminated with di(2-ethylhexyl) phthalate involves degrading di(2-ethylhexyl) phthalate in the contaminated environment, restoring the physicochemical properties of the contaminated environment, and / or restoring the microbial community structure of the contaminated environment.

4. The application according to claim 3, characterized in that, The restoration of the physicochemical properties of the environment contaminated by di(2-ethylhexyl) phthalate includes increasing soil pH, increasing soil organic matter content, and / or increasing soil nitrate nitrogen and / or ammonium nitrogen content; the restoration of the microbial community structure of the environment contaminated by di(2-ethylhexyl) phthalate includes increasing the abundance of Proteobacteria and / or Digestorhynchus, and / or decreasing the abundance of Actinomycetes and / or Pseudonocardiales.

5. A biological agent for environmental remediation, characterized in that, The biological agent contains the *Pseudomonas aeruginosa* as described in claim 1. Pseudomonas aeruginosa PS1.

6. The biological agent according to claim 5, characterized in that, The biological agent also contains silkworm excrement.

7. The use of the biological agent according to claim 5 or 6 in the remediation of an environment contaminated with di(2-ethylhexyl) phthalate.

8. The application according to claim 7, characterized in that, The remediation of the environment contaminated with di(2-ethylhexyl) phthalate involves degrading di(2-ethylhexyl) phthalate in the contaminated environment, restoring the physicochemical properties of the contaminated environment, and / or restoring the microbial community structure of the contaminated environment.

9. A method for remediating an environment contaminated by di(2-ethylhexyl) phthalate, characterized in that, Using the Pseudomonas aeruginosa as described in claim 1 ( Pseudomonas aeruginosa Repair by PS1 or the biological agent as described in claim 5 or 6.

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