A phoxim-degrading bacterial strain and a preparation method and application of phoxim-degrading bacterial balls

Abstract

This invention discloses a phoxim-degrading bacterial strain and a method for preparing phoxim-degrading bacterial balls, as well as their applications. The strain preparation method includes selecting rhubarb rhizosphere soil, isolating and purifying bacteria, conducting initial screening and secondary screening of phoxim-degrading bacteria, and preparing the bacterial balls by immobilization with attapulgite. In this invention, RL4, after immobilization with sodium alginate and attapulgite, achieved a 72H phoxim degradation rate of 87.70%, which is 1.43% higher than using free bacteria. In phoxim-contaminated wastewater, RL4, after immobilization with sodium alginate and attapulgite, achieved a 72H phoxim removal rate of 77%, which is 12.4% higher than using free bacteria. Even after five repeated uses, RL4, after immobilization with sodium alginate and attapulgite, still maintained 80% of its phoxim degradation activity, indicating that attapulgite-immobilized phoxim-degrading bacterial balls SA+ATP-RL4 are feasible for in-situ phoxim remediation.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbial remediation technology, specifically relating to a method for preparing and applying a phoxim-degrading strain and phoxim-degrading bacterial balls. Background Technology

[0002] Phoxim (O,O-diethyl-O-(α-cyanobenzyme)thiophosphate) is a widely used broad-spectrum organophosphorus pesticide that primarily inhibits acetylcholinesterase activity, leading to the accumulation of acetylcholine in the postsynaptic membrane and ultimately causing pest death (Lionetto et al., 2013). In China, phoxim is used in agriculture and fisheries, applied by immersion, spraying, or dumping to control various harmful insects and underground pests, with annual application reaching up to 1,000 tons (Huang et al., 2013). However, due to its continued widespread use, this pesticide is frequently detected in food, soil, agricultural wastewater, and river water (Chao and Chen, 2014; He et al., 2016). Phoxim exhibits neurotoxicity to non-target organisms such as mammals and aquatic organisms, posing harmful risks to the environment and human health (Eevers et al., 2017), raising serious concerns about environmental and food safety.

[0003] Biodegradation is considered a cost-effective technology for pesticide residue remediation (Cycoń et al., 2017; Gangola et al., 2022). Neosphingosine (RL-4) is a yellow-stigmotropic strain collected from rhubarb rhizosphere soil during the screening of soil bacteria for degrading organophosphorus pesticides. This strain was able to degrade 86.5% of 20 mg / L phoxim pesticide within 72 hours. They can use phoxim as their sole carbon and energy source for growth. However, free bacterial cells may not be suitable for direct use in phoxim-contaminated wastewater and soil because they must compete with the local soil microbiome. Furthermore, the preparation of fresh cells is complex, cultivation requires optimal conditions, and cells are easily contaminated. Immobilization methods have recently been used in bioremediation processes because immobilization enhances the resistance of microorganisms to stress conditions and improves the efficiency of bioremediation (Dzionek et al., 2016).

[0004] Microbial immobilization methods mainly include adsorption, encapsulation, and covalent bonding. Encapsulation offers advantages such as improved environmental adaptability, high microbial density, and high bacterial activity; the prepared immobilized beads can be stored for extended periods (Nadroglu et al. 2019). Sodium alginate, with its low cost, non-toxicity, high biomass, and high bacterial permeability, has become one of the most common bacterial retention carriers. Combining adsorbent materials with encapsulation materials to immobilize degradable strains is a novel research direction. Adsorbent materials adsorb pollutants, while encapsulation materials protect and enhance the adaptability of degrading strains. These two materials have a synergistic effect. Biochar possesses high porosity and surface area, strong adsorption capacity, and can simultaneously enrich large amounts of pollutants and microorganisms. It is currently the most commonly used natural adsorbent material (Lawal et al. 2019; Patel et al. 2021; Rajapaksha et al. 2015; Vithanage et al. 2014). Attapulgite (ATP), also known as palygorskite, is a clay mineral containing hydrous magnesium silicate with a predominantly layered chain-like transitional structure, belonging to the sepiolite family (Dai et al., 2022; Zghair et al., 2022). ATP exhibits excellent adsorption and carrier properties due to its unique rod-shaped crystal morphology and porous structure. Hu et al. (2022) prepared biochar adsorbents doped with attapulgite (ATP / BC) via calcination. Compared to unmodified biochar, ATP / BC showed an increase in specific surface area of ​​73.53–131.26% and a decrease in average pore size of 1.77–3.60 nm. The degradation rates of SD and SMZ increased by 20.31% and 23.26%, respectively. This demonstrates that ATP possesses a strong adsorption capacity for pollutants. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for preparing a phoxim-degrading strain; The second objective of this invention is to provide a method for preparing attapulgite-immobilized phoxim-degrading bacterial balls; A third objective of this invention is to provide phoxim-degrading strains or phoxim-degrading bacterial balls for the application of degrading phoxim-contaminated wastewater.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a phoxim-degrading strain includes the following steps: S1. Selection of rhubarb rhizosphere soil: Select soil samples from the rhizosphere of Rheum palmatum contaminated with phorate and sieve them. S2. Isolation and purification of bacteria: Weigh the soil sample selected in S1, add sterile water and shake. Dilute the resulting soil suspension and inoculate it onto LB agar medium. Incubate upside down at 28℃ for 5 days. Streak the grown colonies to isolate and purify them to obtain pure culture. Store the culture at 4℃. S3, initial screening of phoxim-degrading bacteria: The bacterial strain obtained from S2 was inoculated onto LB liquid medium and cultured with shaking at 28°C to obtain a bacterial suspension. When the OD600 reached 0.6, the bacterial suspension was collected by centrifugation at 4°C. The bacterial cells were washed with sterile water and resuspended to obtain a bacterial suspension. Sterile Oxford cups were placed on the surface of solid selective medium, and 100 µL of bacterial suspension was injected into the cups. The suspensions were then cultured at 28°C in the dark for 24 hours. After the liquid in the Oxford cups had evaporated naturally, the Oxford cups were removed and cultured at 28°C in the dark for 2-4 days. The diameter of the degradation zone was measured, and the strains with the largest and second largest degradation zone diameters were selected for secondary screening. S4. Secondary screening of phoxim-degrading bacteria: The strain obtained from S3 activation was used to prepare a bacterial suspension using the same method as S3. The bacterial suspension was inoculated into MSM liquid medium at a volume ratio of 3% and cultured at 27 ℃ in the dark with shaking for 72 h to obtain a phoxim-degrading strain. The phoxim-degrading strain was *Sphingosine Monoclonalella*. Novosphingobium The gene sp.), named RL4, was deposited on December 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 29226. Its sequence is shown in SEQ ID No. 1.

[0007] To further realize the present invention, the soil sample weighed in S2 is 1 g, 100 mL of sterile water is added and shaken for 30 min, the soil suspension is prepared according to the 10-fold dilution method, and the inoculation amount is 0.1 mL.

[0008] To further realize the present invention, the oscillation rate in S3 is 1500 rpm, and the bacterial cells are collected by centrifugation at 8000 rpm for 5 min.

[0009] To further realize the present invention, the oscillation rate in S4 is 1500 rpm.

[0010] To further realize the present invention, the formulation of the solid selective culture medium in S3 is as follows: glucose 10 g / L, NH4SO4 0.5 g / L, MgSO4·7H2O g / L, NaCl 0.3 g / L, KCl 0.3 g / L, FeSO4·7H2O 0.03 g / L, MnSO4·4H2O 0.03 g / L, CaCO3 1.0 g / L and phoxim 20 mg / L.

[0011] To further realize the present invention, the formulation of the MSM liquid culture medium in S4 is NH4SO4 2.0 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.01 g / L, FeSO4·7H2O 0.001 g / L, KH2PO4 0.3 g / L, K2HPO4 1.32 g / L and NaCl 1.0 g / L.

[0012] A method for preparing phoxim-degrading bacterial balls using phoxim-degrading strains includes the following steps: S1. Microbial culture: RL4 was inoculated into LB medium and cultured at 28℃ and 150 rpm for 24 h. The cells were obtained by centrifugation and washed with sterile water to prepare a bacterial suspension with an OD600 of 0.6. S2. Preparation of attapulgite-immobilized bacterial balls: The bacterial suspension obtained from S1 was incubated in LB medium at 28°C. When the OD600 reached 0.8, the bacterial cells were collected by centrifugation, washed three times with sterile water, and then inoculated with fresh bacterial cells in sodium alginate solution. After thorough mixing with or without attapulgite, the mixture was added dropwise to CaCl2 solution and cross-linked at 4°C for 12 h to obtain attapulgite-immobilized phoxim-degrading bacterial balls.

[0013] To further realize the present invention, the bacterial cells collected by centrifugation in S2 are centrifuged at 6000 rpm for 5 min.

[0014] To further realize the present invention, the concentration of sodium alginate solution in S2 is 3%, and the mass concentration of fresh bacterial cells inoculated is 3%.

[0015] To further realize the present invention, the amount of attapulgite added in S2 is 1g.

[0016] An application of a phoxim-degrading strain or phoxim-degrading bacterial pellets for degrading phoxim-contaminated wastewater.

[0017] The advantages of this invention compared to the prior art are as follows: The highly efficient phoxim-degrading strain Neosphingomonas in this invention ( Novosphingobium sp .)RL4 is the first report of its kind in China.

[0018] The highly efficient phoxim-degrading strain Neosphingomonas in this invention ( Novosphingobium After sp. RL4 was immobilized with sodium alginate and attapulgite, the degradation rate of 72H phoxim was 87.70%, which was 1.43% higher than that of free bacteria.

[0019] The highly efficient phoxim-degrading strain Neosphingomonas in this invention ( NovosphingobiumAfter being immobilized with sodium alginate and attapulgite, sp. RL4 achieved a 72H phoxim removal rate of 77% in phoxim-contaminated wastewater, which was 12.4% higher than that achieved using free bacteria.

[0020] The highly efficient phoxim-degrading strain Neosphingomonas in this invention ( Novosphingobium After being immobilized with sodium alginate and attapulgite, sp. RL4 still maintained 80% of its phoxim degradation activity after being reused 5 times, indicating that attapulgite-immobilized phoxim-degrading bacterial balls SA+ATP-RL4 are feasible for in-situ phoxim remediation. Attached Figure Description

[0021] Figure 1 For the plates for the degradation of phoxim by the strain, (a) Monkina medium without added phosphorus source, (b) Monkina medium with phoxim as the sole phosphorus source. Figure 2 For strain RL4, (a) a typical colony of strain RL4 grown on LB agar blood culture plane, (b) an optical microscopic image of Gram-stained cells of Novosymgobium sp. RL4, (c) a scanning electron microscopic image of strain RL4, and (d) a phylogenetic tree of 16S rRNA genes of strain RL4 and related bacteria, constructed using the neighbor-joining method in MEGA 7.0 software; Figure 3 The viability of SA / SA+ATP / SA+BC immobilized bacteria during storage; Figure 4 The immobilized strains were (a) SA+ATP-RL4, (b) SA-RL, and (c) SA+BC-RL4. Figure 5 Mass transfer properties and mechanical strength of three types of immobilized beads, (a) mass transfer properties, (b) mechanical strength; Figure 6 SEM analysis of three types of immobilized beads: (ac)SA-RL4, (df)SK-RL4, and (gi)SP-RL4; Figure 7 EDS analysis of three immobilized beads: (a) SA-RL4, (b) SA+ATP-RL4 and (c) SA+BC-RL4; Figure 8 Adsorption equilibrium of 20 mg / L phoxim on three strain-free RL4 immobilized beads; Figure 9 The degradation kinetics of RL4 (A), SA-RL4 (B), SA+ATP-RL4 (C) and SA+BC-RL4 (D) on 20 mg / L phoxim; Figure 10To improve the reusability of SA+AT-RL4 for the degradation of phoxim; Figure 11 The degradation effects of RL4 and SA+AT-RL4 on wastewater. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0023] A method for preparing a phoxim-degrading strain includes the following steps: S1. Selection of rhubarb rhizosphere soil: Select soil samples from the rhizosphere of Rheum palmatum contaminated with phorate and sieve them. S2. Isolation and purification of bacteria: Weigh 1g of soil sample selected from S1, add 100mL of sterile water and shake for 30min. Dilute the resulting soil suspension by a 10-fold dilution method and inoculate it onto LB agar medium with an inoculation amount of 0.1mL. Incubate upside down at 28℃ for 5 days. Strand the grown colonies to isolate and purify them to obtain pure culture strains. Store them in a refrigerator at 4℃. S3, initial screening of phoxim-degrading bacteria: The bacterial strain obtained from S2 was inoculated onto LB liquid medium and cultured with shaking at 1500 rpm at 28°C to obtain a bacterial suspension. When the OD600 reached 0.6, the bacterial suspension was collected by centrifugation at 8000 rpm for 5 min at 4°C. The bacterial cells were washed with sterile water and resuspended to obtain a bacterial suspension. Sterile Oxford cups were placed on the surface of solid selective medium, and 100 µL of bacterial suspension was injected into the cups. The suspensions were cultured at 28°C in the dark for 24 h. After the liquid in the Oxford cups evaporated naturally, the Oxford cups were removed and cultured at 28°C in the dark for 2-4 days. The diameter of the degradation zone was measured, and the strains with the largest and second largest degradation zone diameters were selected for secondary screening. S4. Secondary screening of phoxim-degrading bacteria: The strain obtained from S3 activation was used to prepare a bacterial suspension using the same method as S3. The bacterial suspension was inoculated into MSM liquid medium at a volume ratio of 3%, and cultured at 27 ℃ in the dark with shaking at 1500 rpm for 72 h to obtain a phoxim-degrading strain. The phoxim-degrading strain was *Sphingosine Monoclonalella*. Novosphingobium The gene sp.), named RL4, was deposited on December 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 29226. Its sequence is shown in SEQ ID No. 1.

[0024] The solid selective medium in S3 was formulated as follows: glucose 10 g / L, NH4SO4 0.5 g / L, MgSO4·7H2O g / L, NaCl 0.3 g / L, KCl 0.3 g / L, FeSO4·7H2O 0.03 g / L, MnSO4·4H2O 0.03 g / L, CaCO3 1.0 g / L, and phoxim 20 mg / L.

[0025] The formulation of the MSM liquid medium in S4 is as follows: NH4SO4 2.0 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.01 g / L, FeSO4·7H2O 0.001 g / L, KH2PO4 0.3 g / L, K2HPO4 1.32 g / L and NaCl 1.0 g / L.

[0026] A method for preparing attapulgite-immobilized attapulgite-degrading bacterial balls using phoxim-degrading strains includes the following steps: S1. Microbial culture: RL4 was inoculated into LB medium and cultured at 28℃ and 150 rpm for 24 h. The cells were obtained by centrifugation and washed with sterile water to prepare a bacterial suspension with an OD600 of 0.6. S2. Preparation of attapulgite-immobilized bacterial balls: The bacterial suspension obtained from S1 was incubated in LB medium at 28°C. When the OD600 reached 0.8, the bacterial cells were collected by centrifugation at 6000 rpm for 5 min. After washing three times with sterile water, 3% fresh bacterial cells were inoculated into a 3% sodium alginate solution. 1 g of attapulgite was added and mixed thoroughly. The mixture was then added dropwise to CaCl2 solution and cross-linked at 4°C for 12 h to obtain attapulgite-immobilized phoxim-degrading bacterial balls.

[0027] Experimental Example 1: I. Materials Phosphate (99.5% purity) standard was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Attapulgite clay carrier suspension powder used in this study was provided by [Company Name], screened through a 300-mesh sieve, and stored at pH 8.9. Biochar was prepared by pyrolysis of wheat straw at 700°C for 2 hours under limited oxygen conditions. Before use in this study, the used biochar was pulverized, screened (300 mesh), washed, and dried. The basic properties of the biochar were: pH 9.46, organic carbon content 42.21%, total nitrogen content 8.34%, total phosphorus content 2.31%, total potassium content 16.12%, and ash content 7.23%.

[0028] Inorganic salt medium (MSM) contains 1.32 g K₂HPO₄·3H₂O, 0.3 g KH₂PO₄, 0.1 g MgSO₄·7H₂O, and 1.0 g / L NaCl, with a pH of 7.0. Luria Bertani (LB) medium contains 10.0 g / L tryptone, 5.0 g yeast extract, and 10.0 g / L NaCl, with a pH of 7.2.

[0029] II. Microbial strains and culture conditions RL4 was isolated from the rhizosphere soil of *Rheum palmatum* contaminated with phorate in Huating County, Gansu Province. The isolated strain RL4 was able to grow on LB, nutrient broth, nutrient agar (NA), and MSM medium with phorate as the sole carbon source. It was also found that the strain could grow stably on Monkina medium with phorate as the sole phosphorus source and exhibited a distinct phosphate-solubilizing zone. Figure 1 ).

[0030] The optimal growth conditions for strain RL4 in LB medium were 28°C and pH 7. Cells of strain RL4 were cultured and activated in LB medium at 28°C and 180 rpm for 48 hours. Cell particles of strain RL4 were then obtained by centrifugation at 5000 rpm for 10 minutes and washed twice with sterile aqueous solution for later use. Individual colonies were approximately 2 mm in diameter, moist, yellow, round, raised, and with smooth edges. Figure 2 a). Strain RL4 is a Gram-negative, short rod-shaped bacterium without pores or flagella ( Figure 2 b), the cells are approximately 1.5 µm long and 0.8 µm wide. Figure 2 c). The growth temperature was 20–40 °C, the pH was 5–8, and the NaCl concentration was 0–75 g L−1. The optimal growth period was 33.5–37 °C, pH 7.0, and 10 g L−1 NaCl, as shown in Table 1, supplementing the biochemical characteristics of strain YWX-1. Furthermore, based on the 16S rRNA sequence, strain RL4 showed the highest pairwise similarity to *Sphingosine Monoclonalella* (MZ825298.1). Figure 2 d). Therefore, the isolated strain RL4 was identified as a strain of neosphingosine, namely *Neosphingosinemonas*. Novosphingobium The gene sp.), named RL4, was deposited on December 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 29226.

[0031] 3. Immobilization of strain RL4 (1) Method Cells without RL4 were incubated in LB medium at 28°C. When OD... 600When the concentration reached 0.8, the cells were harvested by centrifugation at 6000 rpm for 5 minutes. Strains RL4 were immobilized using single or combined SA adsorbents (ATP / BC). (1) SA-RL4: Solution A contained 2% (w / v) wetless cells of strain RL4 and 3% (w / v) SA. (2) SA+ATP-RL4: Solution B contained 2% (w / v) wetless cells of strain RL4, 1% (w / v, ATP), and 3% (w / v, SA). SA+ATP-RL4 immobilized beads were obtained by dripping solution B into a 4% CaCl2 (w / v, CaCl2) solution using a syringe needle. (C) SA+BC-RL4: Solution C contained 2% (w / v) wetless cells of strain RL4, 1% (w / v) BC, and 3% (w / v) SA.

[0032] (2) Survival ability of immobilized bacteria during storage The viable cell numbers of neosphingosine RL4 embedded in SA / SA+ATP / SA+BC were 8.58, 8.69, and 8.81 log, respectively. 10 CFU·g -1 Immobilized bacteria were stored at 4°C, and their viability was monitored weekly after storage at 4°C. The number of viable bacteria embedded in the three immobilization methods decreased over time. The viable cell counts of RL4 encapsulated by SA, SA+ATP, and SA+BC at weeks 2 and 4 of storage were 7.99, 8.05, 8.13 and 6.50, 6.83, 6.66 log, respectively. 10 CFU·g -1 After 28 days of storage, the percentage reduction in RL4 cell count embedded in SA, SA+ATP, and SA+BC materials was 24.3%, 22.5%, and 23.3%, respectively. The immobilized bacteria embedded in these three materials exhibited good survival (over 75%) after 28 days of storage at 4°C, likely because the low temperature reduced microbial metabolism, leading to high survival. These results indicate that SA, SA+ATP, and SA+BC are all suitable bacterial immobilization carriers because the number of viable cells in the immobilized bacterial cells does not decrease significantly after storage (see...). Figure 3 ).

[0033] 4. Characterization of immobilized beads The functional group changes of strain RL4 and the support material during immobilization were recorded using a Fourier transform infrared (FTIR) spectrometer (INVENIO S, Brooke, Germany). Strain RL4 and four types of immobilized beads (SA-RL4, SA+ATP-RL4, and SA+BC-RL4) were freeze-dried and ground into powders. 1 mg of the powder sample was mixed with 100 mg of KBr, ground together, and then compressed into pellets. The obtained spectral range was 4000–400 cm⁻¹. -1 .

[0034] The surface area of ​​the immobilized beads was measured using a Brunauer-Emmett-Teller (BET, BELSORP mini II, MicrotracBEL, Japan) instrument. The freeze-dried immobilized bacterial preparation was placed in test tubes and degassed under vacuum at 120°C for 4 hours. Specific surface area and pore size distribution were calculated by measuring the N2 adsorption-desorption isotherm of the samples at 77 K using a BET instrument.

[0035] The binding of strain RL4 to the carrier material and the bead structure were observed using cold field emission scanning electron microscopy (SEM, Tescan CLARA, Czech Republic). The prepared lyophilized immobilized bead samples were cut in half and sprayed with Pt for 60 seconds. The energy dispersive X-ray spectroscopy (EDS, Oxford Xplore, UK) was used to measure the energy dispersive X-ray spectroscopy using a surface scanning model. All negative controls were based on the same immobilized material and did not contain strain RL4.

[0036] The mass transfer and mechanical strength of the immobilized beads were measured according to previous reports (Chen and Lin 2007; Kim et al. 2017). 1 g of immobilized beads was added to an 800 mg / L methylene blue solution and shaken at 100 rpm for 3 hours. The change in absorbance of methylene blue at 665 nm represented the mass transfer performance. Aliquots of 100 immobilized beads were placed in empty 250 mL flasks and shaken at 300 rpm for 48 hours. The rate of undisturbed beads represented the mechanical strength.

[0037] The prepared SA-RL4, SA+ATP-RL4 and SA+BC-RL4 immobilized beads are as follows Figure 4 As shown. The diameters of the three immobilized beads ranged from 3.5 to 5.0 mm. The mass transfer performance was 0.56–0.58 (…). Figure 5 a) SA+BC-RL4 has the best mechanical strength ( Figure 5 b).

[0038] SEM images of the internal and surface structures of the beads and the growth characteristics of bacteria within the beads, such as... Figure 6 As shown. Figure 6The results show that the addition of attapulgite and biochar improved the surface formation and support of the bacterial spheres (after freeze-drying), with biochar showing the best effect, resulting in dense spherical shapes. The cross-section of the spheres is shown below. Figure 6 As shown in the diagram, the beads exhibit a honeycomb structure and cavities within, providing ample space for microbial adhesion and proliferation. The observed honeycomb structure is a regular channel structure, similar to the bead-like structures reported in previous studies. Compared to the control, the addition of biochar and ATP resulted in a larger internal space. The scanning electron microscopy results are consistent with those of Bin Wang et al. (Wang et al., 2019), where bacteria colonized the biochar in the form of cell aggregates, indicating that microbial cells adhere to the material surface in a biofilm-like manner. Based on the encapsulation efficiency and 28-day strain survival rate, it can be seen that all three encapsulation materials enabled high strain density and maintained their biological activity.

[0039] The microstructure and energy dispersive spectra of the three types of immobilized beads are as follows: Figure 7 As shown, the addition of ATP and BC significantly altered the types and proportions of elements. Compared to SA, SA+BC added Fe and Si elements, increasing the proportion of C. Compared to SA, SA+ATP added Fe, Mg, and Si, increasing the proportion of Al. This is consistent with the elements already present in ATP and BC. Compared to CK, the proportion of C in SA+ATP decreased, while the proportion of oxygen increased, which is the opposite of the changes in SA+BC.

[0040] BET data further illustrate the structural differences of these immobilized beads (Table 1). Compared to SA-RL4, the addition of BC and ATP increased the specific surface area by 3.1-fold and 3.16-fold, respectively.

[0041] 5. Degradation of phoxim by immobilized bacterial pellets The degradation kinetics of phoxim were measured in 20 mL tubes containing 20 mg / L phoxim and 1 g immobilized beads. All samples were incubated in a rotating shaker at 28°C and 150 rpm, with sampling every 2 hours. The reaction was terminated by adding 20 mL of acetonitrile. All samples were in triplicate. The residual amount of phoxim in the samples was determined by Agilent high-performance liquid chromatography (HPLC) using an Agilent HPLC Zorbax C18 column (2.1 × 100 mm, 1.7 μm). The injection volume was 10 μL. The mobile phase consisted of 20% water (A) and 80% acetonitrile (B). Phoxim was separated at 30°C using a gradient program, as previously described, with a detection wavelength of 205 nm.

[0042] The degradation of 20 mg / L phoxim by three types of immobilized beads, as shown in the figure Figure 9As shown, 1 g of immobilized microbeads (containing ~OD600=0.03 free cells) can degrade 86.76%-88.39% of 20 mg / L phoxim within 72 h. The data conform to first-order kinetics, and the R values ​​for RL4, SA-RL4, SA+ATP-RL4, and SA+BC-RL4 are shown. 2 The values ​​were 0.95, 0.80, 0.98, and 0.98 h, respectively. Compared with the SEM and FTIR results, SA+ATP-RL4 immobilized beads not only enhanced the binding ability with strain RL4 but also improved the degradation performance.

[0043] 6. Adsorption kinetics of phorate through carrier materials The adsorption kinetics of phoxim were measured by adding 1 g of immobilized bacterial pellets (SA, SA+ATP, and SA+BC) to 20 mL of 20 mg / L phoxim solution. All samples were incubated in a rotary shaker at 25°C and 150 rpm, with periodic sampling until adsorption equilibrium was reached. Phoxim residues were determined by high-performance liquid chromatography (HPLC). All samples were prepared in triplicate.

[0044] The adsorption isotherm equation (1) is used to calculate the adsorption capacity (QCP) of phorate on three carrier materials.

[0045] (1) Where C0 and C T These are the initial and final concentrations of phoxim, respectively. V is the volume, and m is the mass of the immobilized beads.

[0046] SEM images showed that the immobilized beads contained numerous pores, which not only enhanced the colonization of the degrading strains but also improved the adsorption capacity of the pesticide. The adsorption of x phoxim by three types of immobilized beads was measured at 25°C. The adsorption curves fit well with the Langmuir equation, R0. 2 The values ​​were 0.97 (SA), 0.98 (SA+BC), and 0.95 (SA+ATP), respectively. Figure 8 The maximum adsorption capacities of SA, SA+BC, and SA+ATP were 0.089, 0.113, and 0.124 mg / g, respectively.

[0047] 7. Reusability and stability of SA+ATP-RL4 1 g of immobilized SA+ATP-RL4 beads were added to a 20 mg / L phoxim solution and incubated at 28°C in a rotary shaker at 170 rpm for 24 hours. The immobilized beads were recovered and prepared into 20 mL of MSM containing 20 mg / L phoxim. This experiment was repeated until the degradation rate of the immobilized beads was less than 80%.

[0048] After being immobilized on SA+ATP, RL4 retained 80% of its phoxim degradation activity even after 5 repeated uses. Figure 10 This indicates that the immobilized strain SA+ATP-RL4 is feasible for in-situ remediation of phoxim (Shen et al. 2020). The viable cell numbers of neosphingosine RL4 embedded in SA / SA+ATP / SA+BC were 8.58, 8.69, and 8.81 log, respectively. 10 CFU·g -1 Immobilized bacteria were stored at 4°C, and their viability was monitored weekly after storage at 4°C. The number of viable bacteria embedded in the three immobilization methods decreased over time. The viable cell counts of RL4 encapsulated by SA, SA+ATP, and SA+BC at weeks 2 and 4 of storage were 7.99, 8.05, 8.13 and 6.50, 6.83, 6.66 log, respectively. 10 CFU·g -1 After 28 days of storage, the percentage reduction in the number of RL4 cells embedded in SA, SA+ATP, and SA+BC materials was 24.3%, 22.5%, and 23.3%, respectively. The immobilized bacteria embedded in these three materials exhibited good survival (over 75% survival rate) after 28 days of storage at 4°C, which is likely due to the reduced microbial metabolism at low temperatures, leading to high survival rates. Our results indicate that SA, SA+ATP, and SA+BC are all suitable bacterial immobilization carriers because the number of viable cells in the immobilized bacterial cells does not decrease significantly after storage.

[0049] 8. The degradation effects of RL4 and SA+AT-RL4 on wastewater The actual phoxim-contaminated wastewater was collected from a ditch next to a rhubarb planting base in Huating, Gansu, China. The pH of the wastewater was adjusted to 7.5 to eliminate the influence of pH on phoxim hydrolysis. The phoxim degradation rate was measured over 72 hours in a 20 mL Erlenmeyer flask containing approximately 10 mg / L phoxim and 10⁶ (CFU, approximately 1 g immobilized beads) of free RL4 cells or 1 g of attapulgite-immobilized RL4 cells in the wastewater. All samples were in triplicate and incubated at 28°C on a rotating shaker at 150 rpm, with sampling every 72 hours. Phoxim residues were determined by HPLC.

[0050] In polluted water, analysis of the degradation rate of phoxim after 72 hours revealed that the degradation rate in free cells was only 37.96%, while the degradation rate of SA+ATP-RL4 reached 55.11%, indicating that SA+ATP-RL4 exhibits better tolerance in wastewater. Figure 11 ) 9. HPLC method for determining phorate content The residual levels of organophosphorus pesticides in the samples were determined by Agilent high-performance liquid chromatography (HPLC) using an Agilent HPLC Zorbax C18 column (2.1 × 100 mm, 1.7 μm). The injection volume was 10 μL. The mobile phase consisted of 20% water (A) and 80% acetonitrile (B). Phoxim was separated at 30°C using a d-isocratic program, as previously described, with a detection wavelength of 205 nm.

[0051] Strain sequence SEQ ID No. 1:

Claims

1. A method for preparing phoxim-degrading bacterial balls, characterized in that, Includes the following steps: S1. Microbial culture: Neosphingosomalidone ( Novosphingobium sp.) RL4 was inoculated into LB medium and cultured at 28℃ and 150 rpm for 24 h. The cells were obtained by centrifugation at 6000 rpm for 5 min. The cells were washed with sterile water and then prepared into a bacterial suspension with an OD600 of 0.

6. S2. Preparation of attapulgite-immobilized bacterial balls: The bacterial suspension obtained from S1 was incubated in LB medium at 28°C until OD 600 When the concentration reaches 0.8, the bacterial cells are collected by centrifugation, washed three times with sterile water, and then fresh cells of Neosphingomonas RL4 are inoculated into sodium alginate solution. After thorough mixing with or without attapulgite, the mixture is added dropwise to CaCl2 solution and cross-linked at 4°C for 12 h to obtain attapulgite-immobilized phoxim-degrading bacterial balls. The concentration of the sodium alginate solution was 3%, and the mass concentration of the fresh cells inoculated with *Sphingosine Monoclonalella* RL4 was 3%. The amount of the attapulgite added is 1g.

2. The application of the novel sphingosomalidobacterium RL4 as described in claim 1 in the degradation of phoxim wastewater.

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