Pseudomonas sp. and its application in degrading xanthate in combination with veitchia merrillii

Abstract

The application discloses a kind of pseudomonas and its application in combined degradation of xanthate of horsetail, the pseudomonas (Pseudomonas sp.) W50 is preserved in Guangdong Microbial Culture Collection Center on July 31, 2024, and the preservation number is: GDMCC NO.64924. Its 9h degradation rate to 100mg / L BuX is as high as 99%, and it also has a variety of growth-promoting properties, including phosphorus dissolution, potassium release, IAA and GA secretion and cellulose degradation. It can be used for repairing BuX contaminated wastewater, and can play a synergistic effect when combined with horsetail, and the repair effect is more significant, providing a new method for treating BuX pollution and promoting the healthy growth of aquatic plants in mining area, and has broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a Pseudomonas bacterium and its application in the degradation of xanthate by *Gnaphalium affine*. Background Technology

[0002] Xanthate, also known as xanthate (BuX), is a yellow, powdery solid with a pungent odor and is toxic. It is flammable, easily deliquescent, and unstable, decomposing rapidly in the presence of salt. It is readily soluble in water, acetone, and alcohols. It is commonly used as a flotation reagent for various non-ferrous metals, exhibiting high mineral selectivity and cost-effectiveness. Currently, butyl xanthate (BuX) remains the most produced and used collector in the flotation of sulfides and some oxidized minerals. A significant amount of flotation reagents, including BuX, inevitably remain in mineral processing wastewater.

[0003] Bux is a toxic and harmful organic compound that can have toxic effects on aquatic life and human health even at low concentrations. Specifically, Bux can cause growth inhibition, teratogenicity, and even death of aquatic microorganisms, plants, and animals in water bodies. In this context, discharging Bux-containing flotation wastewater into natural waterways appears destined to lead to devastating consequences, such as water loss, soil erosion, and desertification of mining ecosystems. Therefore, the effective treatment of residual Bux in flotation wastewater and the restoration of vegetation in mining areas are crucial for the ecological sustainability of the mining industry.

[0004] Currently, numerous methods exist for removing xanthates from flotation wastewater, including photocatalytic degradation, adsorption, and chemical oxidation. The main drawbacks of these physical and chemical methods are low reusability, high operating costs, and secondary pollution. In contrast, microbial degradation has garnered more attention due to its simplicity, cost-effectiveness, and environmental friendliness, and has been widely used to remove various organic pollutants. However, reports on microbial degradation of xanthates are very limited. This may be attributed to the toxic effects of xanthates on microorganisms, inhibiting their growth and enzyme activity. Furthermore, factors such as unstable strains, difficulty in colonization, and limited opportunities for contact with pollutants at relatively deep sites also limit the effectiveness of microbial remediation.

[0005] Meanwhile, phytoremediation has emerged as an ecologically safe and sustainable alternative for remediating polluted environments, depending on the tolerance of the selected plants to harmful pollutants. *Cyperus alternifolius*, an emerging aquatic plant, possesses advantageous characteristics such as tolerance to extreme conditions and pollutants, making it a promising candidate for phytoremediation. Numerous studies have demonstrated that *Cyperus alternifolius* can remove various harmful pollutants, such as heavy metals, antibiotics, and steroid hormones. However, mining wastewater typically contains high concentrations of toxic substances. Single-plant phytoremediation often suffers from limitations such as long growth cycles, absorption and degradation capabilities, and insufficient plant resistance, making survival difficult. Therefore, this limits the feasibility of phytoremediation, and single plants are not suitable for remediating heavily polluted environments.

[0006] In conclusion, new remediation methods are needed to overcome the above problems in order to improve the remediation effect of mining wastewater. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Pseudomonas bacterium and its application in the degradation of xanthate by *Gnaphalium affine*.

[0008] The first objective of this invention is to provide a strain of Pseudomonas.

[0009] A second object of the present invention is to provide the use of the aforementioned Pseudomonas in the degradation of xanthates or in the preparation of products that have degraded xanthates.

[0010] A third objective of this invention is to provide the use of the aforementioned Pseudomonas in promoting plant growth or in the preparation of products that promote plant growth.

[0011] A fourth object of the present invention is to provide the use of the aforementioned Pseudomonas in the secretion or production of plant hormones or in the preparation of products that secrete or produce plant hormones.

[0012] A fifth object of the present invention is to provide the use of the aforementioned Pseudomonas in the solubilization of phosphorus and / or potassium, or in the preparation of products that solubilize phosphorus and / or potassium.

[0013] A sixth object of the present invention is to provide the use of the aforementioned Pseudomonas in the degradation of cellulose or in the preparation of cellulose-degraded products.

[0014] The seventh objective of this invention is to provide a method for treating xanthate pollution.

[0015] The eighth object of the present invention is to provide a microbial agent.

[0016] The ninth objective of this invention is to provide a method for mitigating the effects of xanthate pollution on plant growth.

[0017] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0018] This invention claims protection for a strain of Pseudomonas sp. W50, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 31, 2024, with accession number GDMCC NO.64924.

[0019] Further protection is required for the following applications:

[0020] The application of the aforementioned Pseudomonas in the degradation of xanthate or in the preparation of products degraded with xanthate.

[0021] The application of the aforementioned Pseudomonas bacteria in promoting plant growth or in the preparation of products that promote plant growth.

[0022] Preferably, the plant is *Gnaphalium affine*.

[0023] The use of the aforementioned Pseudomonas in the secretion or production of plant hormones or in the preparation of products that secrete or produce plant hormones, wherein the plant hormone is at least one of GA or IAA.

[0024] The use of the aforementioned Pseudomonas in the solubilization of phosphorus and / or potassium, or in the preparation of products that solubilize phosphorus and / or potassium.

[0025] The potassium dissolution process involves converting non-water-soluble potassium salts (mineral potassium) into water-soluble potassium; the phosphorus dissolution process involves converting non-water-soluble phosphorus salts (mineral phosphorus) into water-soluble phosphorus.

[0026] The application of the aforementioned Pseudomonas in the degradation of cellulose or in the preparation of cellulose-degraded products.

[0027] The present invention also claims a method for treating xanthate contamination using the aforementioned Pseudomonas bacteria.

[0028] Preferably, the *Pseudomonas* is used in conjunction with *Gnaphalium affine*.

[0029] As a specific implementation method, the aforementioned Pseudomonas bacteria are added to wastewater that requires xanthate pollution treatment, and the wastewater is used to grow windmill grass.

[0030] The present invention also claims protection for a bacterial agent containing the aforementioned Pseudomonas.

[0031] This invention also claims a method for mitigating the effects of xanthate pollution on plant growth by adding the aforementioned Pseudomonas bacteria to the water in the plant's growing environment.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] This invention provides a strain of Pseudomonas sp. W50 with highly efficient Bux degradation function, achieving a degradation rate of up to 99% for 100 mg / L Bux in 9 hours. It also possesses various growth-promoting properties, including phosphorus solubilization, potassium solubilization, secretion of IAA and GA, and cellulose degradation. It can be used to remediate Bux-contaminated wastewater, and its synergistic effect when used in combination with *Gnaphalium affine* further enhances the remediation effect. This invention provides a new method for treating Bux pollution and promoting the healthy growth of aquatic plants in mining areas, and has broad application prospects. Attached Figure Description

[0034] Figure 1 Phylogenetic tree of Pseudomonas W50: morphological characteristics and gene sequence; where A is an image of Pseudomonas W50 colony morphology on LB agar; B is a scanning electron microscope (10000x) image of Pseudomonas W50; and C is the phylogenetic tree of Pseudomonas W50.

[0035] Figure 2 To assess the tolerance of Pseudomonas W50 to different pH levels.

[0036] Figure 3 To assess the temperature tolerance of Pseudomonas W50.

[0037] Figure 4 To assess the tolerance of Pseudomonas W50 to different initial concentrations of BuX.

[0038] Figure 5 UV-Vis spectra of BuX at different reaction times and BuX biodegradation pathways.

[0039] Figure 6 A represents the phosphate solubility of Pseudomonas W50 under different culture times and carbon, nitrogen, and phosphorus sources; B represents the phosphate content of strain W50 under different culture times; C represents the phosphate content of strain W50 under different carbon sources; D represents the phosphate content of strain W50 under different phosphorus sources; E represents the ability of strain W50 to secrete GA and IAA; and F represents the potassium solubilization and cellulose degradation characteristics of strain W50.

[0040] Figure 7 A) Tolerance of *Gnaphalium affine* to different concentrations of BuX; B) Phenotypic diagram of tolerance of *Gnaphalium affine* to BuX concentrations of 0, 50, 100, 300, and 500 mg / L; C) Effect of different concentrations of BuX on the fresh weight of *Gnaphalium affine*; D) Effect of different concentrations of BuX on the plant height of *Gnaphalium affine*; E) Degradation efficiency of *Gnaphalium affine* to different concentrations of BuX.

[0041] Figure 8The results of the co-remediation of BuX-contaminated wastewater by *Gnaphalium affine* and *Pseudomonas sp.* W50 are shown. A represents plant phenotype; B represents BuX degradation efficiency; C represents fresh weight; D represents plant height; and E represents root length.

[0042] Figure 9 The effects of single-bacterial remediation (W50), phytoremediation (Gynostemma pentaphyllum), and phyto-microbial combined remediation (Gynostemma pentaphyllum-W50) on the water quality purification of BuX-contaminated wastewater were evaluated using chemical oxygen demand (COD). A represents the COD content of 50 mg / L BuX-contaminated xanthate wastewater after treatment with Blank, BuX+W50, BuX+Ca, and BuX+Ca+W50, respectively. B represents the colonization of W50 inoculated bacteria on or within the roots of Gynostemma pentaphyllum exposed to BuX under a fluorescence microscope. C represents the bacterial population density at different locations on Gynostemma pentaphyllum. Detailed Implementation

[0043] 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.

[0044] Butyl xanthate (BuX) was purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai), with a purity >95%; chromatographic grade methanol was purchased from Sigma-Aldrich, USA; hydrochloric acid was purchased from Guangzhou Chemical Reagent Co., Ltd., with an analytical purity; all other reagents were analytical grade and purchased from Tianjin Damao Chemical Reagent Co., Ltd.

[0045] Liquid basic salt medium (MSM): K2HPO4 1.5 g / L, KH2PO4 0.5 g / L, (NH4)2SO4 0.5 g / L, MgSO4·7H2O 0.2 g / L and NaCl 1.0 g / L, pH value approximately 7.0.

[0046] LB broth medium: 10 g / L peptone, 5 g / L yeast extract and 5 g / L sodium chloride, pH approximately 7.0.

[0047] Nutrient agar medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 5g / L, agar 15g / L, pH value approximately 7.0.

[0048] The degradation rate is calculated as follows: Degradation rate (%) = 100% × (C0 - C1 / C0)

[0049] In the formula, C1 is the residual concentration of BuX after treatment with degrading bacteria, and C0 is the initial concentration of BuX without treatment.

[0050] Example 1: Isolation and Identification of Strains

[0051] I. Enrichment, Isolation and Purification of Strains

[0052] 1. Experimental Methods

[0053] Add BuX to 100 mL of sterilized MSM medium. After cooling, add more BuX to achieve an initial concentration of 10 mg / L. Add 5 g of contaminated soil collected from the Dabao Mountain mining area in Shaoguan, Guangzhou, and incubate in the dark at 35℃ and 150 rpm for 7 days. On day 7, transfer the culture to fresh MSM medium at a 1% inoculum level, gradually increasing the contaminant concentration for acclimatization. Repeat this process 5 times until the BuX concentration reaches 1000 mg / L. Spread the final culture solution evenly on nutrient agar plates and incubate at a constant temperature for 48 hours. Then, repeatedly streak colonies until a single colony is obtained, which is named strain W50.

[0054] Single bacteria were inoculated into MSM medium containing 100 mg / L BuX and cultured on a shaker at 35°C and 150 rpm for 72 h to detect their degradation effect. The strains were then inoculated into test tube slant culture medium and stored at 4°C for later use.

[0055] 2. Experimental Methods

[0056] A strain capable of using BuX as the sole carbon source was screened and named W50, which can completely degrade BuX at an initial concentration of 100 mg / L in a short period of time.

[0057] II. Observing Colony Characteristics

[0058] 1. Experimental Methods

[0059] The selected strain W50 was inoculated onto a nutrient agar plate and incubated overnight at 35°C. The shape, size, transparency, edge, and color of the strain were observed.

[0060] 2. Experimental Results

[0061] Strains of strain W50 were inoculated onto nutrient agar plates and incubated overnight. Colony morphology was then observed. After 3 days of incubation on nutrient agar plates, colonies of strain W50 were round, pale yellow, translucent, smooth, with a raised center, and a viscous texture. Figure 1 A).

[0062] III. SEM observation of bacterial cell morphology

[0063] 1. Experimental Methods

[0064] Strain W50 was inoculated into LB broth and cultured. After 24 hours of culture, 800 μL of the bacterial culture was transferred to a 1.5 mL centrifuge tube and centrifuged at 8000 rpm for 5 min, discarding the supernatant. The resulting bacterial pellet was washed with sterile physiological saline, centrifuged again, and the supernatant was discarded. This process was repeated three times. 1 mL of 2.5% glutaraldehyde electron microscopy fixative was added to the washed bacterial pellet, vortexed, and incubated overnight at 4°C. After fixation, the pellet was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were collected and washed three times with sterile physiological saline. A gradient dehydration process was then performed, involving the addition of 30%, 50%, 70%, 80%, and 90% ethanol once each, and 100% ethanol twice. Each dehydration treatment required 15 min of incubation. After the final dehydration treatment, the bacterial cells were dropped onto coverslips and freeze-dried. The dried samples were then sputter-coated with gold before being analyzed.

[0065] 2. Experimental Results

[0066] SEM analysis revealed that strain W50 was a long rod-shaped bacterium (1.5–3.0 × 0.5 μm) with a relatively smooth surface, straight or slightly curved, and blunt-rounded ends. Figure 1 B).

[0067] IV. Physiological and Biochemical Identification of Strains

[0068] 1. Experimental Methods

[0069] Physiological and biochemical experiments on strain W50 were conducted with reference to the "Handbook of Common Bacterial Identification" and "Bergey's Manual of Determinative Bacteriology".

[0070] 2. Experimental Results

[0071] Gram staining results showed that strain W50 was a Gram-negative bacterium. The methyl red test, nitrate reduction, starch hydrolysis, urease production, hydrogen sulfide, and acetylmethylmethanol test of strain W50 were all negative, while the catalase, oxidation reaction, ornithine decarboxylase, and lysine decarboxylase tests were positive. The strain could not utilize malonate, lactose, or glucose (Table 1).

[0072] Table 1. Physiological and biochemical characteristics of strain W50

[0073]

[0074] V. Molecular biological identification

[0075] 1. Experimental Methods

[0076] PCR amplification: The 16S rDNA fragment of the strain was amplified using the universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3').

[0077] PCR reaction system: Taq Mixture, 12.5 μL; ddH2O, 9.5 μL; 27F, 1 μL; 1492R, 1 μL; DNA template, 1 μL; total reaction volume 25 μL.

[0078] PCR reaction conditions: 95℃, initial preheating for 5 min; melting at 94℃ for 45 s, annealing at 55℃ for 45 s, extension at 72℃ for 1 min 15 s, 32 cycles, and a final hold at 72℃ for 10 min. After PCR amplification, the size and specificity of the amplified fragments were detected by 1.5% agarose gel electrophoresis, and photographs were taken using a gel imaging system. The PCR amplification products were purified, recovered, and sequenced by Shanghai Sangon Biotech Co., Ltd.

[0079] Phylogenetic tree construction: The 16S rDNA sequence of strain W50 was obtained by sequencing from Sangon Biotech and submitted to the GenBank database. BLAST was used for alignment analysis, and ClustalX software was used to perform homology analysis on highly matched sequences. Neighbor-joining analysis in MEGA 11.0 software was used to construct the phylogenetic tree of the strain.

[0080] 2. Experimental Results

[0081] Using the genomic DNA of strain W50 as a template, PCR amplification was performed on it using 16S rDNA bacterial universal primers. The amplified products were sent to Shanghai Sangon Biotech Co., Ltd. for purification, and the product length was found to be 1078 bp.

[0082] Phylogenetic analysis showed that the 16S rDNA of W50 had a 98% homology with *Pseudomonas juntendi* BML3 NR 180457 (Genbank accession number: PP737842), and also showed high homology with other strains in the same genus. Therefore, strain W50 was preliminarily identified as belonging to the genus *Pseudomonas*. Figure 1 C).

[0083] Pseudomonas sp. W50 was deposited on July 31, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO. 64924), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. It is classified as Pseudomonas sp.

[0084] Example 2: Effects of environmental factors on the degradation of BuX by strain W50

[0085] I. Quantitative Analysis Method of Bux in Culture Medium

[0086] 1. Sample pretreatment methods

[0087] Transfer 1 mL of culture medium to a 1.5 mL centrifuge tube and centrifuge at 8000 rpm for 5 min. After centrifugation, take 300 μL of the supernatant and dilute it 10 times. To prevent BuX degradation, the BuX content should be measured immediately using UV-Vis.

[0088] 2. Detection method for Bux

[0089] The content of BuX in the solution can be calculated from the standard curve by measuring the absorbance of the solution at 301 nm (Chen, S., Gong, W., and Mei, G. (2010) Study on Biodegradation of Alkyl Xanthate Collectors. In: 2010 4th International Conference on Bioinformatics and Biomedical Engineering, pp. 1-6.).

[0090] 3. Bux Standard Curve

[0091] Accurately weigh a measured amount of BuX, dissolve it in pure water, and dilute to a final volume of 1000 mg / L in a brown conical flask to obtain a BuX standard stock solution. The standard stock solution is serially diluted to prepare standard curve solutions with concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, and 25.0 mg / L. By measuring the absorbance of each gradient of the standard curve solution at 301 nm, and using this value as Y and the corresponding solution concentration as X, a standard curve "Y = a + bX" is constructed.

[0092] II. Preparation of W50 bacterial suspension

[0093] A single colony of *Pseudomonas* sp. W50 from Example 1 was picked and cultured overnight (180 rpm / min, 24 h) in LB medium. The supernatant was then removed by centrifugation (3500 rpm, 15 min), and the culture was washed three times with 0.9% physiological saline and resuspended to obtain the W50 bacterial suspension. For degradation assays, unless otherwise specified, OD values ​​were used. 600nm The bacterial suspension was prepared at a concentration of 1.0 for subsequent degradation experiments.

[0094] III. Effect of pH on the degradation of BuX by strain W50

[0095] 1. Experimental Methods

[0096] Add 1 mL of W50 bacterial suspension (OD600 = 1.0) to 50 mL of MSM medium containing 100 mg / L BuX. Adjust the initial pH of this degradation system to 5, 6, 7, 8, and 9, respectively, and then incubate at 150 rpm in a 35°C constant temperature shaker for 10 h in the dark. Samples were taken every 2 h to detect the BuX content in the solution. The uninoculated treatment group served as a blank control, and three replicates were set up for each treatment group.

[0097] 2. Experimental Results

[0098] Depend on Figure 2 It can be seen that strain W50 maintained a degradation efficiency of over 86.8% for BuX under different pH conditions (5-9) within 8 hours, and could completely degrade 100 mg / L BuX within 10 hours within a wide pH range (5-9). Strain W50 can tolerate a certain pH range and has strong environmental adaptability in practical applications.

[0099] IV. Effect of Temperature on the Degradation of BuX by Strains W50

[0100] 1. Experimental Methods

[0101] Add BuX to a 100 mL Erlenmeyer flask containing 50 mL of sterile MSM medium to achieve an initial concentration of 100 mg / L. The initial pH of the degradation system is 7.0. Inoculate with W50 bacterial suspension (OD600 = 1.0) at a 2% inoculation rate. Place the Erlenmeyer flasks in shakers at 20, 25, 30, 35, and 40 °C and incubate at 150 rpm in the dark for 24 h. Samples are taken every 3 h to detect the residual BuX content. The uninoculated treatment serves as a blank control, and each group is divided into three replicates.

[0102] 2. Experimental Results

[0103] from Figure 3As can be seen, at temperatures of 35 or 40°C, 100 mg / L BuX can be completely degraded in 9 hours, at 30°C it takes 12 hours, and at 20 or 25°C it takes 24 hours to completely degrade 100 mg / L BuX. W50 can degrade high concentrations of BuX in a short time even at low temperatures, demonstrating significant practical application value.

[0104] V. Effect of initial concentration of substrate BuX on the degradation of BuX by strain W50

[0105] 1. Experimental Methods

[0106] BuX was added to 50 mL of sterile MSM medium to achieve initial concentrations of 100, 300, 500, 700, and 1000 mg / L, respectively, with an initial pH of 7.0. 1 mL of W50 bacterial suspension (OD600 = 1.0) was then inoculated, and the medium was incubated at 35°C and 150 rpm in the dark for 12 h. BuX concentrations were measured at 2-hour intervals. Uninoculated treatments served as blank controls, and each group was divided into three replicates.

[0107] 2. Experimental Results

[0108] The results are as follows Figure 4 The results showed that when the initial concentration of BuX was 100–1000 mg / L, strain W50 achieved a degradation rate of 77.9–100% for different concentrations of BuX in MSM medium within 9 hours, with the optimal concentration being 100 mg / L. Strain W50 was able to completely degrade BuX within 12 hours at initial concentrations of 100–1000 mg / L, indicating that strain W50 can tolerate extremely high concentrations of BuX and can fully utilize BuX as the sole carbon and energy source for growth, without exhibiting a significant lag effect during the degradation process.

[0109] Example 3: Degradation kinetics of BuX by strain W5

[0110] I. Experimental Methods

[0111] Different amounts of BuX were added to 100 mL of sterile MSM medium to achieve final BuX concentrations of 100, 300, 500, 700, and 1000 mg / L. The resulting Pseudomonas sp. W50 bacterial suspension (as described in Example 2) was inoculated and cultured under the optimal conditions determined in Example 2 (temperature 35°C, pH 5, inoculation volume 2 mL (OD600nm = 1.0)) with shaking (150 rpm). Samples were taken at 0, 2, 4, 6, 8, 10, and 12 h, and the residual BuX in each bacterial suspension was determined using UV light. Degradation kinetic curves for different BuX concentrations were plotted. No inoculation was used as a control, and each treatment was performed in triplicate.

[0112] II. Experimental Results

[0113] The results are shown in Table 2. Based on the degradation kinetics and half-life models, the degradation kinetics and half-life of *Pseudomonas sp.* W50 at different initial BuX concentrations were determined. The half-life of *Pseudomonas sp.* W50 for BuX degradation was positively correlated with the substrate concentration. The half-lives for 100, 300, 500, 700, and 1000 mg / L BuX were 3.925 h, 3.977 h, 6.159 h, 6.207 h, and 6.636 h, respectively. *Pseudomonas sp.* W50 exhibited a wide range of initial BuX concentrations for degradation, a short degradation half-life, and high BuX tolerance and degradation efficiency.

[0114] Table 2 Degradation kinetic parameters of BuX at different initial concentrations

[0115]

[0116] Example 4: Intermediates and biomineralization pathways of BuX degradation by strain W5

[0117] I. Experimental Methods

[0118] Determination of BuX characteristic peaks: Prepare a 1 mg / L BuX solution, and then perform a full-band scan using a UV spectrophotometer, with a scanning range of 185–350 nm. Determine the UV wavelength corresponding to BuX based on the characteristic peaks in the UV spectrum.

[0119] The *Pseudomonas* sp. W50 bacterial suspension from Example 2 was inoculated into sterilized MSM medium supplemented with an initial concentration of 100 mg / L BuX. The initial pH of the degradation system was 5, the temperature was 35°C, the inoculation volume was 2 mL (OD600nm = 1.0), and the culture was shaken at 150 rpm. Samples were taken periodically for pretreatment. The same medium containing the same concentration of BuX without inoculation served as a control. Each treatment was repeated in triplicate. The culture was diluted tenfold and scanned using UV-Vis in the wavelength range of 185–350 nm to detect intermediate degradation products of BuX.

[0120] II. Experimental Results

[0121] The UV spectrum showed that the products of BuX degradation by Pseudomonas sp. W50 exhibited absorption peaks at 301 nm, 226 nm, and 206 nm. Specifically, 226 nm and 301 nm are characteristic absorption peaks of BuX, while 206 nm is the characteristic absorption peak of CS2. As the degradation reaction progressed, the intensity of the BuX characteristic peak continuously decreased. The characteristic peak of CS2 briefly appeared after 6 hours of reaction and then disappeared. No other characteristic peaks of possible BuX intermediate products were detected, indicating that CS2 is the main product of BuX biodegradation. Figure 5 ).

[0122] Example 5: Analysis of the plant growth-promoting characteristics of Pseudomonas sp. W50

[0123] I. Experimental Methods

[0124] The obtained W50 strain was tested for plant growth-promoting traits (phosphorus solubilization, gibberellin secretion, IAA secretion, siderophore production, nitrogen fixation, potassium solubilization, and cellulose degradation) using conventional methods, as follows:

[0125] Phosphate-solubilizing activity was assessed using NBRIP medium, following the method described by Mehta and Nautiyal (2001). To quantify phosphate solubility, a W50 suspension was inoculated into NBRIP liquid medium and incubated in the dark for 4 days using a rotary shaker (30°C, 180 rpm). Every 12 hours, the culture was centrifuged at 3500 rpm for 15 minutes, and 1 ml of the supernatant was mixed with 2 ml of molybdenum antimony chromogenic reagent, then diluted to 25 ml with distilled water. After reacting in the dark for 30 minutes, the optical density of the supernatant mixture was measured at 700 nm. Three parallel treatments were set up, with an uninoculated treatment as a control. Furthermore, the effects of carbon, nitrogen, and phosphorus sources on the ability of W50 to solubilize phosphate in the liquid medium were investigated using single-factor control variables.

[0126] Following the method of Xiao et al. (1997), the gibberellin production capacity of strain W50 was determined with slight modifications. Only the bacterial concentration was changed through serial dilutions from 5 to 25 μg / L; all other conditions remained unchanged. Following the protocol proposed by Bric et al. (1991), the Salkowski colorimetric method was used to screen bacterial isolates for IAA production. Bacterial isolates were inoculated into King's B liquid medium containing 0.05 g / L tryptophan and incubated for 24 h. The culture was then centrifuged at 10,000 rpm for 5 min, and 50 μL of supernatant was added along with 100 μL of Salkowski reagent. The color change was observed. The presence of a red color confirmed IAA production. For quantification, the optical density (OD) was recorded at 530 nm, and the IAA content was calculated using an IAA standard curve.

[0127] As described by Schwyn and Neilands (1987), strain W50 was inoculated onto CAS plates and incubated at 30°C for 18–24 h. The appearance of an orange halo on the plate indicated that the strain possessed siderophore-producing activity.

[0128] The method described by Zhang et al. (2023) was used to determine the nitrogen-fixing / potassium-solubilizing ability of the strain. W50 was inoculated into LB broth and vortexed (30°C, 150 rpm, 24 h) to obtain a suspension. 5 μL of this suspension was then inoculated onto Assumption solid medium or potassium-solubilizing solid medium and incubated at 30°C for 3 days. The presence of colonies indicated that the strain possessed nitrogen-fixing or potassium-solubilizing ability.

[0129] The ability to degrade cellulose was detected by using Congo red staining (Li et al., 2016) to form a clear zone around the colonies on CMC-Na agar plates.

[0130] II. Experimental Results

[0131] like Figure 6 As shown, strain W50 exhibits a variety of plant growth-promoting traits, including phosphate solubility, GA secretion, IAA production, potassium solubility, and cellulose degradation, but it does not possess the ability to fix nitrogen or produce siderophores.

[0132] Among them, the phosphate-solubilizing ability of Pseudomonas sp. W50 showed a trend of first increasing and then decreasing over time. At 72 hours, strain W50 reached its highest phosphate-solubilizing ability of 269.5 mg / L. Figure 6 A in the middle.

[0133] The optimal carbon, nitrogen, and phosphorus sources for W50 phosphorus solubility are glucose, peptone, and tricalcium phosphate, respectively. When glucose is used as the sole carbon source, the highest phosphorus solubility can reach 227.93 mg / L. Figure 6 (B in the text) When peptone is used as the sole nitrogen source, the lysed phosphorus content can reach a maximum of 234.68 mg / L, which is 27.97%–922.13% higher than that of other nitrogen sources. Figure 6 (C in the text). Strain W50 prefers tricalcium phosphate as a phosphorus source, with a highest soluble phosphorus content of 246.07 mg / L. Figure 6 (D in the middle).

[0134] like Figure 6 The E values ​​in the data show that the GA and IAA secreted by strain W50 reached their highest values ​​of 93.86 mg / L and 105.21 mg / L at 48 h and 24 h, respectively.

[0135] like Figure 6The F-values ​​in the data indicate that strain W50 possesses various plant growth-promoting characteristics, including potassium solubilization, siderophore production, and cellulose degradation.

[0136] Example 6: Tolerance of *Gnaphalium affine* to different Bux concentrations

[0137] I. Experimental Methods

[0138] After simple sterilization of the roots of *Gnaphalium affine* with 5% sodium hypochlorite solution, *Gnaphalium affine* plants of similar size and with well-developed root systems were selected and transferred to a sterile water incubator containing 5 liters of 1× Hogrange solution for one week of acclimatization. After one week, BuX was added to achieve concentrations of 0, 50, 100, 300, and 500 mg / L for further cultivation of the *Gnaphalium affine* plants for one week. Three replicates were set up for each concentration, and the BuX degradation efficiency (measured by UV spectra at 301 nm), root length, plant height, and fresh weight of each treatment group were recorded periodically.

[0139] II. Experimental Results

[0140] The results are as follows Figure 7 As shown, with increasing BuX concentration, the yellowing of leaves in *Gnaphalium affine* gradually worsened, and root development was inhibited. This indicates that BuX has a certain degree of biotoxicity to *Gnaphalium affine*, and even at low concentrations (50 mg / L), it can inhibit the growth and development of *Gnaphalium affine*.

[0141] Degradation efficiency of *Gnaphalium affine* at different Bux concentrations was measured, revealing that *Gnaphalium affine* exhibits a certain degree of Bux tolerance, with the highest degradation efficiency observed on the first day, followed by a stabilization. At a Bux concentration of 50 mg / L, *Gnaphalium affine* demonstrated higher Bux degradation efficiency than other Bux concentrations, reaching a peak efficiency of 75.76% on day 5 at this concentration. Compared to the control group (0 mg / L Bux), plant fresh weight, plant height, and root length were significantly reduced.

[0142] Example 7: Co-treatment of BuX contaminated wastewater with Pseudomonas sp. W50 and Cyperus alternifolius

[0143] I. Experimental Methods

[0144] The pretreatment, such as the sterilization method and cultivation process of the windmill grass, was the same as in Example 6. A total of 6 treatment groups were set up, as detailed in Table 3, with 3 replicates for each group. Each treatment group lasted for one week, and the residual BuX content in each treatment group was measured periodically.

[0145] At the end of the exposure period, the water quality purification parameters (COD) of each sample group (Blank, BuX+W50, BuX+Ca and BuX+Ca+W50) were measured, and the biomass (fresh weight, plant height and root length) of the windmill grass was counted.

[0146] Table 3 Design of the Combined Remediation Experimental Treatment Group

[0147]

[0148] II. Experimental Results

[0149] Figure 8 Results A showed that strain W50 promoted the growth of *Gnaphalium affine*, and the addition of strain W50 significantly alleviated the biotoxicity of BuX (50 mg / L) to *Gnaphalium affine*. Measurements of residual BuX in the water revealed that the treatment groups BuX+W50, BuX+Ca, and BuX+Ca+W50 all reduced the BuX content. Figure 8 (B) Among them, the best biodegradation effect of BuX was achieved by BuX+Ca+W50, which demonstrates the feasibility of strain W50 and *Gnaphalium affine* in remediating BuX pollution.

[0150] Biomass, including fresh weight, of strain W50 under BuX stress combined with *Gnaphalium affine*. Figure 8 C) Plant height ( Figure 8 (D) and root length ( Figure 8 The levels of E in the samples were significantly higher than those in the single treatment with *Gnaphalium affine*. This indicates that this combined remediation technique is more effective than single phytoremediation, exhibiting higher BuX removal efficiency and promoting *Gnaphalium affine* growth.

[0151] The COD content of Blank, BuX+W50, BuX+Ca, and BuX+Ca+W50 was determined. The results are as follows: Figure 9 As shown in Table 4, BuX+W50, BuX+Ca, and BuX+Ca+W50 can all reduce the degree of water pollution, and the treatment group with only strain W50 added has the best effect. Figure 9 (A) However, in practical applications, single-microbial remediation is greatly affected by the environment, and microorganisms are difficult to maintain stability and reproduce. This study combined strain W50 with *Gnaphalium affine*, which greatly promoted the active colonization of strain W50 in the rhizosphere and roots (…). Figure 9 The inclusion of B and C in the data improves the feasibility of on-site bioremediation of BuX contamination.

[0152] Table 4: COD content of each treatment in the combined remediation experiment of *Gymnocladus orientalis* and W50

[0153]

Claims

1. A strain of Pseudomonas ( Pseudomonas sp.)W50, characterized in that, It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 31, 2024, with accession number GDMCC NO. 64924.

2. The use of the *Pseudomonas* strain according to claim 1 in simultaneously degrading xanthate, promoting plant growth, secreting or producing plant hormones, solubilizing phosphorus and potassium, and degrading cellulose, characterized in that, The plant hormones mentioned are GA and IAA.

3. The use of the *Pseudomonas* strain according to claim 1 in the preparation of the product, characterized in that, The product is one that simultaneously has the functions of degrading xanthate, promoting plant growth, secreting or producing plant hormones, solubilizing phosphorus and potassium, and degrading cellulose. The plant hormones are GA and IAA.

4. A method for treating xanthate pollution, characterized in that, Using the pseudomonads described in claim 1 as the sole strain, combined with *Gnaphalium affine*.

5. A microbial agent, characterized in that, Contains the Pseudomonas bacillus as described in claim 1.

6. A method for mitigating the impact of xanthate pollution on plant growth, characterized in that, The Pseudomonas aeruginosa of claim 1 was added to the water in the plant's growing environment, and the windmill grass was planted.

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