Pseudomonas and application of pseudomonas in degradation of xanthate by combination of pseudomonas and lysimachia christinae

Abstract

The invention discloses Pseudomonas sp. W50 and an application of the Pseudomonas sp. W50 in degradation of xanthate by combining the Pseudomonas sp. W50 with Cynanchum otophyllum. The Pseudomonas sp. W50 is preserved in Guangdong Microbial Culture Collection Center on July 31, 2024, and the preservation number of the Pseudomonas sp. W50 is GDMCC NO.64924. The Pseudomonas sp. W50 is preserved in the Guangdong Microbial Culture Collection Center on July 31, 2024. The degradation rate of 100mg / L BuX in 9h reaches up to 99%, and the strain also has various growth promoting characteristics of dissolving phosphorus, dissolving potassium, secreting IAA and GA and degrading cellulose. The BuX-polluted wastewater remediation agent can be used for remediation of BuX-polluted wastewater, can play a synergistic interaction role when being combined with pinkflower clover herb, has a more remarkable remediation effect, provides a new method for treatment of BuX pollution and promotion of healthy growth of aquatic plants in mining areas, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and more specifically to a pseudomonad and application thereof in degrading xanthate in combination with windmill grass. Background Art

[0002] Xanthate, also known as xanthate (BuX), is a yellow powder solid with a pungent odor and is toxic. It burns when ignited, is easily deliquesced by water, is unstable in nature, decomposes rapidly when exposed to salt, and is easily soluble in water, acetone, and alcohol. It is commonly used as a flotation reagent for various non-ferrous metals, with high mineral selectivity and cost-effectiveness. At present, butyl xanthate (BuX) is still the largest collector in terms of production and usage in the flotation of sulfides and some oxidized minerals. A large amount of flotation reagents, including BuX, will inevitably remain in the mineral processing wastewater.

[0003] BuX is a toxic and hazardous organic compound that can have toxic effects on aquatic organisms and human health even at very low concentrations. Specifically, BuX can cause growth inhibition, teratogenicity, and even death in aquatic microorganisms, plants, and animals in water bodies. In this context, the discharge of flotation wastewater containing BuX into natural waters seems destined to lead to destructive consequences such as water loss, soil erosion, and desertification of mine ecosystems. Therefore, the effective treatment of residual BuX in flotation wastewater and the restoration of vegetation in mining areas are crucial to the ecologically sustainable development of the mining industry.

[0004] At present, there are many methods to remove xanthate from flotation wastewater, including photocatalytic degradation, adsorption, chemical oxidation, etc. The disadvantages of the above physical and chemical methods are mainly low reuse rate, high operating cost and secondary pollution. In contrast, microbial degradation has received more attention due to its simple operation, affordability and environmental friendliness, and has been widely used to remove various organic pollutants. However, reports on microbial degradation of xanthate are very limited. This may be attributed to the toxic effect of xanthate on microorganisms, which inhibits their growth and enzyme activity. However, the effectiveness of microbial remediation is also limited by factors such as strain instability, difficulty in colonization, and less opportunity to contact pollutants in relatively deep sites.

[0005] At the same time, phytoremediation has emerged as an ecologically safe and sustainable alternative method for remediating polluted environments, which depends on the tolerance of the selected plants to harmful pollutants. Cyperus alternifolius is an emerging aquatic plant with favorable properties of tolerance to extreme conditions and pollutants, which makes it a successful candidate plant in phytoremediation. A large number of studies have shown that Cyperus alternifolius is able to remove a variety of harmful pollutants, such as heavy metals, antibiotics, and steroid hormones. However, mining wastewater usually contains high concentrations of toxic substances. Single plant remediation usually suffers from long growth cycles, limited absorption and degradation capabilities, and insufficient resistance of most plants, making it difficult to survive. Therefore, this limits the feasibility of phytoremediation, and single plants are not suitable for the remediation of heavily polluted environments.

[0006] In summary, new remediation methods are needed to overcome the above problems and improve the remediation effect of mining wastewater. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a Pseudomonas sp. and its application in degrading xanthate in combination with windmill grass.

[0008] The first object of the present invention is to provide a strain of Pseudomonas.

[0009] The second object of the present invention is to provide the use of the Pseudomonas in degrading xanthate or preparing products of degraded xanthate.

[0010] The third object of the present invention is to provide the use of the Pseudomonas in promoting plant growth or preparing products that promote plant growth.

[0011] The fourth object of the present invention is to provide the use of the Pseudomonas in secreting or producing plant hormones or preparing products that secrete or produce plant hormones.

[0012] A fifth object of the present invention is to provide the use of the Pseudomonas in solubilizing phosphate and / or potassium, or in preparing a product solubilizing phosphate and / or potassium.

[0013] The sixth object of the present invention is to provide the use of the Pseudomonas in degrading cellulose or preparing products of degraded cellulose.

[0014] The seventh object of the present invention is to provide a method for treating xanthate pollution.

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

[0016] The ninth object of the present invention is to provide a method for alleviating the impact of xanthate pollution on plant growth.

[0017] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0018] The present invention claims protection for a strain of Pseudomonas sp. W50, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on July 31, 2024, with the deposit number: GDMCC NO.64924.

[0019] The following applications are further claimed:

[0020] The application of the Pseudomonas in degrading xanthate or preparing products of degrading xanthate.

[0021] The application of the Pseudomonas in promoting plant growth or preparing products promoting plant growth.

[0022] Preferably, the plant is windmill grass.

[0023] The use of the Pseudomonas in secreting or producing plant hormones or preparing products that secrete or produce plant hormones, wherein the plant hormone is at least one of GA and IAA.

[0024] The pseudomonas is used in solubilizing phosphate and / or potassium, or in preparing products of solubilizing phosphate and / or potassium.

[0025] The potassium dissolution is to convert the water-insoluble salt of potassium (mineral potassium) into water-soluble potassium; the phosphorus dissolution is to convert the water-insoluble salt of phosphorus (mineral phosphorus) into water-soluble phosphorus.

[0026] The application of the Pseudomonas in degrading cellulose or preparing products of degraded cellulose.

[0027] The present invention also claims a method for treating xanthate pollution, which utilizes the Pseudomonas.

[0028] Preferably, the Pseudomonas is used in combination with Herba Pinelliae.

[0029] As a specific implementation method, the Pseudomonas is added to sewage that needs to be treated for xanthate pollution, and the sewage is used to plant windmill grass.

[0030] The present invention also claims to protect a bacterial agent containing the Pseudomonas.

[0031] The present invention also claims a method for alleviating the impact of xanthate pollution on plant growth, which comprises adding the Pseudomonas to the water in the plant growth environment.

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

[0033] The present invention provides a strain of Pseudomonas sp. W50 with a high-efficiency BuX degradation function, which has a degradation rate of up to 99% for 100 mg / L BuX in 9 hours, and also has multiple growth-promoting properties including phosphorus dissolution, potassium dissolution, IAA and GA secretion, and cellulose degradation. The strain can be used to repair wastewater polluted by BuX, and can be used in combination with windmill grass to achieve synergistic synergy, with a more significant repair effect, providing a new method for controlling BuX pollution and promoting the healthy growth of aquatic plants in mining areas, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The phylogenetic tree of the morphological characteristics of Pseudomonas W50 and its gene sequence; A is a picture of the colony morphological characteristics of Pseudomonas W50 on the LB plate; B is a scanning electron microscope (10,000 times) image of Pseudomonas W50; C is a phylogenetic tree of Pseudomonas W50.

[0035] Figure 2 This is the tolerance of Pseudomonas W50 to different pH values.

[0036] Figure 3 This is the tolerance of Pseudomonas W50 to different temperatures.

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

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

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

[0040] Figure 7 A is the tolerance of windmill grass to different concentrations of BuX; A is the phenotype diagram of windmill grass tolerance to 0, 50, 100, 300, and 500 mg / L BuX concentrations; B is the effect of different concentrations of BuX on the fresh weight of windmill grass; C is the effect of different concentrations of BuX on the plant height of windmill grass; D is the effect of different concentrations of BuX on the root length of windmill grass; E is the degradation efficiency of windmill grass to different concentrations of BuX.

[0041] Figure 8The results of the combined remediation of BuX-contaminated wastewater by Pinellia ternata and Pseudomonas sp. W50; A is the plant phenotype; B is the BuX degradation efficiency; C is the fresh weight; D is the plant height; and E is the root length.

[0042] Fig. 9 Figure 3: Water purification effects of single bacteria remediation (W50), phytoremediation (windmill grass) and plant-microorganism combined remediation (windmill grass-W50) on BuX-contaminated wastewater (assessed by chemical oxygen demand, COD); A is the COD content of 50 mg / L BuX xanthate wastewater after treatment with Blank, BuX+W50, BuX+Ca and BuX+Ca+W50, respectively; B is the colonization of the inoculated bacteria W50 on or in the roots of windmill grass exposed to BuX under a fluorescence microscope; C is the bacterial population density at different positions of windmill grass. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

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

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

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

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

[0048] The calculation method of degradation rate is: degradation rate (%) = 100% × (C0-C1 / C0)

[0049] Where 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] 1. Enrichment, separation and purification of strains

[0052] 1. Experimental methods

[0053] Add BuX to 100mL of sterilized MSM medium, and add BuX after cooling to make its initial concentration reach 10mg / L. Add 5g of collected contaminated soil from the mining area (taken from Dabaoshan mining area in Shaoguan, Guangzhou) to it, and culture it in a shaker at 35℃ and 150rpm in the dark for 7d. On the 7th day of culture, transfer it to a new MSM medium with an inoculum of 1%, gradually increase the concentration of pollutants for acclimatization, transfer it 5 times in a row, and finally raise the BuX concentration to 1000mg / L. Take the last culture solution and evenly spread it on the nutrient agar plate. After constant temperature culture for 48h, pick the colony and repeatedly streak it until a single colony is obtained, named strain W50.

[0054] A single bacterium was inoculated into MSM medium with 100 mg / L BuX, and cultured in a shaker at 35°C and 150 rpm for 72 h. The degradation effect was detected, and the strain was inoculated into a test tube slant medium and stored in a refrigerator at 4°C for use.

[0055] 2. Experimental methods

[0056] A strain that can use BuX as the sole carbon source was screened and named W50. It can completely degrade BuX with an initial concentration of 100 mg / L in a short period of time.

[0057] 2. Observe the characteristics of colonies

[0058] 1. Experimental methods

[0059] The screened strain W50 was inoculated onto a nutrient agar plate, inverted in an incubator at 35°C for overnight culture, and the shape, size, transparency, edge and color of the strain were observed.

[0060] 2. Experimental results

[0061] The strain W50 was inoculated on a nutrient agar plate and its colony morphology was observed after overnight culture. The colonies of the strain W50 inoculated on the nutrient agar plate and cultured for 3 days were round, light yellow, translucent, smooth in surface, convex in the middle, and sticky in texture ( Figure 1 A).

[0062] 3. SEM observation of bacterial morphology

[0063] 1. Experimental methods

[0064] The strain W50 was inoculated in LB broth medium for cultivation. 800 μL of the bacterial solution after 24 hours of cultivation was taken into a 1.5 mL centrifuge tube, and the supernatant was discarded after centrifugation at 8000 rpm for 5 minutes. The obtained bacterial precipitate was washed with sterile saline, centrifuged and the supernatant was discarded, and this operation was repeated 3 times. 1 mL of 2.5% glutaraldehyde electron microscope fixative was added to the washed bacteria, vortexed and mixed, and allowed to stand overnight in a 4°C refrigerator. After fixation, centrifuged at 8000 rpm for 5 minutes, the supernatant was discarded, the bacteria were collected and washed with sterile saline 3 times. Then, gradient dehydration was performed, that is, 30%, 50%, 70%, 80%, and 90% ethanol were added once, and 100% ethanol was added twice. Each dehydration treatment required standing for 15 minutes. After the last dehydration treatment, the bacteria were dropped on a cover glass and placed in a freeze dryer for freeze drying. The dried samples were sprayed with gold and then observed on the machine.

[0065] 2. Experimental results

[0066] SEM observation showed that strain W50 was a long rod (1.5-3.0×0.5 μm) with a relatively smooth surface, straight or slightly curved, and blunt-ended amastigotes ( Figure 1 B).

[0067] 4. Physiological and biochemical identification of strains

[0068] 1. Experimental methods

[0069] Physiological and biochemical experiments of strain W50 were carried out with reference to the Manual 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 acetylmethylcarbinol tests of strain W50 were all negative, while the catalase, oxidation reaction, ornithine decarboxylase, and lysine decarboxylase tests were positive. The strain was unable to utilize malonate, lactose, and glucose (Table 1).

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

[0073]

[0074] 5. Molecular Biology Identification

[0075] 1. Experimental methods

[0076] PCR amplification: The 16S rDNA fragment of the strain was amplified using bacterial universal 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. The total reaction system is 25 μL.

[0078] PCR reaction conditions: 95℃, initial preheating for 5min; melting at 94℃, 45s, annealing at 55℃, 45s, extension at 72℃, 1min15s, 32 cycles, and finally 72℃ for 10min. After PCR amplification, the size and specificity of the amplified fragments were detected by 1.5% agarose gel electrophoresis, and photographed in a gel imaging system. The PCR amplification products were purified, recovered and sequenced by Shanghai Bioengineering Company.

[0079] Construction of phylogenetic tree: The 16S rDNA sequence of strain W50 was obtained by sequencing by Sangon Company and submitted to the GenBank database. BLAST was used for comparison analysis, and ClustalX software was used for homology analysis of related sequences with high matching degree. Neighborhood-joining method in MEGA 11.0 software was used for relationship analysis to construct the phylogenetic tree of the strain.

[0080] 2. Experimental results

[0081] The genomic DNA of strain W50 was used as a template and PCR amplification was performed using 16S rDNA bacterial universal primers. The amplified products were sent to Shanghai Shenggong Biotechnology Co., Ltd. for purification, and the detected product lengths were 1078 bp.

[0082] Phylogenetic tree analysis showed that the 16S rDNA of W50 was 98% homologous to Pseudomonas juntendi BML3 NR 180457 (Genbank accession number: PP737842), and also had high homology with other strains of the genus. Therefore, it was preliminarily determined that strain W50 belonged to the genus Pseudomonas sp. Figure 1 C).

[0083] Pseudomonas sp. W50 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 31, 2024. Its collection number is: GDMCC NO.64924. The collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City. It is classified and named Pseudomonas sp.

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

[0085] 1. Quantitative analysis of BuX in culture medium

[0086] 1. Sample pretreatment method

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

[0088] 2. Detection method of BuX

[0089] The content of BuX in the solution can be calculated based on 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 certain amount of BuX, dissolve it in pure water and dilute it to a 50mL brown conical flask to obtain a BuX standard stock solution with a final concentration of 1000mg / L. Dilute the standard stock solution step by step to prepare standard solutions with concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, and 25.0mg / L, respectively. By measuring the absorbance of the standard solution of each gradient at 301nm, with it as Y and the corresponding solution concentration as X, a standard curve "Y=a+bX" is prepared.

[0092] 2. Preparation of W50 bacterial suspension

[0093] A single colony of Pseudomonas sp. W50 from Example 1 was selected and cultured in LB medium overnight (180 rpm / min, 24 h), then centrifuged to remove the supernatant (3500 r / min, 15 min), rinsed three times with 0.9% saline and resuspended to obtain W50 bacterial suspension. In the degradation assay, OD was used unless otherwise specified. 600nm The subsequent degradation experiments were carried out using a bacterial suspension of 1.0.

[0094] 3. Effect of pH on the degradation of BuX by strain W50

[0095] 1. Experimental methods

[0096] 1 mL of W50 bacterial suspension (OD600 = 1.0) was added to 50 mL of MSM medium containing 100 mg / L BuX, and the initial pH of the degradation system was adjusted to 5, 6, 7, 8, and 9, respectively, and then placed in a 35°C constant temperature oscillator at 150 rpm in the dark for 10 hours. Samples were taken every 2 hours to detect the content of BuX in the solution, and the treatment group without bacteria was used as a blank control. Three parallels were set for each treatment group.

[0097] 2. Experimental results

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

[0099] 4. Effect of temperature on the degradation of BuX by strain W50

[0100] 1. Experimental methods

[0101] BuX was added to a 100 mL conical flask containing 50 mL of sterilized MSM medium to an initial concentration of 100 mg / L. The initial pH of the degradation system was 7.0, and W50 bacterial suspension (OD600 = 1.0) was inoculated at 2% inoculation. The conical flasks were placed in an oscillator at 20, 25, 30, 35, and 40°C, and cultured at 150 rpm in the dark for 24 hours. Samples were taken every 3 hours to detect the residual BuX content, and the non-inoculated treatment was used as a blank control. Three parallels were set for each group.

[0102] 2. Experimental results

[0103] from Figure 3It can be seen that when the temperature is 35 or 40℃, 100mg / L BuX can be completely degraded in 9 hours, 12 hours are required at 30℃, and 24 hours are required to completely degrade 100mg / L BuX when the temperature reaches 20 or 25℃. W50 can degrade high concentrations of BuX in a short time even in a low temperature environment, and has good practical application value.

[0104] 5. 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 sterilized MSM medium to an initial concentration of 100, 300, 500, 700 and 1000 mg / L, respectively. The initial pH of the system was 7.0. 1 mL of W50 bacterial suspension (OD600 = 1.0) was inoculated and then placed in a constant temperature oscillator at 35 ° C and 150 rpm in the dark for 12 hours. Samples were taken at intervals of 2 hours to determine the BuX concentration. The uninoculated treatment was used as a blank control. Three parallels were set for each group.

[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, the degradation rate of strain W50 on different concentrations of BuX in MSM medium reached 77.9-100% within 9 hours, and the optimal concentration was 100 mg / L; strain W50 could completely degrade BuX within 12 hours when the initial concentration of BuX was 100-1000 mg / L, indicating that strain W50 can tolerate extremely high concentrations of BuX, and can make full use of BuX as the only carbon source and energy source for growth, and there was no obvious hysteresis effect in the degradation process.

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

[0110] 1. Experimental Methods

[0111] Different amounts of BuX were added to 100 mL of sterilized MSM medium to make the final concentrations of BuX 100, 300, 500, 700 and 1000 mg / L, respectively, and the Pseudomonas sp. W50 bacterial suspension of Example 2 was inoculated. The culture was shaken (150 rpm) under the optimal conditions determined in Example 2 (temperature 35°C, pH 5, inoculation amount 2 mL (OD600nm = 1.0)), and samples were taken at 0, 2, 4, 6, 8, 10 and 12 h, respectively, and the residual amount of BuX in each bacterial solution sample was determined by ultraviolet, and the degradation kinetic curves of BuX at different concentrations were drawn. With no inoculation as the control, 3 replicates were set for each treatment.

[0112] 2. Experimental Results

[0113] The results are shown in Table 2. According to the degradation kinetic fitting model and the half-life model fitting, the degradation kinetic equation and half-life results of Pseudomonas sp. W50 for different initial BuX concentrations were obtained. The half-life of BuX degradation by Pseudomonas sp. W50 was positively correlated with the substrate concentration. The half-lives corresponding to 100, 300, 500, 700 and 1000 mg / L BuX were 3.925h, 3.977h, 6.159h, 6.207h and 6.636h, respectively. Pseudomonas sp. W50 has a wide initial concentration range for the degradation of BuX, 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] 1. Experimental Methods

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

[0119] The Pseudomonas sp. W50 bacterial suspension of Example 2 was inoculated into a sterilized MSM medium supplemented with BuX at an initial concentration of 100 mg / L. The initial pH of the degradation system was 5, the temperature was 35° C., the inoculation amount was 2 mL (OD600nm=1.0), and the culture was shaken at 150 rpm. Samples were taken regularly for pretreatment, and the same medium without inoculation of the bacterial suspension and the same concentration of BuX was used as a control. Three replicates were set for each treatment. The culture solution was diluted ten times and then scanned using UV-Vis in the wavelength range of 185 to 350 nm to detect the intermediate degradation products of BuX.

[0120] 2. Experimental Results

[0121] It can be seen from the UV spectrum that the product of BuX degradation by Pseudomonas sp. W50 has absorption peaks at 301nm, 226nm and 206nm. Among them, 226nm and 301nm are the characteristic absorption peaks of BuX, and 206nm is the characteristic absorption peak of CS2. As the degradation reaction proceeds, the intensity of the BuX characteristic peak continues to decrease. When the reaction reaches 6h, the characteristic peak of CS2 appears briefly, and then the peak disappears. In addition, no other characteristic peaks of possible BuX intermediates were detected. It can be seen that CS2 is the main product after BuX biodegradation ( Figure 5 ).

[0122] Example 5 Analysis of plant growth promoting properties of Pseudomonas sp. W50

[0123] 1. 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. The specific methods are as follows:

[0125] Phosphate solubilization activity was assessed using NBRIP medium as described by Mehta and Nautiyal (2001). To quantify phosphate solubilization, W50 suspensions were inoculated in NBRIP liquid medium and incubated in the dark on a rotary shaker (30°C, 180 rpm) for 4 days. 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 antichromogen, and then the volume was made up to 25 ml with distilled water. After 30 minutes of reaction in the dark, the optical density of the supernatant mixture was measured at 700 nm. Three parallel treatments were set up, with the non-inoculated treatment as the control. In addition, single-factor control variables were used to study the effects of carbon source, nitrogen source and phosphorus source on the ability of W50 to solubilize phosphate in liquid media.

[0126] The ability of strain W50 to produce gibberellins was determined by slightly modifying the method of Xiao et al. (1997). Only the concentration of the bacterial solution was replaced according to a gradient dilution of 5 to 25 μg / L, and other conditions remained unchanged. According to the scheme proposed by Bric et al. (1991), the IAA production of bacterial isolates was screened using the Salkowski colorimetric method. After inoculating the bacterial isolate in King's B liquid medium containing 0.05 g / L tryptophan and incubating for 24 hours, the culture was centrifuged at 10,000 rpm for 5 minutes, 50 μL of supernatant and 100 μL of Salkowski reagent were added, and the color change was observed. The presence of red confirms the production of IAA. For quantification, the optical density (OD) was recorded at 530 nm, and the IAA content was calculated using the IAA standard curve.

[0127] As described by Schwyn and Neilands (1987), the W50 strain was inoculated onto a CAS plate and incubated at 30°C for 18-24 h. If an orange halo appeared on the plate, it indicated that the strain had the ability to produce siderophores.

[0128] The method described by Zhang et al. (2023) was used to determine the nitrogen fixation / potassium solubilization ability of the strain. W50 was inoculated into LB broth, and the suspension was obtained by rotary shaking (30°C, 150 rpm, 24 h). 5 μL was inoculated into Axubei solid medium or potassium solubilization solid medium and incubated at 30°C for 3 days. The formation of colonies indicates that the strain has the ability to fix nitrogen or solubilize potassium.

[0129] The Congo red staining method ( Li et al., 2016 ) was used to detect the cellulose degradation ability by forming a transparent circle around the colonies on the CMC-Na plate medium.

[0130] 2. Experimental Results

[0131] like Figure 6 As shown, strain W50 possessed multiple plant growth-promoting traits, including phosphate solubilization, GA secretion, IAA production, potassium solubilization, and cellulose degradation, but lacked the ability to fix nitrogen and produce siderophores.

[0132] Among them, the phosphate solubilization capacity of Pseudomonas sp. W50 showed a trend of first increasing and then decreasing with the extension of time. At 72h, the phosphate solubilization capacity of strain W50 reached a maximum of 269.5mg / L ( Figure 6 A).

[0133] The optimal carbon, nitrogen and phosphorus sources for W50 phosphorus dissolution are glucose, peptone and tricalcium phosphate. When glucose is the only carbon source, the phosphorus dissolution content can reach up to 227.93 mg / L ( Figure 6 When peptone is used as the only nitrogen source, the dissolved phosphorus content can reach up to 234.68 mg / L, which is 27.97% to 922.13% higher than other nitrogen sources ( Figure 6 C in the figure). When strain W50 selected phosphorus sources, it preferred tricalcium phosphate, and the highest dissolved phosphorus content could reach 246.07 mg / L ( Figure 6 D).

[0134] like Figure 6 As shown in E, the GA and IAA secretion contents of strain W50 reached the maximum values ​​of 93.86 mg / L and 105.21 mg / L at 48 h and 24 h, respectively.

[0135] like Figure 6The results of F in the strains showed that strain W50 possessed multiple plant growth-promoting properties such as potassium solubilization, siderophore production and cellulose degradation.

[0136] Example 6 Tolerance of Pinus armandii to different BuX concentrations

[0137] 1. Experimental Methods

[0138] After sterilizing the roots of the windmill grass with a 5% sodium hypochlorite solution, the windmill grass with similar size and good root system was selected and transferred to a sterile water incubator containing 5 liters of 1× Hoagland solution for one week of adaptation. After one week, BuX was added to the concentrations of 0, 50, 100, 300 and 500 mg / L for the cultivation of windmill grass for one week. Three replicates were set for each concentration, and the BuX degradation efficiency (detection of ultraviolet spectrum at a wavelength of 301 nm), plant root length, plant height and fresh weight of each treatment group were regularly recorded.

[0139] 2. Experimental Results

[0140] The results are as follows Figure 7 As shown in the figure, with the increase of BuX concentration, the leaves of windmill grass became more and more yellow, and the root development was inhibited. This shows that BuX has certain biological toxicity to windmill grass, and even at a low concentration (50 mg / L), it will inhibit the growth and development of windmill grass.

[0141] By measuring the degradation efficiency of different BuX concentrations of windmill grass, it was found that windmill grass has a certain tolerance to BuX, especially on the first day, the efficiency of BuX degradation was the highest, and then it tended to be stable. When the BuX concentration was 50mg / L, the efficiency of BuX degradation of windmill grass was higher than that of other BuX concentrations. On the 5th day, the BuX degradation efficiency of windmill grass at this concentration could reach 75.76%. Compared with the control group (0mg / L BuX), the fresh weight, plant height and root length of the plants were significantly reduced.

[0142] Example 7 Pseudomonas sp. W50 and Pinus armigera combined to remediate BuX-contaminated wastewater

[0143] 1. Experimental Methods

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

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

[0146] Table 3 Treatment group design of combined restoration experiment

[0147]

[0148] 2. Experimental Results

[0149] Figure 8 The results in A showed that strain W50 could promote the growth of windmill grass, and the addition of strain W50 significantly alleviated the biological toxicity of BuX (50 mg / L) to windmill grass. By measuring the residual BuX content in the water, it was found that the treatment groups BuX+W50, BuX+Ca and BuX+Ca+W50 could reduce the BuX content ( Figure 8 Among them, the best biodegradation effect of BuX was found in BuX+Ca+W50, which indicated the feasibility of strain W50 and windmill grass in repairing BuX pollution.

[0150] Biomass of strain W50 and windmill grass combined under BuX stress, including fresh weight ( Figure 8 C in), plant height ( Figure 8 D) and root length ( Figure 8 The E in the figure was significantly increased compared with that of the single phytoremediation. It can be seen that this combined remediation technology has a better effect than single phytoremediation, has a higher BuX removal efficiency and can promote the growth of phytoremediation.

[0151] The COD content of Blank, BuX+W50, BuX+Ca and BuX+Ca+W50 was measured. Fig. 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 ( Fig. 9 A in the figure). However, in practical applications, single microbial remediation is greatly affected by the environment, and it is difficult for microorganisms to exist and reproduce stably. In this study, strain W50 was combined with windmill grass, which greatly promoted the active colonization of strain W50 in the rhizosphere and roots ( Fig. 9 B and C), thereby improving the feasibility of on-site bioremediation of BuX contamination.

[0152] Table 4: COD content of each treatment in the windmill grass-W50 joint restoration experiment

[0153]

Claims

1. A strain of Pseudomonas sp. W50, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection Center on July 31, 2024, with the deposit number: GDMCC NO.64924.

2. Use of the Pseudomonas according to claim 1 in degrading xanthate or preparing products degraded from xanthate.

3. Use of the Pseudomonas according to claim 1 in promoting plant growth or preparing products that promote plant growth.

4. Use of the Pseudomonas according to claim 1 in secreting or producing plant hormones or preparing products that secrete or produce plant hormones, characterized in that: The plant hormone is at least one of GA and IAA.

5. Use of the Pseudomonas according to claim 1 in solubilizing phosphate and / or potassium, or in preparing a product of solubilizing phosphate and / or potassium.

6. Use of the Pseudomonas according to claim 1 in degrading cellulose or preparing products of degraded cellulose.

7. A method for treating xanthate pollution, characterized in that: Utilize the Pseudomonas described in claim 1.

8. The method according to claim 7, characterized in that Utilize the Pseudomonas described in claim 1 in combination with windmill grass.

9. A bacterial agent, characterized in that Contains the Pseudomonas according to claim 1.

10. A method for alleviating the effect of xanthate pollution on plant growth, characterized in that: The Pseudomonas according to claim 1 is added to water in a plant growth environment.

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