An immobilized bacterial agent and its application in degrading butyl xanthate and benzohydroxamic acid pollution

Abstract

The present invention discloses an immobilized bacterial agent and its application in degrading butyl xanthate and benzohydroxamic acid pollution. The preparation method of the immobilized bacterial agent is characterized in that it comprises the following steps: after mixing biochar with Pseudomonas and copper-greedy bacteria composition, fully adsorbing to obtain product 1; fully mixing sodium alginate aqueous solution with product 1 to obtain product 2; dropping CaCl2 solution into product 2 drop by drop, fully solidifying, and purifying to obtain. The immobilized bacterial agent of the present invention can make the embedded Pseudomonas and copper-greedy bacteria flora remain relatively stable in contaminated water and soil, improve their tolerance to adverse environmental conditions, avoid competition with other microorganisms, thereby being able to more efficiently degrade BuX and BHA in water and soil, weaken the adverse effects on aquatic and soil ecosystems, and ensure agricultural safety production.

Description

Technical Field

[0001] The present invention relates to the technical field of pollution remediation, and more particularly to an immobilized bacterial agent and application thereof in degrading butyl xanthate and benzohydroxamic acid pollution. Background Art

[0002] Flotation, the most effective and widely used method in ore processing, inevitably involves the use of flotation reagents. These reagents are not fully utilized during flotation, and most of them remain in the water. Flotation wastewater is often discharged into the surrounding environment without treatment, causing frequent pollution.

[0003] Among them, butyl xanthate (BuX) and benzohydroxamic acid (BHA) are two typical flotation collectors. These agents separate valuable ores from worthless minerals by adjusting the surface properties of minerals, thereby achieving the purpose of flotation separation.

[0004] To improve the recovery rate of the target metal during flotation, these agents are often added in excess. However, only a portion of these agents is actually consumed, with the excess discharged with the flotation wastewater. Existing wastewater treatment technologies are unable to completely remove high concentrations of flotation agents from water bodies, resulting in high levels of BuX and BHA in wastewater discharged from mineral processing plants. High concentrations of BuX and BHA in water bodies can cause problems such as foul odor and increased COD levels. Even at extremely low concentrations, their presence can pose significant risks to aquatic life, such as affecting embryonic development, causing fish deformities, and inhibiting algae growth. Discharge of BuX- and BHA-contaminated water into the environment can further pollute surrounding rivers and be used for agricultural irrigation by downstream residents, posing a serious threat to agricultural safety. BuX and BHA can also affect human health.

[0005] Existing studies have mostly focused on removing BuX and BHA through physical and chemical methods. However, considering the overall cost, environmental protection, and renewability, bioremediation methods are more cost-effective and eco-friendly. Currently, no studies have reported on the remediation of BuX and BHA combined pollution using microorganisms. Given the complexity of actual polluted environments, the degradation efficiency of degrading bacteria is highly susceptible to adverse environmental conditions and can be significantly reduced. Therefore, improving the bioavailability of BuX and BHA in contaminated water by degrading bacteria, thereby enhancing the remediation efficacy of functional microorganisms and effectively remediating BuX and BHA combined pollution, is of great significance for ensuring agricultural safety and human health. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide an immobilized bacterial agent and its application in degrading butyl xanthate and benzohydroxamic acid pollution.

[0007] The first object of the present invention is to provide a composition.

[0008] The second object of the present invention is to provide a bacterial agent.

[0009] The third object of the present invention is to provide a method for preparing an immobilized bacterial agent.

[0010] The fourth object of the present invention is to provide an immobilized bacterial agent prepared by any of the above preparation methods.

[0011] A fifth object of the present invention is to provide a use of the composition in preparing a product for simultaneously degrading butyl xanthate and benzohydroxamic acid, or in simultaneously degrading butyl xanthate and benzohydroxamic acid.

[0012] The sixth object of the present invention is to provide the application of the bacterial agent or the immobilized bacterial agent

[0013] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0014] The present invention claims a composition comprising Pseudomonas ( Pseudomonas sp.) W50 and Cupribotium ( Cupriavidus sp.)HY21,

[0015] The Pseudomonas sp. was deposited in Guangdong Provincial Microbiological Culture Collection Center on July 31, 2024, with the deposit number: GDMCC NO. 64924;

[0016] The copper-greedy bacteria was deposited in the Guangdong Provincial Microbial Culture Collection Center on September 11, 2024, and its preservation number is: GDMCC No: 65117.

[0017] Preferably, the amount of Pseudomonas and Cupribotium is 0.5-2:0.5-2 in volume ratio, with an OD 600nm It is a mixture of 0.8 to 1.2 bacterial suspensions.

[0018] More preferably, the amount of Pseudomonas and Cupriavidus is 1:1 in volume ratio. 600nm A mixture of 1% bacterial suspension.

[0019] The invention also claims a bacterial agent prepared by using the composition.

[0020] And a preparation method of an immobilized bacterial agent, comprising the following steps:

[0021] S1. After the biochar is mixed with the composition, the adsorption is fully performed to obtain product 1;

[0022] S2. The sodium alginate aqueous solution is thoroughly mixed with product 1 to obtain product 2;

[0023] S3. Product 2 is added dropwise to a CaCl2 solution until fully solidified and purified.

[0024] Preferably, in product 1, the concentration of biochar is 0.005 to 0.045 g / mL.

[0025] Preferably, in product 1, the concentration of biochar is 0.005 to 0.015 g / mL.

[0026] More preferably, the concentration of biochar in product 1 is 0.01 g / mL.

[0027] Preferably, in product 2, the concentration of sodium alginate is 0.02 to 0.08 g / mL.

[0028] Preferably, in product 2, the concentration of sodium alginate is 0.03-0.05 g / mL.

[0029] More preferably, in product 2, the concentration of sodium alginate is 0.04 g / mL.

[0030] Preferably, the concentration of the CaCl2 solution is 0.01 to 0.04 g / mL.

[0031] Preferably, the concentration of the CaCl2 solution is 0.015 to 0.025 g / mL.

[0032] More preferably, the concentration of the CaCl2 solution is 0.02 g / mL.

[0033] Preferably, the sufficient curing is curing in a refrigerator at 4-8°C for 12-14 hours.

[0034] More preferably, the sufficient curing is curing in a refrigerator at 4°C for 12 hours.

[0035] Preferably, the purification is washing the fixed product with physiological saline.

[0036] Preferably, the biochar preparation method comprises the following steps: preparing corn straw powder, calcining at 450-550° C. for 3-5 hours, washing, preparing powder, sieving, sterilizing, and cooling.

[0037] More preferably, the washing is performed by sequentially washing with 0.1-0.2 M HCl and pure water.

[0038] More preferably, the sieving is through a 80-100 mesh sieve.

[0039] More preferably, the sterilization is carried out at 121-130°C for 20-30 min.

[0040] Preferably, the composition is the Pseudomonas OD 600nm The bacterial suspension and the OD of the copper-greedy bacteria were 0.8 to 1.2. 600nm It is a mixture of 0.8 to 1.2 bacterial suspensions.

[0041] More preferably, the bacterial suspension is prepared by culturing the Pseudomonas or Cupriavidus at 28-37°C for 12-24 hours to obtain activated bacteria; the activated bacteria are cultured in LB liquid culture medium at 150-180 r / min and 30-35°C for 12-24 hours; after centrifugation at 3-5°C and 4500-5000 r / min for 5-10 minutes, the bacteria are washed with 0.9-1.2% sterile saline and resuspended to prepare the suspension.

[0042] The immobilized bacterial agent prepared by any of the preparation methods also falls within the protection scope of the present invention.

[0043] The use of the composition in preparing a product for simultaneously degrading butyl xanthate and benzohydroxamic acid, or in simultaneously degrading butyl xanthate and benzohydroxamic acid, also falls within the protection scope of the present invention.

[0044] The application of the bacterial agent or the immobilized bacterial agent is one or more of the following:

[0045] Preparation of products for the simultaneous degradation of butyl xanthate and benzohydroxamic acid;

[0046] Application in the simultaneous degradation of butyl xanthate and benzohydroxamic acid;

[0047] Application of butyl xanthate and benzohydroxamic acid in remediation of contaminated soil.

[0048] Application of butyl xanthate and benzohydroxamic acid in remediation of contaminated water bodies.

[0049] Preferably, the enzymatic activity of soil catalase and / or urease is increased.

[0050] Preferably, the soil is enhanced to increase the abundance of bacterial communities.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The immobilized bacterial agent of the present invention can keep the embedded Pseudomonas and Cupribotium flora relatively stable in polluted water and soil, improve their tolerance to adverse environmental conditions, avoid competition with other microorganisms, and thus more efficiently degrade BuX and BHA in water and soil, weaken the adverse effects on aquatic and soil ecosystems, and ensure safe agricultural production.

[0053] (2) When the concentrations of BuX and BHA in the composite polluted water were as high as 100 mg / L, by adding the composite bacterial agent of the present invention to the water, BuX and BHA in the water could be completely removed in only 9 and 30 h, respectively, which was significantly higher than the treatment group using free bacteria alone.

[0054] (3) The composite bacterial agent capable of degrading BuX and BHA of the present invention has a wide range of applications and can effectively reduce the content of BuX and BHA in water bodies contaminated by BuX and BHA under extreme pH, temperature or heavy metal stress conditions, thereby reducing their harm to the water environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The colony morphology of the strains on nutrient agar plates; A is strain W50; B is strain HY21.

[0056] Figure 2 The morphological characteristics of the strains under SEM; A is strain W50; B is strain HY21.

[0057] Figure 3 Electrophoresis of PCR amplification products. M: DNA molecular weight standard; 1: W50 amplification product; 2: HY21 amplification product.

[0058] Figure 4 This is the phylogenetic tree of strain W50 based on 16S rDNA sequence.

[0059] Figure 5 This is the phylogenetic tree of strain HY21 based on the 16S rDNA sequence.

[0060] Figure 6 The chromatograms of quantitative ion (A) and qualitative ion (B) of BHA.

[0061] Figure 7 The degradation kinetic curves (A) and fitting curves (B) of strain HY21 for different initial concentrations of BHA.

[0062] Figure 8 is the tolerance of strain W50 to different initial BuX concentrations.

[0063] Figure 9 This is the antagonism between strain W50 and strain HY21.

[0064] Figure 10 Degradation of BuX (A) and BHA (B) by mixed bacterial consortium FB in single and combined pollution systems;

[0065] Figure 11 This is the SEM image of biochar.

[0066] Figure 12 FT-IR image of biochar.

[0067] Figure 13 The appearance (A) and particle size (B) of the immobilized bacterial agent.

[0068] Figure 14 The surface morphology and internal structure of the immobilized bacterial agent; A and B are the surface morphology; C and D are the internal structure.

[0069] Figure 15 FT-IR images of biochar, SB and IB.

[0070] Figure 16 Degradation effects of each treatment group on BuX and BHA; A is the degradation effect on BuX; B is the degradation effect on BHA.

[0071] Figure 17 These are immobilized bacterial agents prepared with different sodium alginate contents; the sodium alginate contents in A to D are 1%, 2%, 3% and 4%, respectively.

[0072] Figure 18 The degradation effects of immobilized bacteria with different sodium alginate contents on BHA and BuX.

[0073] Figure 19 The degradation effects of immobilized bacteria with different biochar contents on BHA and BuX.

[0074] Figure 20 The degradation effect of immobilized bacteria at different CaCl2 concentrations on BHA and BuX

[0075] Figure 21 Degradation ability of immobilized bacteria under strong acid and strong alkali conditions; degradation of BuX (A) and BHA (B) by each treatment group under strong acid conditions (pH = 5); degradation of BuX (C) and BHA (D) by each treatment group under strong alkaline conditions (pH = 9).

[0076] Figure 22 Figure 5 shows the degradation ability of immobilized bacteria at high and low temperatures; the degradation of BuX (A) and BHA (B) by each treatment group under high temperature conditions (40 ℃); the degradation of BuX (C) and BHA (D) by each treatment group under low temperature conditions (20 ℃).

[0077] Figure 23 Cd 2+ Degradation of BuX (A) and BHA (B) by each treatment group at a concentration of 5 mg / L.

[0078] Figure 24 The degradation of BuX (A) and BHA (B) by immobilized bacteria after 5 cycles.

[0079] Figure 25 The degradation effects of immobilized bacteria on BuX and BHA after storage for different days.

[0080] Figure 26 Changes in soil BuX concentration in each treatment group at different restoration periods; A is group S1; B is group S2.

[0081] Figure 27 Changes in soil pH in each treatment group during different restoration periods; A is group S1; B is group S2.

[0082] Figure 28 Changes in soil catalase activity in each treatment group at different restoration periods; A is group S1; B is group S2.

[0083] Figure 29 Changes in soil urease activity in each treatment group at different restoration periods; A is group S1; B is group S2.

[0084] Figure 30 These are principal coordinate analysis diagrams of each treatment group; A is for group S1; B is for group S2.

[0085] Figure 31 is the relative abundance of soil bacterial communities at the phylum level in each treatment group.

[0086] Figure 32 is the relative abundance of soil bacterial communities at the genus level in each treatment group

[0087] Figure 33 is the relative abundance of Pseudomonas in each treatment group. DETAILED DESCRIPTION

[0088] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0089] Butyl xanthate (BuX) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Aladdin) with a purity of >95%; Benzohydroxamic acid (BHA) was purchased from Sigma-Aldrich, USA, with a purity of >99%; Chromatographic grade methanol was purchased from Sigma-Aldrich, USA; Hydrochloric acid was purchased from Guangzhou Chemical Reagent Co., Ltd. with analytical purity; All other reagents were purchased from Tianjin Damao Chemical Reagent Co., Ltd.

[0090] Liquid basal 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 approximately 7.0.

[0091] LB broth medium: peptone 10 g / L, yeast extract 5 g / L and sodium chloride 5 g / L, pH approximately 7.0.

[0092] Nutrient agar medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, agar 15 g / L, pH approximately 7.0.

[0093] The soil samples used to isolate degrading bacteria in this study were taken from the Dabaoshan mining area in Shaoguan, Guangzhou.

[0094] Example 1 Enrichment and separation and purification of strains

[0095] 1. Experimental Methods

[0096] Sterilize 100 mL of MSM medium in a 250 mL conical flask. After cooling, add BuX or BHA to an initial concentration of 10 mg / L. Add 5 g of contaminated soil collected from a mining area (Dabaoshan mining area, Shaoguan, Guangzhou) and incubate in the dark at 35°C and 150 rpm in a shaker. On the seventh day, transfer the inoculum to fresh MSM medium at a 10% inoculum size and gradually increase the contaminant concentration for acclimatization. Repeat this transfer five times.

[0097] The final culture solution was evenly spread on a nutrient agar plate. After constant temperature incubation for 48 hours, colonies were picked and repeatedly streaked until a single colony was obtained. The single bacteria were inoculated into MSM medium supplemented with 100 mg / L BHA or BuX and incubated in a shaker at 35°C and 150 rpm for 72 hours. The degradation effect was tested and the strain was inoculated into a test tube slant culture medium and stored in a refrigerator at 4°C until use.

[0098] 2. Experimental Results

[0099] After gradient acclimation, a strain capable of using BuX as its sole carbon source was identified, named W50, and another strain capable of using BHA as its sole carbon source was named HY21. Both strains were able to completely degrade BuX or BHA, respectively, at an initial concentration of 100 mg / L, within a short period of time.

[0100] Example 2 Identification of strain morphological characteristics

[0101] 1. Observe the characteristics of the colony

[0102] 1. Experimental methods

[0103] The strains W50 and HY21 screened in Example 1 were inoculated onto nutrient agar plates, respectively, and inverted in an incubator at 35° C. for overnight culture. The shape, size, transparency, edge, and color of the strains were observed.

[0104] 2. Experimental results

[0105] After overnight culture, the colony morphology of the two strains was observed. On the nutrient agar plate, the strain W50 was round, light yellow, translucent, with a smooth surface, a raised center, and a sticky texture ( Figure 1 A in the figure); strain HY21 showed milky white, irregular, opaque, and rough surface ( Figure 1 B in ).

[0106] 2. SEM observation of bacterial morphology

[0107] 1. Experimental methods

[0108] Strains W50 and HY21 were inoculated into LB broth. After 24 hours of culture, 800 μL of the culture fluid was transferred to a 1.5 mL centrifuge tube and centrifuged at 8000 rpm for 5 minutes, after which the supernatant was discarded. The resulting bacterial pellet was washed with sterile saline, centrifuged, and the supernatant discarded. This process was repeated three times. 1 mL of 2.5% glutaraldehyde electron microscopy fixative was added to the washed cells, vortexed, and incubated overnight at 4°C. After fixation, the cells were centrifuged at 8000 rpm for 5 minutes, the supernatant discarded, and the cells were collected and washed three times with sterile saline. Dehydration was then performed using a gradient of 30%, 50%, 70%, 80%, and 90% ethanol, followed by two additions of 100% ethanol. Each dehydration step was followed by 15 minutes of immersion. After the final dehydration step, the cells were placed on a coverslip and lyophilized in a freeze dryer. The dried samples were then sprayed with gold and examined.

[0109] 2. Experimental results

[0110] SEM observations showed that strain W50 was long rod-shaped (1.5-3.0 × 0.5 μm) with a relatively smooth surface ( Figure 2 A in the figure); strain HY21 is short rod-shaped (0.8-2.0×0.5 μm) with a rough surface ( Figure 2 B in ).

[0111] 3. Physiological and biochemical identification of strains

[0112] 1. Experimental methods

[0113] Physiological and biochemical experiments of strains W50 and HY21 were performed with reference to the Manual of Identification of Common Bacteria and Bergey's Manual of Determinative Bacteriology.

[0114] 2. Experimental results

[0115] Gram staining results showed that both strains were Gram-negative bacteria. The W50 methyl red test, nitrate reduction, starch hydrolysis, urease production, hydrogen sulfide, and acetylmethylcarbinol tests were all negative, while the catalase, oxidation reaction, ornithine decarboxylase, and lysine decarboxylase tests were positive. The strains were unable to utilize malonate, lactose, and glucose (Table 1).

[0116] Table 1 Physiological and biochemical characteristics of strain W50

[0117]

[0118] Note: “+” indicates that it can be used or positive; “-” indicates that it cannot be used or negative.

[0119] The strain HY21 tested negative for methyl red, nitrate reduction, catalase, starch hydrolysis, hydrogen sulfide, and acetylmethylcarbinol, but was positive for oxidation, ornithine decarboxylase, lysine decarboxylase, and urease production. The strain was unable to utilize lactose and glucose but could utilize malonate (Table 2).

[0120] Table 2 Physiological and biochemical characteristics of strain HY21

[0121]

[0122] 4. Molecular Biological Identification of Strains

[0123] 1. Experimental methods

[0124] PCR amplification: The 16S rDNA fragments of strains W50 and HY21 were amplified using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), respectively.

[0125] 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.

[0126] PCR reaction conditions included an initial preheating at 95°C for 5 min, followed by 32 cycles of melting at 94°C for 45 s, annealing at 55°C for 45 s, and extension at 72°C for 1 min 15 s, with a final hold at 72°C for 10 min. After PCR amplification, the size and specificity of the amplified fragments were determined by electrophoresis on a 1.5% agarose gel and photographed using a gel imaging system. The PCR products were then purified and sequenced by Shanghai Bioengineering Co., Ltd.

[0127] Phylogenetic tree construction: 16S rDNA sequences of the two strains were obtained by sequencing at Sangon and submitted to the GenBank database. BLAST alignment analysis was performed, and homology analysis was performed using ClustalX software for closely matching related sequences. Neighborhood-joining analysis was performed using MEGA 11.0 software to construct a phylogenetic tree.

[0128] 2. Experimental results

[0129] The genomic DNA of strain W50 and strain HY21 were used as templates for PCR amplification using 16S rDNA bacterial universal primers. The electrophoresis results of the amplified products were shown in Figure 2. Figure 3 The amplified products were sent to Shanghai Sangon Biotechnology Co., Ltd. for purification and sequencing. The 16S rDNA sequencing results of strain W50 and strain HY21 were respectively submitted to the NCBI Genbank database for BLAST comparison. The sequences with high homology to the target strain in the BLAST comparison analysis results were aligned for multiple sequences, and the phylogenetic trees of strain W50 and strain HY21 were constructed by the neighbor-joining method ( Figure 4 and Figure 5 ).

[0130] The phylogenetic tree results showed that the 16S rDNA of strain W50 was similar to Pseudomonas juntendi BML3 NR180457 (Genbank accession number: PP737842) has the highest homology with the genus ( Pseudomonas ) Other strains also have high homology. Combined with the physiological and biochemical identification results of the W50 strain, it is preliminarily determined that the strain W50 belongs to the genus Pseudomonas ( Pseudomonas ); 16S rDNA of strain HY21 and Cupriavidus basilensis strain N1129 (MN691130.1) has the highest homology with the genus ( Cupriavidus) other strains also have high homology. Combined with the physiological and biochemical identification results of the above strain HY21, it is preliminarily determined that the strain HY21 belongs to the genus Cupriavidus ( Cupriavidus )

[0131] The strain W50 and strain HY21 were deposited separately: strain W50 was named Pseudomonas ( Pseudomonas sp.) W50, deposited in Guangdong Provincial Microbial Culture Collection Center on July 31, 2024, with the deposit number: GDMCC NO. 64924, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; strain HY21 is named: Copper-Greedy Bacteria ( Cupriavidus sp.) HY21, and was deposited in Guangdong Provincial Microbiological Culture Collection on September 11, 2024. Its deposit number is: GDMCC No: 65117, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0132] Example 3 Degradation effect of strain HY21 on BHA

[0133] 1. Experimental Methods

[0134] At pH 5.5, temperature 30.5°C, inoculum size 1.0 (OD 600 Under degradation conditions (2% volume fraction), the bacterial suspension was inoculated into 50 mL of MSM medium. BHA concentrations in the medium were set at 50, 100, 150, and 200 mg / L, respectively. The medium was shaken at 150 rpm in a constant temperature shaker for 48 hours. Samples were taken at 0, 6, 12, 24, 30, 36, and 48 hours after degradation, and the remaining BHA concentration in the culture medium was measured. Degradation kinetics of strain HY21 at different initial BHA concentrations were plotted. A control group was not inoculated, and triplicates were set for each treatment group.

[0135] The method for detecting the concentration of BHA is:

[0136] 1. Sample pretreatment method

[0137] Transfer 1 mL of the culture medium to be tested to a 1.5 mL centrifuge tube and centrifuge at 8000 rpm for 5 minutes. Dilute 100 μL of the supernatant 100-fold with methanol-water (50 / 50, v / v). Filter through a 0.22 μm microporous membrane and collect the filtrate into a vial for determination of BHA content using LC-MS / MS.

[0138] 2. LC-MS / MS detection method for BHA

[0139] An LC-MS / MS detection method was established using an AB Sciex 5500MD high performance liquid chromatography-tandem mass spectrometer.

[0140] Chromatographic conditions: The chromatographic column was Water Xbridge C18 (3.0 mm × 50 mm, 5 μm); the mobile phase A was water; the mobile phase B was methanol; the column flow rate was 0.40 mL / min; the column temperature was 40 °C; the injection volume was 5 μL; and the mobile phase gradient elution program was as shown in Table 3.

[0141] Table 3 Mobile phase gradient elution program

[0142]

[0143] Mass spectrometry conditions:

[0144] Ion source: electrospray ionization source negative ion (ESI-); electrospray voltage: -4500 V; nebulizer gas temperature: 500°C; nebulizer gas pressure: 55 psi; auxiliary gas pressure: 50 psi; curtain gas pressure: 35 psi; collider pressure: 6 psi; scanning mode: multiple reaction monitoring (MRM). The mass spectrometry parameters of BHA are shown in Table 4. The quantitative ion and qualitative ion chromatograms are shown in Figure 4. Figure 4 shown.

[0145] Table 4

[0146]

[0147] 3. BHA standard curve

[0148] Add 10 μL of the BHA standard solution (1000 mg / L) to an injection vial and dilute with 990 μL of chromatography-grade methanol to obtain a 10 mg / L BHA stock standard solution. Prepare solutions of 50, 75, 150, 300, 600, and 1200 ng / mL using a gradient dilution method using a 50 / 50, v / v methanol-water solution. Measure the peak area corresponding to each concentration using the BHA LC-MS / MS method described above. Determine the peak area Y and the corresponding standard solution concentration X, and plot a standard curve ("Y = a + bX").

[0149] 2. Experimental Results

[0150] The chromatograms of quantitative ion (A) and qualitative ion (B) of BHA are shown in Figure 6 .

[0151] The results are as follows Figure 7As shown in Figure A, the results show that strain HY21 can tolerate a BHA concentration range of 50 to 200 mg / L. A strain of Klebsiella BHA-degrading bacteria has been reported to only achieve a degradation rate of 85.04% for an initial concentration of 100 mg / L BHA after 12 days of culture (Hu Chun, Wang Can, Gong Wenqi et al. Study on the microbial degradation of three hydroxamic acid collectors [J]. Hubei Agricultural Sciences, 2013, 52(11): 2505-2507). In comparison, strain HY21 can tolerate higher concentrations of BHA and has a higher BHA degradation efficiency.

[0152] Example 4 Degradation effect of strain W50 on BuX

[0153] 1. Experimental Methods

[0154] BuX was added to 50 mL of sterilized MSM medium to make the initial concentrations reach 100, 300, 500, 700 and 1000 mg / L, respectively. The initial pH of the system was 7.0. 2% W50 bacterial suspension (OD 600 = 1.0), then incubate in a constant-temperature shaker at 35°C, 150 rpm, in the dark for 12 h. Samples were taken every 2 h to determine the BuX concentration. The uninoculated treatment served as a blank control. Three replicates were set up for each group.

[0155] The method for detecting the concentration of BuX is:

[0156] 1. Sample pretreatment method

[0157] Transfer 1 mL of culture medium to a 1.5 mL centrifuge tube and centrifuge at 8000 rpm for 5 minutes. After centrifugation, dilute 300 μL of the supernatant 10-fold to obtain the test solution. Immediately measure the BuX content using UV-Vis spectrophotometry (to prevent BuX degradation).

[0158] 2. Detection method of BuX

[0159] The absorbance of the test solution at 301 nm was measured and the BuX content in the solution was calculated based on the standard curve.

[0160] 3. BuX standard curve

[0161] Accurately weigh a certain amount of BuX and dissolve it in pure water to a final volume of 50 mL to obtain a BuX standard stock solution with a final concentration of 1000 mg / L. Dilute the standard stock solution serially to prepare calibration solutions with concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, and 25.0 mg / L. Measure the absorbance of each calibration solution at 301 nm, using this as Y and the corresponding solution concentration as X, to construct a standard curve (Y = a + bX).

[0162] 2. Experimental Results

[0163] The results show Figure 8 The results showed that strain W50 completely degraded BuX within 12 hours at initial concentrations of 100 to 1000 mg / L, demonstrating that strain W50 can tolerate extremely high BuX concentrations and exhibits no significant hysteresis during the degradation process. Excessive addition of BuX during flotation often results in high BuX concentrations in the discharged flotation wastewater. Therefore, strains that can tolerate high BuX concentrations are needed. Strain HY21 has excellent application prospects in treating high-concentration BuX wastewater.

[0164] Example 5 Construction of mixed bacterial flora

[0165] 1. Experimental Methods

[0166] Strains W50 and HY21 isolated in Example 1 were streaked onto nutrient agar using the cross method. After streaking, the culture medium was inverted and placed in a 35°C constant temperature incubator for 24 hours. The growth of the strains at the intersection was observed to determine whether a clear zone of inhibition appeared.

[0167] 2. Experimental Results

[0168] The results are as follows Figure 9 As shown, the results of cross-striping of strain W50 and strain HY21 showed that the strains at the intersection grew well, and there was no growth inhibition between the two strains. Therefore, strain W50 and strain HY21 can be used to construct a bacterial community.

[0169] Strain W50 and strain HY21 were inoculated into LB medium and cultured overnight (150-180 rpm, 30-35°C for 12-24 h). When the strains grew to the logarithmic phase, they were centrifuged at 4500 rpm for 10 min to obtain bacterial pellets. The bacterial pellets were washed three times with sterile physiological washing, resuspended in physiological saline, and the OD was adjusted. 600 to 1.0, and then mixed at a ratio of 1:1 (V / V) to obtain a mixed bacterial community (i.e., isolated bacterial community, FB).

[0170] Example 6 Degradation Effect of Bacteria in Single or Composite Pollution Systems

[0171] 1. Experimental Methods

[0172] 50 mL of MSM culture medium containing only 100 mg / L BuX or BHA, or both, was prepared and inoculated with the mixed bacterial colony obtained in Example 5 (i.e., the isolated bacterial colony, FB) at a volume ratio of 2%. The conical flask was placed in a constant temperature of 35°C and 150 rpm and shaken for 48 h. Samples were taken at regular intervals to determine the residual BuX and BHA contents in the solution at different time points (using the same method as in Examples 3 and 4).

[0173] 2. Experimental Results

[0174] The results are as follows Figure 10 The results showed that it took 9 h and 30 h for the mixed bacterial community in the single pollution system to completely degrade BuX or BHA with an initial concentration of 100 mg / L, respectively, while the time required for complete degradation of BuX and BHA in the composite system was prolonged to 24 h and 48 h, respectively.

[0175] Example 7 Preparation and Characterization of Biochar

[0176] 1. Preparation of Biochar

[0177] Washed and dried corn stalks were pulverized into a powder, placed in a crucible, covered, and calcined at 500°C in a muffle furnace under a nitrogen atmosphere for 4 hours. The biochar obtained after calcination was then demineralized by washing with 0.1 M HCl and then washed with deionized water to thoroughly remove residual acid and ash on the biochar surface. Finally, the biochar was ground into a fine powder, passed through a 100-mesh sieve, and sterilized at 121–130°C for 20–30 minutes. After cooling to room temperature, it was stored in a dry environment until ready for use.

[0178] 2. Scan the surface of biochar using SEM

[0179] 1. Experimental methods

[0180] The biochar was washed with sterile saline, the supernatant discarded, and this process repeated three times. 1 mL of 2.5% glutaraldehyde electron microscopy fixative was added to the washed culture, vortexed to mix, and allowed to stand in a refrigerator at 4°C for 20 min. After fixation, the supernatant was discarded, and the culture was collected and washed three times with sterile saline. Dehydration was then performed using a gradient of ethanol concentrations of 30%, 50%, 70%, 80%, and 90% (volume fraction) once, followed by 100% (volume fraction) ethanol twice. Each dehydration treatment was followed by 15 minutes of immersion. After the final dehydration treatment, the cells were dropped onto a coverslip and freeze-dried in a freeze dryer. The dried samples were then sprayed with gold and examined.

[0181] 2. Experimental results

[0182] Through SEM results ( Figure 11) The biochar surface exhibits a porous structure with large pores and no apparent collapse. This structural feature provides a suitable growth environment for microorganisms, encouraging their colonization. It also increases the biochar's specific surface area, providing more active sites for pollutant adsorption.

[0183] 3. FT-IR scanning of biochar

[0184] 1. Experimental methods

[0185] After the biochar is fully freeze-dried, an appropriate amount of potassium bromide powder is added and ground and mixed. After being pressed into tablets, the tablets are placed in a Fourier transform infrared spectrometer and scanned in the range of 4000 ~ 400 cm-1.

[0186] 2. Experimental results

[0187] The FT-IR results of biochar are shown in Figure 12. In the FT-IR graph, infrared absorption peaks of various functional groups are observed, such as 3428 cm -1 (-OH), 1576 cm -1 (-C=O), 1384 cm -1 (-COOH), and 1081 cm -1 (-CO), the above results indicate that the prepared biochar contains rich oxygen-containing functional groups, which increases the active sites for biochar to adsorb pollutants and can provide nutrients for microorganisms to promote their growth and reproduction.

[0188] Example 8 Preparation of immobilized bacterial agent and degradation effect

[0189] 1. Preparation method

[0190] The preparation of immobilized bacteria (IB) adopts the adsorption-embedding method, which mainly includes adsorption, mixing and embedding, three steps:

[0191] Adsorption: 0.5 g of the biochar prepared in Example 7 was weighed and placed in a conical flask. After sterilization, the flask was cooled. 50 mL of the mixed bacterial flora FB (obtained in Example 5) was poured into the biochar and shaken in a shaker at 35°C for 2 h to perform adsorption as the adsorption system.

[0192] Mixing: Weigh 2g of sodium alginate and dissolve it in 50mL of water. Heat and stir until completely dissolved, then sterilize at 121°C for 20min. After cooling to room temperature, pour the adsorbed system into the sodium alginate solution and stir the mixed solution on a magnetic stirrer for 30min to ensure thorough mixing.

[0193] Embedding: Use a syringe to drip the mixed solution dropwise into a 0.02 g / mL CaCl₂ solution at a constant speed to embed the immobilized beads. After embedding, place the beads in a 4°C refrigerator to continue solidifying for 12 hours. After 12 hours, wash the beads three times with sterile saline to remove excess CaCl₂ on the surface. Store the beads in a 4°C refrigerator to obtain the immobilized bacterial agent.

[0194] At the same time, immobilized beads (SB) without added bacteria were prepared: that is, sterile physiological saline was directly added to the biochar during adsorption (instead of the mixed bacterial community FB obtained in Example 5), and the remaining steps were consistent with the immobilized bacterial agent preparation process.

[0195] 2. Apparent properties of immobilized bacteria (IB)

[0196] 1. Experimental methods

[0197] (1) Observe the shape of the inoculum with the naked eye, take a portion of the inoculum and weigh it, and measure the diameter of the inoculum with a vernier caliper.

[0198] (2) Determination of mass transfer efficiency: Add the pellets to 10 mL of 0.01% methylene blue solution at a ratio of 10%, place in a constant temperature oscillator at 30°C and 150 rpm for 24 hours, and observe the change in the absorbance of the solution at 665 nm before and after the addition of the immobilized bacterial agent.

[0199] (3) Mechanical strength determination: Add 10 immobilized bacteria of uniform size into 20 mL of pure water, shake in a shaking incubator at 220 rpm / min for 5 days, and observe the breakage of the bacteria.

[0200] 2. Experimental results

[0201] Finished products of immobilized bacteria agents such as Figure 13 Its apparent properties are shown in Table 5. The results showed that the immobilized bacteria agent was regular spherical and had high mass transfer efficiency and mechanical strength.

[0202] Table 5 Apparent properties of immobilized bacterial agents

[0203]

[0204] 3. SEM observation of the surface morphology and internal structure of immobilized bacteria (IB)

[0205] 1. Experimental methods

[0206] The immobilized bacterial culture (IB) was washed with sterile saline, the supernatant discarded, and this process repeated three times. 1 mL of 2.5% glutaraldehyde electron microscopy fixative was added to the washed culture, vortexed to mix, and incubated in a refrigerator at 4°C for 20 min. After fixation, the supernatant was discarded, and the culture was collected and washed three times with sterile saline. Subsequently, a gradient dehydration process was performed, with each of 30%, 50%, 70%, 80%, and 90% (volume fraction) ethanol added once, followed by 100% (volume fraction) ethanol twice. Each dehydration treatment was followed by 15 min of immersion. After the final dehydration treatment, the cells were dropped onto a coverslip and freeze-dried in a freeze dryer. The dried samples were then gold-sprayed and examined for surface morphology and internal structure.

[0207] 2. Experimental results

[0208] The surface morphology characteristics of the immobilized bacteria are as follows Figure 14 As shown in the figure, Figure A shows the overall morphology of the immobilized bacterial agent, which presents a regular spherical shape; Figure B is the surface morphology of the immobilized bacterial agent. Since the spheres shrink after gradient dehydration with ethanol, wrinkles appear on the surface, but a large number of strains can still be observed to be embedded in them; Figures C and D are scans of the cross section of the immobilized bacterial agent to observe its internal structure; in Figure C, it can be seen that there are a large number of honeycomb pores inside the spheres. The existence of this structure can increase the porosity and specific surface area of ​​the carrier, reduce the diffusion resistance, and is conducive to the transmission of oxygen and nutrients, providing a better microenvironment for the growth and metabolism of microorganisms, thereby accelerating their degradation of pollutants; in Figure D, a large number of bacteria were observed to be attached to the pores inside the carrier, which indicates that the embedded bacteria are growing well in the immobilized carrier.

[0209] 4. FT-IR analysis of immobilized bacterial agent (IB)

[0210] 1. Experimental methods

[0211] After the immobilized beads (SB) and immobilized bacterial agent (IB) without bacterial flora were fully freeze-dried, they were ground and mixed with appropriate amount of potassium bromide powder, pressed into tablets and placed in a Fourier infrared spectrometer. 1 The FT-IR spectrum was obtained by scanning within a certain range to analyze the composition of its functional groups.

[0212] 2. Experimental results

[0213] According to the FT-IR results ( Figure 15 ), 3428 cm -1 The absorption peak at 2972 ​​cm is generated by OH stretching vibration. The absorption intensity of this peak is enhanced after the biochar is combined with sodium alginate, indicating that a large number of hydrogen bonds are generated between the -OH groups of biochar and sodium alginate.-1 The new peak at 1418 cm in SB is attributed to the stretching vibration of CH, indicating that the biochar is firmly combined with the sodium alginate matrix due to the tight connection formed by hydrogen bonds. -1 、1618 cm -1 The absorption peaks at 1081 cm-1 are characteristic peaks of sodium alginate, representing the symmetric and antisymmetric stretching vibrations of COO-, respectively. -1 and 1034 cm -1 This corresponds to the deformation of -CO and the vibration of -COC in sodium alginate. Compared with IB, all the functional groups possessed by SB are present in IB, and the intensity of the absorption peak is significantly enhanced, which is closely related to the formation of hydrogen bonds during the material composite process, indicating that the materials are well bonded.

[0214] 5. Degradation effect of immobilized bacteria (IB) on BuX and BHA

[0215] 1. Experimental methods

[0216] 2 g of the prepared immobilized bacterial agent (IB), 2 g of the unencapsulated immobilized beads (SB), and 1 mL of the free bacterial colony (FB) prepared in Example 5 were added to 50 mL of MSM culture medium containing 100 mg / L BuX and BHA. The culture was shaken at 35°C and 150 rpm for 24 hours. Samples were taken at 12 and 24 hours of incubation, and the BuX and BHA contents were determined (using the same methods as in Examples 3 and 4). Three replicates were set up for each group. The control group consisted of MSM culture medium containing only 100 mg / L BuX and BHA, without inoculation.

[0217] 2. Experimental results

[0218] The results are as follows Figure 16 As shown, the degradation rates of pollutants ranked from fastest to slowest as follows: IB > FB > SB. In the IB group, 100 mg / L of BuX and BHA were completely degraded in just 9 and 30 hours, respectively, while in the FB group, it took 24 and 48 hours to completely degrade BuX and BHA, respectively. The removal rates in the SB group after 48 hours were only 50.45% and 22.25%, respectively. These results indicate that the preparation of immobilized bacterial agents significantly improved the degradation rates of BuX and BHA in a complex polluted system. The pollutant concentrations in the SB group were lower than in the CK group, indicating that the immobilized material had a certain adsorption effect on the pollutants. Comparison between the IB and SB treatments indicates that the reduction in pollutant concentrations in the IB group was primarily due to the degradation effect of the bacterial strain.

[0219] Example 9 Effect of Sodium Alginate Content on the Degradation of BuX and BHA by Immobilized Bacteria

[0220] 1. Experimental Methods

[0221] The immobilized bacterial agent (IB) was prepared according to the method of Example 1. The specific method is as follows:

[0222] Adsorption: same as Example 8;

[0223] Mixing: Dissolve 1g (1%), 2g (2%), 3g (3%), and 4g (4%) of sodium alginate in 50 mL of water. Heat and stir until completely dissolved, then sterilize at 121°C for 20 minutes. After cooling to room temperature, pour the adsorbed system into the sodium alginate solution and stir the mixture on a magnetic stirrer for 30 minutes to thoroughly mix.

[0224] Embedding: Same as Example 8.

[0225] The degradation of BuX and BHA by the immobilized bacterial agent prepared and tested according to the method of Example 6 was investigated.

[0226] 2. Experimental Results

[0227] The apparent morphology of the immobilized bacterial agents prepared at different sodium alginate contents is as follows: Figure 17 As shown in the figure, the immobilization beads prepared when the sodium alginate content is 2%, 3%, and 4% are all regular spheres, but the increase in sodium alginate content will lead to an increase in the viscosity of the material, and the difficulty of its preparation will also increase accordingly. When the sodium alginate content is 1%, the beads are irregular in shape and are very easy to break during use.

[0228] according to Figure 18 The degradation results for BuX and BHA in the culture medium show that at 1% and 2% sodium alginate content, the degradation rate of BuX exceeded 90% after 12 hours of incubation, and over 80% of BHA was degraded by 24 hours. When the sodium alginate content reached 3% and 4%, the degradation rate of BuX decreased slightly, reaching 88.62% and 88.99%, respectively, while the degradation rate of BHA decreased significantly, to 53.02% and 49.14%. This indicates that increasing the sodium alginate content leads to a decrease in the degradation efficiency of the immobilized bacteria for both pollutants, possibly due to a decrease in the mass transfer efficiency of the pellets.

[0229] Therefore, the immobilized bacterial agent prepared with 2% sodium alginate has the best apparent morphology and degradation effect on pollutants.

[0230] Example 9 Effect of biochar content on the degradation of BuX and BHA by immobilized bacteria

[0231] 1. Experimental Methods

[0232] The immobilized bacterial agent (IB) was prepared according to the method of Example 8. The specific method is as follows:

[0233] Adsorption: 0.5 g (i.e., 0.5% content), 1 g (i.e., 1% content), 1.5 g (i.e., 1.5% content), and 2 g (i.e., 2% content) of the biochar prepared in Example 7 were weighed and placed into a conical flask. After sterilization, the flask was cooled. 50 mL of the mixed bacterial community FB (obtained in Example 5) was poured into the biochar. The conical flask was then placed in a shaker at 35°C for 2 h for adsorption as an adsorption system.

[0234] Mixing: Same as Example 8;

[0235] Embedding: Same as Example 8.

[0236] The degradation of BuX and BHA by the immobilized bacterial agent prepared and tested according to the method of Example 6 was investigated.

[0237] 2. Experimental Results

[0238] There is no obvious difference in the morphology of immobilized bacteria with different biochar contents.

[0239] from Figure 19 As can be seen in the figure, when the biochar content is 0.5%, the degradation rates of both BuX and BHA pollutants reach the highest, at 97.23% and 96.32% respectively. When the biochar content exceeds 0.5%, the degradation rate of pollutants decreases significantly.

[0240] Example 9 Effect of CaCl2 Concentration on the Degradation of BuX and BHA by Immobilized Bacteria

[0241] 1. Experimental Methods

[0242] The immobilized bacterial agent (IB) was prepared according to the method of Example 8. The specific method is as follows:

[0243] Adsorption: same as Example 8;

[0244] Mixing: Same as Example 8;

[0245] Embedding: The mixed solution was dripped dropwise at a constant speed into 0.02g / mL, 0.03g / mL, 0.04g / mL, and 0.05g / mL CaCl₂ solutions through a 50 mL syringe to form immobilized microspheres. After embedding, the microspheres were placed in a 4°C refrigerator to allow them to solidify for 12 hours. After 12 hours, the microspheres were washed three times with sterile saline to remove excess CaCl₂ from the surface. The microspheres were then stored in a 4°C refrigerator to obtain the immobilized bacterial agent.

[0246] The degradation of BuX and BHA by the immobilized bacterial agent prepared and tested according to the method of Example 6 was investigated.

[0247] 2. Experimental Results

[0248] The results are as follows Figure 20 As shown, CaCl2 concentration has little effect on the degradation of BuX, but significantly affects the degradation of BHA. As CaCl2 concentration increases, the degradation rates of BuX increase to 94.28%, 96.05%, 95.87%, and 85.02%, respectively, while the degradation rates of BHA increase to 97.63%, 55.02%, 83.37%, and 47.56%, respectively. Considering the degradation of both pollutants, a CaCl2 concentration of 2 g / ml is the most suitable.

[0249] Example 10 Degradation ability of immobilized bacterial agents under strong acid and strong base

[0250] 1. Experimental Methods

[0251] The pH of the MSM culture medium was accurately adjusted to 5 and 9 respectively using a pH meter, and BuX and BHA were added to the aliquoted MSM culture medium so that the concentrations of both pollutants reached 100 mg / L.

[0252] 2% (volume ratio) of the free bacterial community (FB) prepared in Example 5, 2 g of the immobilized bacterial agent (IB) prepared in Example 8, and 2 g of the uninoculated immobilized beads (SB) prepared in Example 8 were added to 50 mL of MSM culture medium, respectively. After inoculation, the conical flask was placed at a constant temperature of 35 ° C and shaken at 150 rpm for 48 h. Samples were taken at different time points to determine the concentrations of BuX and BHA. Three replicates were set for each treatment group, and the blank group was MSM culture medium (CK) without inoculation.

[0253] 2. Experimental Results

[0254] like Figure 21 As shown, IB can effectively degrade BuX and BHA in both strong acid and strong base environments. At pH 5, complete degradation of BuX and BHA in the IB group took only 6 hours and 24 hours, respectively. When the pH was increased to 9, the degradation times were 12 hours and 0 hours, respectively.

[0255] The FB group was able to achieve highly efficient degradation of BuX and BHA in both strong acid and strong alkaline environments. At a pH of 5, FB only took 9 h and 30 h to completely degrade BuX and BHA in the composite system. When the pH reached 9, the degradation rates of BuX and BHA were only 58.21% and 23.22% after culturing for 24 h and 48 h, respectively.

[0256] In the SB group, the pH was 5 or 9, and the BHA removal rates were 25.78% and 18.46% at 48 h, respectively. Compared with them, the BuX removal rates at 24 h were quite different. The BuX in the former was completely removed, while the BuX removal rate in the latter was only 39.39% at the same time.

[0257] The results show that the immobilized bacterial agent prepared by the adsorption-encapsulation method has a significantly improved removal ability of BuX and BHA in an adverse pH environment under the protection of the biochar-sodium alginate carrier.

[0258] Example 11 Degradation ability of immobilized bacterial agents at high and low temperatures

[0259] 1. Experimental Methods

[0260] 2% (volume ratio) of the free bacterial group (FB) prepared in Example 5, 2 g of the immobilized bacterial agent (IB) prepared in Example 8, and 2 g of the uninoculated immobilized beads (SB) prepared in Example 8 were added to 50 mL of MSM culture medium. The blank control (CK) was not inoculated with bacteria. The initial pH of the degradation system was 7. The temperatures of the constant temperature shaker were set to 20°C and 40°C, respectively. The conical flask was then placed in it and reacted at 150 rpm for 48 h. Samples were taken at regular intervals to observe the changes in the concentrations of BuX and BHA in the solution.

[0261] 2. Experimental Results

[0262] Depend on Figure 22 It can be seen that no matter under high or low temperature conditions, the IB group was the first to completely degrade BuX and BHA.

[0263] IB degraded BHA in just 30 hours at either 40°C or 20°C. At 40°C, IB completely degraded BuX in just 6 hours. While high temperatures accelerated the degradation of BuX, compared to CK, the bacterial strain was the primary factor in the degradation process. When the temperature dropped to 20°C, the lower temperature inhibited BuX degradation, but IB still reached the endpoint within 9 hours.

[0264] At 40°C, the degradation rates of BuX and BHA were 96.61% and 70.35% after FB degradation for 24 and 8 hours, respectively. However, at 20°C, the degradation rates of BuX and BHA decreased significantly, reaching only 57.1% and 8.6% within the same time period.

[0265] The above results show that both high and low temperature conditions can have a significant inhibitory effect on the activity of free bacterial communities, and the prepared immobilized bacterial agent improves the temperature tolerance of the bacterial community and has broad development prospects in the remediation of actual contaminated sites.

[0266] Example 12 Degradation ability of immobilized bacterial agents under heavy metal stress

[0267] 1. Experimental Methods

[0268] Cd was added to 50 ml of MSM medium containing 100 mg / L BuX and 100 mg / L BHA. 2+ , so that its concentration reaches 5 mg / L, the initial pH of the system is 7, and then 2% (volume ratio) of the free bacterial group (FB) prepared in Example 5, 2 g of the immobilized bacterial agent (IB) prepared in Example 8, and 2 g of the uninoculated immobilized beads (SB) prepared in Example 8 are respectively inoculated therein. The reaction is carried out at 35°C and 150 rpm for 48 h. Each group is repeated three times. The blank control group (CK) is not subjected to other inoculation treatments. Samples are taken at regular intervals to determine the remaining BuX and BHA contents in the solution.

[0269] 2. Experimental Results

[0270] Depend on Figure 23 It can be seen that Cd 2+ Under stress, the degradation effect of immobilized strains on pollutants was significantly better than that of free bacteria. 2+ At a concentration of 5 mg / L, IB achieved a BuX degradation rate of over 99% within 9 hours, while the degradation rate of the FB group was only 37.6% within the same period. This difference was even more pronounced in the degradation of BHA: IB completely degraded a 100 mg / L BHA solution within 30 hours, while FB only degraded 28.8% of the BHA after 48 hours of culture.

[0271] Example 13 Recycling of Immobilized Bacteria

[0272] 1. Experimental Methods

[0273] BuX and BHA were added to 50 mL of MSM culture medium with an initial pH of 7, achieving a final concentration of 100 mg / L. Two grams of immobilized bacterial culture was then weighed and added to the medium. The sealed conical flask was then placed in a 35°C incubator for 48 hours with shaking. Samples were taken at intervals, and the changes in BuX and BHA concentrations over time were recorded. After 48 hours, the immobilized spheres were separated from the culture medium, washed three times with sterile saline, and transferred to fresh culture medium. Culture was repeated under the above conditions, and this process was repeated multiple times until the immobilized spheres ruptured.

[0274] 2. Experimental Results

[0275] result Figure 24As shown, repeated use leads to a loose internal structure of the immobilized beads, a continuous decrease in the number of entrapped live cells, and the active sites of the immobilized material become occupied, reducing its adsorption capacity for pollutants. Therefore, as the number of cycles increases, the time required for the immobilized bacteria to completely degrade the pollutants also increases. The mechanical strength of the immobilized beads gradually decreases after repeated recycling, until they break after the fifth cycle. However, after five cycles, the immobilized beads were still able to completely degrade BuX and BHA at an initial concentration of 100 mg / L within 48 hours, indicating that the immobilized bacterial community is more stable and has good recyclability.

[0276] Example 14 Storage stability of immobilized bacterial agent

[0277] 1. Experimental Methods

[0278] The immobilized bacterial agent prepared in Example 8 was stored in a refrigerator at 4°C for 0, 10, 30, 50, 70, and 90 days. It was then inoculated into 50 mL of MSM culture medium (pH = 7) with an initial concentration of 100 mg / L for both BuX and BHA at 35°C and 150 rpm. Samples were taken after 24 and 48 hours of reaction to determine the BuX and BHA contents, respectively.

[0279] 2. Experimental Results

[0280] The degradation effect of immobilized bacteria on BuX and BHA after storage for a certain period of time is as follows: Figure 25 As shown in Figure 2 , the freshly prepared immobilized bacterial agent achieved a 100% degradation rate for both BuX and BHA. After 70 days of storage at 4°C, the degradation rate remained above 98%. After 90 days of storage, the degradation rate of BuX remained above 98%, while the degradation rate of BHA decreased to 84.3%. These results demonstrate that the immobilized bacterial agent prepared by the adsorption-encapsulation method can maintain good microbial activity even after long-term storage at low temperatures.

[0281] Example 15 Remediation of composite contaminated soil using immobilized bacterial agents

[0282] 1. Preparation of simulated contaminated soil

[0283] The test soil was divided into two parts, one part was not treated (S1), and Cd(NO3)2 solution was added to the other part (S2) to make Cd 2+ Once the final concentration reaches 5 mg / kg, the soil is aged in a well-ventilated area for one month. BuX and BHA are then added slowly, in portions, and thoroughly stirred until the concentrations of both groups reach 50 mg / kg. The resulting composite contaminated soils are then aged in the dark for two weeks.

[0284] 2. Remediation of composite contaminated soil

[0285] The aged contaminated soils S1 and S2 were divided into four equal portions, each containing 200 g of soil, and placed in 500 mL plastic boxes. To each soil group, 5% (w / w) of the immobilized bacterial agent (IB) prepared in Example 8, 5% (w / w) of the uninoculated immobilized pellets (SB) prepared in Example 8, and 5% (v / v) of the free bacterial flora (FB) prepared in Example 5 were added. The final portion was supplemented with 5% sterile water as a blank control (CK). Three replicates were set up for each treatment group. During the remediation of the composite contaminated soil, water was regularly added to maintain a moisture content of approximately 20%. Samples were collected on days 0, 3, 5, 7, and 14 of remediation. Specific groupings are shown in Table 6 below.

[0286] Table 6 Grouping of remediation of composite contaminated soil

[0287]

[0288] Example 15 Effect of Immobilized Bacteria on BuX Removal in Composite Contaminated Soil

[0289] 1. Experimental Methods

[0290] The BuX content of the soil of each sample of Example 14 was detected by the following method:

[0291] To prevent BuX decomposition, freeze-dry a portion of the soil as soon as possible after sampling. Accurately weigh 10 g of dried soil into a 50 mL centrifuge tube and add 4 mL of ammonia-methanol solution (30 / 70, v / v). Shake at 2500 rpm for 8 minutes to achieve complete extraction. After shaking, centrifuge at 8000 rpm for 10 minutes. Transfer the supernatant to a new centrifuge tube and extract the precipitate again according to the above steps. After two centrifugations, combine the supernatants. Add 50 mg of C18 scavenger to the combined supernatant, vortex for 1 minute to fully adsorb impurities, and centrifuge again at 8000 rpm for 10 minutes. Pass the supernatant through a 0.22 μm filter membrane, and adjust the filtrate pH to approximately 10.5 with formic acid. Detect BuX using the method described in Example 4.

[0292] 2. Experimental Results

[0293] The results are as follows Figure 26 As shown in the figure, BuX content in the CK group will gradually decrease because BuX itself is unstable and its structure is relatively simple, making it easily utilized by some indigenous microorganisms. At 14 days after restoration, the BuX content in the CK group in S1 and S2 was 25.95 and 29.18 mg / kg, respectively. The higher BuX content in S2 is due to the Cd 2+It will chelate with BuX, inhibiting the natural degradation of BuX, while also increasing its toxic effects on indigenous microorganisms and interfering with their utilization of BuX.

[0294] At 14 days after restoration, the BuX contents in the FB groups of S1 and S2 were 10.82 and 20.54 mg / kg, respectively. This shows that the addition of free bacteria did promote the degradation of BuX in the soil, but Cd 2+ The presence of free bacteria significantly inhibited the degradation of BuX. Although the SB group was not affected by the combined pollution, the adsorption of BuX reached saturation after 14 days of repair, and the BuX removal rate reached about 60%.

[0295] Compared with the above groups, the IB group with the immobilized bacterial agent effectively removed BuX from the soil in both S1 and S2, with the BuX removal rate exceeding 90% after 14 days. These results indicate that the immobilized bacterial agent prepared via the adsorption-encapsulation method significantly mitigated the toxic effects of pollutants on the microbial community, enhanced the microbial community's tolerance to the environment, and accelerated the microbial community's removal rate of pollutants.

[0296] Example 15 Effect of Immobilized Bacteria on pH of Composite Contaminated Soil

[0297] 1. Experimental Methods

[0298] The soil pH of each sample in Example 14 was measured as follows: 2 g of air-dried soil was placed in a 50 mL centrifuge tube and 5 mL of CO₂-free deionized water was added at a ratio of 1:2.5 (w / v). The tube was sealed and shaken at 180 rpm for 30 minutes. After standing for 45 minutes, the pH of the supernatant was measured using a pH meter. The average of the three measurements was used to determine the soil pH.

[0299] 2. Experimental Results

[0300] Figure 27 It can be seen that the initial soil pH was weakly acidic, and the pH of S1 after FB restoration for 14 days was higher than that of CK group. 2+ The presence of biochar in the soil not only limits the decomposition of pollutants but also inhibits their removal by the microbial community. At this point, there was no significant difference in pH between FB and CK. The soil pH in both the IB and SB groups continued to rise throughout the remediation process, indicating that the alkalinity of biochar can neutralize slightly acidic soils to a certain extent, thereby raising their pH.

[0301] Example 16 Effect of immobilized bacterial agents on enzyme activity in complex polluted soil

[0302] 1. Pretreatment of soil samples

[0303] After sampling, part of each sample in Test Example 14 was air-dried and passed through a 200-mesh sieve for determination of enzyme activity.

[0304] 2. Effects of immobilized bacterial agents on catalase activity in composite-contaminated soils

[0305] 1. Experimental methods

[0306] (1) Construct a standard curve for catalase activity: take 0, 1, 2, 3, 4, and 5 mL of 0.3% (v / v) H2O2 solution in a 50 mL volumetric flask, add 5 mL of sulfuric acid (1.5 mol / L), dilute to the mark with deionized water, then add 1 mL of saturated potassium aluminum sulfate solution, mix well, and measure the absorbance of the solution at 240 nm.

[0307] (2) Take 2 g of the pretreated soil sample and place it in a 50 mL centrifuge tube. Accurately pipette 40 mL of deionized water and 5 mL of 0.3% H2O2 solution into the sample. Oscillate the sample at 200 rpm on an oscillator for 20 min. Immediately add 1 mL of saturated potassium aluminum sulfate solution, mix well, and filter the mixture into a conical flask containing 5 mL of sulfuric acid (1.5 mol / L).

[0308] (3) Measure the OD240nm of the filtrate using UV-Vis.

[0309] This experiment requires the establishment of soil-free and substrate-free controls.

[0310] 2. Experimental results

[0311] The results are as follows Figure 28 As shown, soil catalase activity in the S1 group generally showed a downward and then upward trend throughout the remediation process. On the third day, enzyme activity decreased, indicating that BuX and BHA inhibited soil catalase activity. The two degrading bacteria in the immobilized microbial agent require time to adapt to their new environment. Afterward, they can utilize BuX and BHA in the soil as a carbon source, promoting their own growth and reproduction while also reducing the concentration of pollutants in the environment. Consequently, catalase activity in the FB and IB groups continued to increase after three days, reaching 2.14 and 2.47 mg / g, respectively, by the 14th day of remediation. The most significant change in enzyme activity was observed in the IB group, where soil catalase activity increased by 33.36% compared to the CK group. The increased catalase activity in the SB group may be due to the adsorption of pollutants by the immobilized material, which weakened the impact of pollutants on the activity of indigenous microorganisms and increased soil catalase activity.

[0312] 2. Effects of immobilized bacterial agents on urease activity in composite-contaminated soil

[0313] 1. Experimental methods

[0314] (1) Construct a standard curve for urease activity: Accurately pipette 0, 1, 3, 5, 7, 9, 11, and 13 mL of a 0.1 mg / mL ammonia standard solution into a 50 mL volumetric flask and add water to 20 mL. Then, add 4 mL of sodium phenolate solution and 1.5 mL of sodium hypochlorite solution, mix well, and let it stand for 20 minutes. After color development, dilute to the scale and measure the absorbance at 578 nm within 60 minutes.

[0315] (2) Weigh 1 g of the pretreated soil sample into a 50 mL conical flask and add 0.2 mL of toluene to eliminate microbial interference. After standing for 15 min, add 4 mL of citrate buffer solution (pH 6.7) and 2 mL of 10% (w / w) urea solution to the conical flask, mix well, and place in a constant temperature shaker at 37 °C and 150 rpm for 24 h. After incubation, filter it and transfer 0.2 mL of the filtrate to a 10 mL colorimetric tube. Then, add 0.8 mL of sodium phenolate solution and 0.6 mL of sodium hypochlorite solution in sequence. After color development for 20 min, dilute to 10 mL with water.

[0316] (3) Measure the absorbance of the solution at a wavelength of 578 nm within 60 minutes.

[0317] At the same time, soil-free and matrix-free controls were set up to eliminate the influence of the original ammonia in the soil samples on the results.

[0318] 2. Experimental results

[0319] The results are as follows Figure 29 As shown in Figure 2, after 14 days of restoration, the urease activities of CK, SB, FB and IB in group S1 were 0.19, 0.40, 0.42 and 0.41 mg / (g•h), respectively, which can increase the soil urease activity. 2+ stress, the urease activity of the FB group was only 0.20 mg / (g•h), while the urease activity of the IB group reached 0.33 mg / (g•h). This result shows that the addition of immobilized bacteria helps to increase soil urease activity, catalyze urea hydrolysis, promote soil nitrogen cycle, and improve soil fertility.

[0320] Example 17 Effect of immobilized bacterial agents on bacterial community structure in complex contaminated soil

[0321] 1. Experimental Methods

[0322] Twenty-four soil samples treated on day 14 in Example 14 were collected and sent to Guangzhou Jidi'ao Biotechnology Co., Ltd. for 16S rRNA amplicon sequencing. PCR amplification of the V3-V4 region of the bacterial 16S rRNA gene was performed using specific primers (341F: 5'-CCTACGGGNGGCWGCAG-3'; 806R: 5'-GGACTACHVGGGTATCTAAT-3'). The amplified products were extracted and purified, and then sequenced using the Illumina platform of Guangzhou Jidi'ao Biotechnology Co., Ltd.

[0323] 2. Experimental Results

[0324] 1. Changes in soil bacterial community diversity

[0325] Based on the 16S rRNA sequencing results, the Alpha diversity of the soil bacterial community in each treatment group was analyzed at 14 days after restoration, and the results are shown in Table 7.

[0326] Table 7 Alpha diversity index of bacteria:

[0327]

[0328] Note: The data in the table are the mean ± standard deviation of 3 parallel samples. The same letter at the end of the data in the same column indicates no significant difference (P>0.05).

[0329] Shannon diversity index: an indicator that comprehensively measures species richness and individual distribution uniformity, often used to assess the diversity of ecosystems; Simpson diversity index: a diversity index that focuses on reflecting the relative richness of species, often used to describe the differences in the number of individuals between species; Chao1: a method for estimating the number of unobserved species based on sample data, suitable for situations where the sample size is small or the species distribution is uneven; Abundance-based Coverage Estimator (ACE): a method for estimating the total number of species in the entire community based on the individual number distribution of species in the sample; Coverage: refers to the proportion of species observed in the sample to the actual total number of species, used to evaluate the representativeness of the sample and the completeness of the survey.

[0330] The Coverage index represents the sequencing depth, and its values ​​are all greater than 0.98, indicating that the sequencing results can reflect the actual situation of the microorganisms in the sample.

[0331] The Chao1 and ACE indices of the CK group in S2 were higher than those of the CK group in S1, while the Shannon and Simpson indices were higher in the CK group in S1.2+ The presence of heavy metals exacerbates biological toxicity, strains that cannot tolerate heavy metal stress cannot survive, the diversity of indigenous microorganisms decreases, and the abundance of dominant strains increases.

[0332] In S1, the diversity of SB and FB groups decreased compared with that of CK group, but their Chao1 and ACE indices increased, indicating that the addition of free bacteria and immobilized bacterial agents can increase the abundance of bacterial communities.

[0333] The various indexes of the FB group in S2 increased, indicating that the free bacterial community can tolerate a certain concentration of heavy metals. The addition of free bacterial community can stimulate the activity of indigenous microorganisms, thereby increasing their diversity and abundance.

[0334] The various indexes of the treatment group inoculated with immobilized bacteria were the highest in S1 compared with other groups, indicating that the materials used to prepare the immobilized bacteria provided a more stable growth environment for microorganisms, and the addition of foreign bacteria could stimulate the growth of indigenous bacteria. The immobilized bacteria prepared by combining the two helped to increase the diversity and abundance of soil bacterial communities.

[0335] 2. Beta diversity analysis

[0336] Beta diversity analysis is to compare the bacterial community composition in different treatment groups and describe the similarity of species composition structure by the distance between treatment groups. The closer the distance, the more similar the composition structure. Figure 30 The PcoA diagram is a Bray-Curtis distance calculated based on the OUT of each treatment group. It shows that in S1, the SB and FB groups are close to each other, but farther away from the other groups, indicating that the two treatments can change the community structure of indigenous microorganisms, but the bacterial community structure in the soil after the two treatments is not much different. In S2, CK, SB, and FB are all close to each other, indicating that Cd 2+ Under stress, the addition of free bacteria or immobilized microbial agents to the composite contaminated soil had little effect on the original bacterial community structure. However, the IB group in both S1 and S2 remained relatively distant from the other groups, indicating that the addition of immobilized microbial agents significantly altered the soil bacterial community structure.

[0337] 3. Changes in soil bacterial community abundance

[0338] (1) Door level

[0339] The results are as follows Figure 31At the phylum level, the bacterial composition of treatments S1 and S2 was largely consistent, with the dominant phyla including Proteobacteria (20.6-42.49%), Bacteroidota (14.23-32.42%), Actinobacteriota (7.93-16.73%), Chloroflexi (8.95-13.36%), Acidobacteriota (5.89-9.66%), and Firmicutes (3.26-10.66%). The proportions of each dominant phylum varied slightly between treatments, with Proteobacteria and Bacteroidota being the two most dominant phyla.

[0340] Bacteroidota was the most abundant phylum in the CK and FB groups, accounting for 25.42% and 32.42% in the CK group and 27.68% and 26.98% in the FB groups for S1 and S2, respectively. However, the proportion of this phylum decreased to 14.23% and 17.81% in the IB groups for S1 and S2, respectively. In the treatments with immobilized microorganisms, Proteobacteria became the predominant phylum, accounting for 31.82% and 42.49%, respectively. Compared to the CK group, the relative abundance of this phylum increased by 54.24% and 71.74%, respectively. These results suggest that Proteobacteria plays a key role in the degradation of BuX and BHA, and that the addition of immobilized microorganisms significantly increased the relative abundance of this phylum in the soil.

[0341] (2) Genus level

[0342] Figure 32The relative abundance of each genera in the soil bacterial communities of different treatment groups at the genus level was shown. The dominant genera mainly included Sphingomonas (9.71-15.96%), Cupriavidus (0.15-10.01%), Ramlibacter (0.86-3.46%), Pseudomonas (0.06-9.22%), The genus Flavisolibacter (12.5-27.84%) of the Bacteroidota, the genus Bacillus (2.04-9.47%) of the Firmicutes, the genus Micromonospora (1.87-6.11%) and the genus Streptomyces (0.69-1.78%) of the Actinobacteriota.

[0343] like Figure 33 As shown in the figure, the proportion of Pseudomonas in the FB group of S1 and S2 was only 0.18% and 0.17%, respectively. In comparison, the relative abundance of Pseudomonas in the IB group increased significantly, accounting for 2.07% and 9.22% in S1 and S2, respectively. This result shows that the immobilized bacterial agent can effectively prevent the embedded microorganisms from being lost to the external environment by embedding them in a certain area of ​​space, and significantly increase their abundance. In S1, the competition among the bacterial communities was fierce, resulting in a low relative abundance of Pseudomonas, while in S2, due to Cd 2+ The toxic effect of Cd caused the abundance of strains that could not tolerate heavy metal stress to decrease, and Pseudomonas became the dominant genus due to its high tolerance to Cd, and its relative abundance was further increased.

[0344] Cluster analysis of the top nine bacterial genera in relative abundance showed that in addition to the increase in the relative abundance of the genera containing the bacteria contained in the immobilized bacterial agent, the addition of the immobilized bacterial agent can also stimulate the growth of certain indigenous bacteria and increase the relative abundance of these genera in the soil bacterial community.

[0345] Compared to the CK group, the abundance of Cupriavidus, Micromonospora, Streptomyces, and Sphingomonas increased in the IB group, with the most significant change in Cupriavidus. Studies have shown that Cupriavidus strains are highly effective in degrading pollutants containing N-heterocyclic structures, suggesting that the increased abundance of this genus may be due to its ability to degrade BHA in the soil.

[0346] Micromonospora and Streptomyces belong to the phylum Actinobacteriota. Actinomycetes, as one of the plant growth-promoting rhizobacteria (PGPR), have been widely confirmed to promote plant growth and improve soil fertility.

[0347] Most Sphingomonas bacteria have excellent ability to degrade aromatic compounds. In addition, relevant studies have shown that this genus is also a PGPR with great application potential. It promotes plant growth by regulating plant physiological metabolism and recruiting beneficial rhizosphere bacteria.

[0348] In summary, immobilized bacterial agents can weaken the toxic effects of pollutants on indigenous microorganisms, provide a suitable growth environment for beneficial bacteria, and make them the dominant bacterial genera in the soil. The enrichment of beneficial bacteria can improve the soil environmental quality to a certain extent.

Claims

1. A composition, characterized in that Contains Pseudomonas Pseudomonas sp.) and Cuproplastes ( Cupriavidus sp.), The Pseudomonas sp. was deposited in Guangdong Provincial Microbiological Culture Collection Center on July 31, 2024, with the deposit number: GDMCC NO. 64924; The copper-greedy bacteria was deposited in the Guangdong Provincial Microbial Culture Collection Center on September 11, 2024, and its preservation number is: GDMCCNo: 65117.

2. A bacterial agent, characterized in that The composition according to claim 1 is used for preparation.

3. A method for preparing an immobilized bacterial agent, characterized in that: The following steps are involved: S1. The biochar is mixed with the composition according to claim 1 and adsorbed to obtain product 1; S2. The sodium alginate aqueous solution is thoroughly mixed with product 1 to obtain product 2; S3. Product 2 is added dropwise to a CaCl2 solution until fully solidified and purified.

4. The preparation method according to claim 3, characterized in that In product 1, the concentration of biochar is 0.005-0.045 g / mL.

5. The preparation method according to claim 3, characterized in that In product 2, the concentration of sodium alginate is 0.02-0.08 g / mL.

6. The preparation method according to claim 3, characterized in that The concentration of CaCl2 solution is 0.01~0.04g / mL.

7. The preparation method according to claim 3, characterized in that The biochar preparation method comprises the following steps: preparing corn straw powder and calcining the powder at a high temperature of 450-550° C. for 3-5 hours.

8. The immobilized bacterial agent prepared by the preparation method according to any one of claims 3 to 7.

9. Use of the composition according to claim 1 in preparing a product for simultaneously degrading butyl xanthate and benzohydroxamic acid, or in simultaneously degrading butyl xanthate and benzohydroxamic acid.

10. Use of the bacterial agent according to claim 2 or the immobilized bacterial agent according to claim 8, characterized in that: The application is one or more of the following: Preparation of products for the simultaneous degradation of butyl xanthate and benzohydroxamic acid; Application in the simultaneous degradation of butyl xanthate and benzohydroxamic acid; Application in remediation of soil contaminated by butyl xanthate and benzohydroxamic acid; Application of butyl xanthate and benzohydroxamic acid in remediation of contaminated water bodies.

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