Electrical resistant polymer-degrading bacterial agent for treating poly-containing oily sludge and preparation method thereof

Abstract

The application provides a polymer-degrading bacteria agent for treating poly-containing oily sludge and a preparation method thereof. The preparation method comprises the following steps: laying the homogenized poly-containing oily sludge in an electrochemical biological coupling treatment device, the device comprising electrode plates on both sides, the electrode plates on both sides generating a voltage to act on the poly-containing oily sludge; after the device is powered on, the original microorganisms in the poly-containing oily sludge are subjected to voltage domestication; the poly-containing oily sludge subjected to voltage domestication is inoculated into a culture medium to be cultured, separated and purified, so as to obtain at least one target strain; ecological construction is performed according to the obtained at least one target strain, so as to obtain the polymer-degrading bacteria agent. Through voltage domestication, the high-efficiency bacteria agent capable of performing microbial degradation in the reaction device under different voltage conditions is obtained. The bacteria agent has strong adaptability to the poly-containing sludge, can tolerate certain toxicity, and has good degradation effect on the polymer molecules in the poly-containing sludge.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to an electrostatic polymer-degrading bacterial agent for treating polymeric oily sludge and its preparation method. Background Technology

[0002] With the development of the petroleum industry, many oilfields have entered the high water-cut development stage, posing challenges to the efficient recovery of oil resources. Polymer flooding is widely used due to its significant improvement in oil recovery, but it also generates a large amount of polymer-containing oily sludge. This sludge has a complex composition, mainly consisting of crude oil, fine sand, salt-resistant polymers (such as HPAM), and chemical additives. It has high viscosity and easy settling characteristics, making it difficult to effectively solve the problem using traditional treatment methods.

[0003] Currently, oily sludge containing polymers is mainly treated using biological or electrochemical technologies. Biological treatment technologies are gaining increasing attention due to their high safety, low energy consumption, and environmental friendliness. Microorganisms secrete degrading enzymes (such as amidases and laccases) to transform complex pollutants into harmless substances. Electrochemical methods, as an important branch of sludge reduction technology, also demonstrate remarkable potential. Through the interaction of an electric field and low current, they trigger redox reactions, decomposing pollutants and promoting water-oil separation, thereby effectively reducing sludge volume.

[0004] However, given the complexity and unique characteristics of polymer-containing oily sludge, especially the presence of salt-resistant polymers such as HPAM, higher demands are placed on the screening and cultivation of degrading strains. Therefore, it is necessary to find and cultivate salt-resistant polymer-degrading bacteria that possess both high-efficiency degradation capabilities and tolerance to electric field environments to support the effective implementation of electrochemical-biological coupling technology. Summary of the Invention

[0005] This application provides an electrostatic polymer-degrading bacterial agent for treating polymeric oily sludge and its preparation method.

[0006] In a first aspect, this application provides a method for preparing an electrostatically resistant polymer-degrading bacterial agent for treating polymeric oily sludge, comprising:

[0007] In an electrochemical-biological coupling treatment device, homogenized aggregated oily sludge is laid. The electrochemical-biological coupling treatment device includes electrode plates on both sides, and the electrode plates on both sides generate voltage to act on the aggregated oily sludge.

[0008] After the electrochemical-biological coupling treatment device is powered on, it performs voltage acclimatization on the original microorganisms in the aggregated oily sludge.

[0009] The voltage-acclimated oily sludge was inoculated into a culture medium for cultivation, separation, and purification to obtain at least one target strain;

[0010] Ecological construction is carried out based on the obtained at least one target strain to obtain the polymer-degrading bacterial agent.

[0011] Furthermore, the step of inoculating the voltage-acclimated oily sludge into a culture medium for cultivation, isolation, and purification to obtain at least one target strain, including:

[0012] The voltage-acclimated oily sludge was inoculated into an enrichment medium and cultured under the following conditions: temperature 20-35℃, shaking speed 120-150rpm, culture time 24-48 hours.

[0013] The bacterial solution in the enrichment medium is screened out by dilution and spreading on a solid separation medium to identify single colonies that grow independently. The solid separation medium includes a salt-resistant polymer.

[0014] Single colonies were picked from the solid separation medium and inoculated into liquid LB medium for culture. The selection principle for single colonies was based on colony phenotype.

[0015] The single-colony bacterial suspension in the liquid LB medium was purified by streak plating to obtain at least one of the target strains.

[0016] Furthermore, the voltage acclimatization period is 7-10 days. During the voltage acclimatization process, nutrient salt solution is sprayed at preset intervals, stirred evenly, and spread out.

[0017] Furthermore, the voltage for voltage conditioning is less than 30V.

[0018] Furthermore, the target strain includes one or more of Alcaligenes faecalis, Microbacterium oxysporum, Bacillus cereus, and Aspergillus sclerotiorum.

[0019] Furthermore, the ecological construction includes: mixing and culturing multiple target strains in a preset ratio.

[0020] Furthermore, the preparation method further includes:

[0021] The biomass of the polymer-degrading microbial agent is increased by using a seed culture medium, wherein the seed culture medium, in addition to the liquid-based LB medium, also includes inorganic salts and trace elements, wherein the inorganic salts are selected from at least one of dipotassium hydrogen phosphate, sodium acetate, and triammonium citrate, and the trace elements are selected from at least one of magnesium ions, manganese ions, and iron ions.

[0022] Furthermore, the seed culture medium comprises: 0.5-2.0% yeast extract, 1-2% peptone, 0.25-0.35% triammonium citrate, 0.05-0.15% dipotassium hydrogen phosphate, 0.5-1.0% sodium chloride, 0.01-0.05% magnesium sulfate, 0.1-0.5% sodium thiosulfate, and 1L distilled water.

[0023] Secondly, this application provides a polymer-degrading microbial agent, which is prepared by the method for preparing an electrostatic polymer-degrading microbial agent for treating polymeric oily sludge as described in any one of the first aspects.

[0024] Thirdly, a method for applying a polymer-degrading microbial agent is provided, wherein the polymer-degrading microbial agent as described in the second aspect is added to polymer-containing oily sludge and degraded by electricity in an electrochemical-biological coupling treatment device.

[0025] This application provides an electrostatically resistant polymer-degrading bacterial agent for treating polymer-containing oily sludge and its preparation method. The preparation method includes: laying homogenized polymer-containing oily sludge in an electrochemical-biological coupling treatment device, the device including electrode plates on both sides, the electrode plates generating a voltage applied to the polymer-containing oily sludge; after the electrochemical-biological coupling treatment device is energized, voltage acclimation is performed on the native microorganisms in the polymer-containing oily sludge; the voltage-acclimated polymer-containing oily sludge is inoculated into a culture medium for cultivation, separation, and purification to obtain at least one target strain; ecological construction is performed based on the obtained at least one target strain to obtain the polymer-degrading bacterial agent. By acclimating the highly efficient polymer-degrading bacteria among the native microorganisms in polymer-containing sludge with an electric field, a highly efficient bacterial agent capable of microbial degradation under different voltage conditions in a reaction device is obtained. This bacterial agent has strong adaptability to polymer-containing sludge, can tolerate certain toxicity, and has a good degradation effect on polymer molecules in polymer-containing sludge. Attached Figure Description

[0026] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0027] Figure 1 This is a technical roadmap of the present invention;

[0028] Figure 2 Diagram of an electrochemical-biological coupling technology device;

[0029] Figure 3 This image shows the colony morphology of electropolymer-resistant fungi on an isolation medium.

[0030] Figure 4 The polymer degradation rate of different bacterial agents;

[0031] Figure 5 COD degradation rate of different microbial agents;

[0032] Figure 6 The effect of trace elements on bacterial biomass;

[0033] Figure 7 The effect of inorganic salts on bacterial biomass;

[0034] Figure 8 This is a scanning electron microscope image of the original aggregated oily sludge;

[0035] Figure 9 This is a scanning electron microscope image of the electrochemically treated oily sludge containing polymers.

[0036] Figure 10 This is a scanning electron microscope image of polymeric oily sludge after treatment by an electrochemical-biological coupling process.

[0037] The accompanying drawings illustrate specific embodiments of this application, which will be described in more detail below. These drawings and descriptions are not intended to limit the scope of the concept in any way, but rather to illustrate the concept of this application to those skilled in the art through reference to particular embodiments. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0039] Polymer-containing oily sludge (such as sludge generated during oil extraction or refining) is complex and unique, containing a large amount of polymers, especially high-molecular-weight polyacrylamide (HPAM), a salt-resistant polymer. HPAM is a polymeric flooding agent commonly used in oil extraction. It has strong water solubility and stability, making it difficult to degrade in water bodies, which poses a significant challenge to sludge treatment and environmental restoration.

[0040] The presence of HPAM and other salt-resistant polymers in sludge makes traditional biodegradation methods ineffective. HPAM has a highly stable chemical structure and maintains good physicochemical properties even under high salt and high temperature conditions, posing a significant obstacle to microbial degradation. Even in environments with severe organic pollution, these polymers are difficult for traditional microbial communities to degrade. Due to its high water solubility, HPAM often remains in wastewater or sludge for extended periods, posing a potential environmental hazard.

[0041] For sludge, electrochemical methods, as an important branch of volume reduction technology, have also demonstrated remarkable potential. This technology cleverly utilizes an electric field and low current to initiate redox reactions in the sludge system, precisely decomposing organic and inorganic pollutants. Furthermore, it promotes water-oil separation through electrochemical reactions, significantly reducing sludge volume and achieving the volume reduction goal. The core advantage of electrochemical methods lies in their direct action at the molecular level. By disrupting the molecular structure of pollutants, they achieve highly efficient removal. Studies have confirmed their outstanding performance in treating pollutants such as polycyclic aromatic hydrocarbons, petroleum hydrocarbons, heavy metals, and semi-volatile chlorides in soil and wastewater.

[0042] This application couples microbial treatment with electrochemical technology to construct an electrochemical-biological coupling system, which can fully leverage the advantages of both to achieve a synergistic effect in the treatment of polymer-containing oily sludge. The electrochemical-biological coupling system generates current through electrode electrochemical reactions, driving microbial metabolism to degrade pollutants or improving the degradation efficiency of microorganisms. In this technology, microorganisms not only need to possess degradation capabilities but also need to be able to grow and metabolize normally under the influence of an electric field. Therefore, how to screen and cultivate microorganisms that can both degrade salt-resistant polymers and tolerate the electric field environment becomes crucial.

[0043] The technical solution of this application and how the technical solution of this application solves the above-mentioned technical problems are described in detail below with specific embodiments. These specific embodiments can be combined with each other, and the same or similar concepts or processes may not be described again in some embodiments. The embodiments of this application will now be described with reference to the accompanying drawings.

[0044] The first aspect of this application provides a method for preparing an electrostatic polymer-degrading bacterial agent for treating polymeric oily sludge, comprising:

[0045] S1. The collected raw aggregated oily sludge is homogenized and then laid in an electrochemical-biological coupling treatment device. The electrochemical-biological coupling treatment device includes electrode plates on both sides, and the electrode plates on both sides generate voltage to act on the aggregated oily sludge.

[0046] Optionally, the electrochemical coupling treatment device also includes a leak-proof layer to prevent sludge leakage.

[0047] S2. After the electrochemical-biological coupling treatment device is powered on, it performs voltage acclimatization on the original microorganisms in the aggregated oily sludge.

[0048] S3. The voltage-acclimated oily sludge containing aggregates was inoculated into a culture medium for cultivation, separation and purification to obtain at least one target strain.

[0049] In this step, the isolation process requires screening for salt-resistant polymer-bearing bacterial strains; therefore, the isolation medium includes salt-resistant polymers. The polymers that need to be degraded in polymer-containing oily sludge may differ, depending on the type of polymer used in polymer flooding. Therefore, when screening for target strains, the salt-resistant polymer added to the isolation medium is the polymer that needs to be degraded; the specific type of polymer is not limited here.

[0050] It should be noted that the original microorganisms of aggregated oily sludge from different regions or obtained by different treatment methods may be different. Therefore, the target strains isolated may also be different.

[0051] S4. Based on at least one target strain obtained, construct an ecosystem to obtain a polymer-degrading bacterial agent.

[0052] In one implementation, each target strain selected can be used individually as a polymer-degrading agent.

[0053] In one implementation, two target strains are mixed as a polymer-degrading agent.

[0054] In one implementation, multiple target bacterial strains are mixed as polymer-degrading agents. Specifically, the obtained multiple bacteria are mixed according to a preset ratio, which is related to the polymer content and oil content in the polymer-containing oily sludge. Different strains have different abilities to decompose polymers.

[0055] The method for preparing an electrostatic polymer-degrading bacterial agent for treating polymer-containing oily sludge provided in this embodiment obtains a highly efficient bacterial agent capable of microbial degradation under high voltage conditions by acclimating the highly efficient polymer-degrading bacteria among the native microorganisms in the polymer-containing sludge to an electric field. This bacterial agent has strong adaptability to polymer-containing sludge, can tolerate certain toxicity, and has a good degradation effect on polymer molecules in the polymer-containing sludge.

[0056] Furthermore, during the voltage acclimatization period of 7-10 days, a nutrient solution is sprayed at preset intervals, stirred evenly, and spread evenly. The nutrient solution comprises 0.1-0.5% glucose, 0.1-0.5% potassium dihydrogen phosphate, 0.1-0.5% sodium chloride, 0.1-0.5% sodium nitrate, 0.1-0.5% ammonium sulfate, and 0.1-0.5% magnesium sulfate. The preset time can be set to 24 hours, 12 hours, or 20 hours; no specific limitation is made here.

[0057] Furthermore, the voltage for voltage acclimatization is less than 30V. In one implementation, different voltages can be used during the acclimatization process, for example, acclimatizing for a certain period of time using low voltage (0-5V), medium voltage (5-10V), and high voltage (10-20V), respectively. By acclimatizing with different voltages, the acclimatized microorganisms can be adapted to different voltage ranges.

[0058] Furthermore, in step S3, the target strain is cultured, isolated, and purified to obtain at least one target strain, including the following steps:

[0059] The voltage-acclimated, oily sludge containing aggregates was inoculated into an enrichment medium and cultured under the following conditions: temperature 20-35℃, shaking speed 120-150 rpm, and culture time 24-48 hours. In one specific implementation, the enrichment medium included: 15-30% (volume fraction) nutrient salt solution, 1-2.5 g / L yeast extract, and 1 L distilled water.

[0060] The bacterial suspension in the enrichment medium is screened for individually growing single colonies on a solid separation medium by dilution and plating. The solid separation medium includes a salt-resistant polymer. In one embodiment, the separation medium includes: 500-1000 mg / L of salt-resistant polymer, 15-30% (volume fraction) of nutrient salt solution, 1-2.5 g / L of yeast extract, and 1.8-2.0% agar powder added to the solid medium, along with 1 L of distilled water.

[0061] Single colonies are picked from the solid separation medium and inoculated into liquid basal LB medium for culture. The selection principle for single colonies is based on colony phenotype. In one implementation, the basal LB medium includes: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, and 1.8-2.0% agar powder and 1 L distilled water added to the solid medium.

[0062] Single colony culture in liquid LB medium was purified by streak plating to obtain at least one target strain.

[0063] Furthermore, after obtaining the target strain, it is necessary to cultivate it to increase its biomass. Different inorganic salts and trace elements have different effects on the proliferation of the strain. The inorganic salts are selected from at least one of dipotassium hydrogen phosphate, sodium acetate, and triammonium citrate, and the trace elements are selected from at least one of magnesium ions, manganese ions, and iron ions.

[0064] The second aspect of this application provides a polymer-degrading microbial agent, which is prepared by a method for preparing an electrostatic polymer-degrading microbial agent for treating polymeric oily sludge as described in any of the first aspects.

[0065] The third aspect of this application provides a method for applying a polymer-degrading microbial agent, wherein any polymer-degrading microbial agent as described in the first aspect is added to polymer-containing oily sludge, and the sludge is degraded by passing electricity through an electrochemical-biological coupling treatment device.

[0066] The following describes how to prepare polymer-degrading bacterial agents using specific implementation methods.

[0067] Implementation Method 1:

[0068] Figure 1 This is a technical roadmap of the present invention, combined with Figure 1 This embodiment describes a method for preparing an electro- and salt-resistant polymer-degrading bacterial agent specifically designed for polymeric oily sludge based on electrochemical-biological coupling technology. The preparation process involves the following steps:

[0069] Step 1: After homogenizing the raw, polymeric, oily sludge collected on-site, add it to the electrochemical-biological coupling treatment device, and install the positive and negative electrode plates, such as... Figure 2 .

[0070] Figure 2 This is a diagram of an electrochemical-biological coupling technology device.

[0071] Step 2: Prepare a nutrient solution. The nutrient solution contains 0.1-0.5% glucose, 0.1-0.5% potassium dihydrogen phosphate, 0.1-0.5% sodium chloride, 0.1-0.5% sodium nitrate, 0.1-0.5% ammonium sulfate, and 0.1-0.5% magnesium sulfate.

[0072] Step 3: Acclimate the native microorganisms in the aggregated oily sludge using an electric field. Connect the device to a DC power source and set the voltage to 0-5V, medium (5-10V), or high (10-20V). The electric field acclimation period is 7-10 days. During the acclimation process, spray an appropriate amount of nutrient salt solution every 24 hours, stir evenly, and spread it out.

[0073] Step 4: Determine the polyacrylamide content of sludge from different acclimation cycles. Select 5-10g of samples with high degradation rates, inoculate them into 100-250mL of sterile water (with glass beads), shake on a shaker at 120-150rpm at 20-35℃ for 30-120min, let stand for 10-30min, centrifuge at 12000rpm for 5min, and freeze the solid phase at -80℃ for bacterial and fungal diversity analysis.

[0074] Step 5: Analyze the microbial composition and predict its function, identifying microbial genera and species with resistance to salt-polymer degradation. Based on these results, screen strains and optimize the screening and isolation media. Microorganisms with resistance to salt-polymer degradation include oligotrophic bacteria, alkali-producing bacteria, microbes, Aspergillus, Bacillus, and Aureobacterium. These bacteria are typically round with smooth surfaces, yellow or white colonies, and regular edges. Based on this morphology, strains with resistance to salt-polymer degradation are screened, isolated, and purified.

[0075] In this step, microbial diversity analysis was performed on the aggregated oily sludge acclimated under the voltage with the best treatment effect to identify the enriched microbial species, providing phenotypic data to support subsequent strain screening. The screened strains were then subjected to biochemical identification and first-generation sequencing.

[0076] Step Six: Culture Medium Preparation

[0077] Enrichment medium: 15-30% (volume fraction) nutrient salt solution, 1-2.5 g / L yeast extract, 1 L distilled water;

[0078] Separation medium: Salt-resistant polymer (internal product used on-site, molecular weight approximately 16 million) 500-1000 mg / L, nutrient salt solution 15-30% (volume fraction), yeast powder 1-2.5 g / L, solids require the addition of 1.8-2.0% agar powder, and 1 L of distilled water;

[0079] Basic LB medium: 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, and 1.8-2.0% agar powder should be added to the solids, along with 1 L of distilled water.

[0080] Step 7: Inoculate 5-10 g / L of sludge acclimated to 10-30V voltage into enrichment medium and shake on a shaker at 120-150 rpm at 20-35℃ for 24-48 hours. Inoculate 5% (volume fraction) of bacterial solution into liquid separation medium and incubate at 90-150 rpm at 20-35℃ for 3-5 days.

[0081] Step 8: Screen the bacterial suspension from Step 7 using the dilution and plating method. Placing the bacterial suspension diluted to 10⁴, 10⁵, and 10⁶ onto solid separation medium and incubating in a constant temperature incubator at 20-35℃ for 2-3 days.

[0082] Step 9: Pick out the single colonies from the solid separation medium in Step 8 and inoculate them into liquid basal LB medium. Incubate at 90-150 rpm and 20-35℃ for 1-2 days. The selection principle for single colonies is based on the colony phenotype in Step 5.

[0083] Step 10: Purify the single colony culture from Step 9 using the streak plate method. After inoculating the culture with an inoculation loop, streak the culture in solid LB medium in sections. The culture conditions are to incubate in a constant temperature incubator at 20-35℃ for 2-3 days.

[0084] Step 11: Repeat steps 9 and 10 4-5 times to obtain a single pure bacterium.

[0085] Step 12: Examine all purified single strains under an optical microscope to observe bacterial morphology and perform Gram staining. Most bacteria are bacilli, while the fungi have a velvety surface morphology that develops wrinkles as the colony grows. The surface spores are dark brown and produce pigment.

[0086] Step 14: Test the polymer degradation efficiency of the screened electro- and salt-resistant polymer-degrading bacteria. Select strains with good degradation efficiency for first-generation sequencing.

[0087] Step 15: The 16S rRNA gene sequence of the purified bacteria and the 18S rRNA gene sequence of the fungi were determined by first-generation sequencing. After comparison and analysis with the NCBI database, the microbial genera and species with high polymer degradation efficiency are Stenotrophomonas acidaminiphila, Alcaligenes faecalis, Microbacterium oxydans, Citrobacter freundii, Ochrobactrum lupini, Lysinibacillus macroides, and Aspergillus sclerotiorum.

[0088] Step 15: Ecological construction of the selected strains is carried out, and the efficacy of different combinations is verified to obtain a highly efficient and electrostatically resistant polymer-degrading agent for the deep treatment of polymer-containing sludge.

[0089] Step 16: Improve the biomass of the inoculum by optimizing the seed culture medium. Optimized seed culture medium: 0.5-2.0% yeast extract, 1-2% peptone, 0.25-0.35% triammonium citrate, 0.05-0.15% dipotassium hydrogen phosphate, 0.5-1.0% sodium chloride, 0.01-0.05% magnesium sulfate, 0.1-0.5% sodium thiosulfate, and 1L distilled water.

[0090] Implementation Method Two:

[0091] The difference between this embodiment and embodiment one is that the microbial agent ecological construction in step fifteen adopts a dual-microbial mixed mode, the specific process of which is as follows:

[0092] The selected single bacterial strains were inoculated into LB liquid medium and cultured for 10-16 hours until the logarithmic growth phase. They were then combined in pairs at a 1:1 volume ratio and added to polymer medium containing 1000-2000 mg / L at a total addition amount of 5%. The cultures were then incubated at 90-150 rpm and 20-35℃ for 4-5 days.

[0093] The other steps and parameters are the same as in Specific Implementation Method 1.

[0094] Implementation Method 3:

[0095] The difference between this embodiment and embodiment one or two is that the microbial agent ecological construction in step fifteen adopts a multi-microbial mixed mode, the specific process of which is as follows:

[0096] The selected single bacterial strains were inoculated into LB liquid medium and cultured for 10-16 hours until the logarithmic growth phase. In a multi-strain mixed mode, with each strain in the same proportion, the strains were added to a polymer medium containing 1000-2000 mg / L at a total addition amount of 5% (volume fraction) and cultured at 90-150 rpm and 20-35℃ for 4-5 days.

[0097] The other steps and parameters are the same as in specific implementation method one or two.

[0098] For example, this application identifies the degrading strains in the polymer-containing oily sludge collected on-site using the methods in steps four and five of the above embodiment one. The identification results are shown in Table 1.

[0099] Table 1

[0100]

[0101]

[0102] "+": positive; "-": negative; " / ": not detected.

[0103] The microbial strains identified in the results are mostly Gram-negative bacteria, obligate aerobic or aerobic, and predominantly bacilli. Related studies have reported their metabolic effects on organic pollutants such as polycyclic aromatic hydrocarbons (PAHs) and aromatic hydrocarbons. Therefore, strains capable of polymer decomposition can be identified from the results, and their colony morphology can be queried to select the desired strains.

[0104] Figure 3 This image shows the colony morphology of electropolymer-resistant fungi on an isolation medium. Different colony morphologies can be used for strain screening.

[0105] After voltage acclimation, three types of bacteria and one type of fungus were screened from the sludge. The three types of bacteria were Alcaligenes faecalis (P1), Bacillus cereus (P2), and Microbacterium oxysporum (P3), and the one type of fungus was Aspergillus sclerotiorum (P4).

[0106] Therefore, in Embodiment 1, polymer-degrading bacterial agents of four single bacteria (P1, P2, P3, P4) can be obtained; in Embodiment 2, a mixed bacterial agent of three bacteria (P1:P2, P1:P3, P2:P3) can be obtained, with each of the above mixing ratios being 1:1; in Embodiment 3, a mixed bacterial agent of one bacterium (P1:P2:P3, mixing ratio 1:1:1) and a bacterial agent of a complete mixture of bacteria and fungi (P1:P2:P3:P4, mixing ratio 1:1:1:1) can be obtained.

[0107] Polymer degradation efficiency tests were conducted on the above-mentioned microbial agents at a dosage of 1-5% of the strain. The agents were cultured in a shaker at 20-35℃ and 90-150rpm for 4-5 days. The degradation efficiency of microorganisms on polymers under these different microbial agent ecological conditions was compared, and the polymer content and COD content were determined.

[0108] Method for determining polymer content: Starch-cadmium iodide spectrophotometry

[0109] ① Plotting the standard curve: Prepare a 100 mg / L polymer standard solution using deionized water. Add 5 mL of sodium acetate buffer (25 g sodium acetate trihydrate, 0.75 g aluminum sulfate hydrate, pH adjusted to 4 with acetic acid, and volume brought to 1000 mL) to nine 50 mL colorimetric tubes. Then add 0, 0.3, 0.5, 0.8, 1.0, 1.5, 2.0, 2.5, and 3.0 mL of the standard solution respectively, dilute to 35 mL, add 1 mL of saturated bromine water to each tube, react for 15 min, and then add 5 mL of the standard solution. 1% sodium formate was added, and the reaction was allowed to proceed for 5 minutes. Then, 5 mL of a starch-cadmium iodide solution (11 g of cadmium iodide dissolved in 400 mL of deionized water, boiled for 10 minutes, diluted to 800 mL, 2.5 g of soluble starch added, dissolved, filtered through filter paper, and brought to a final volume of 1000 mL) was added. The solution was diluted to the graduation mark, and the color development time was 15 minutes. The absorbance was measured at a wavelength of 590 nm using a 1 cm cuvette. A standard curve was plotted with solution concentration on the x-axis and absorbance on the y-axis. R0 2 ≥0.990.

[0110] ② Sample Measurement: Filter the sample containing precipitate using filter paper. Take 2 mL of sample, 25 mL of deionized water, 5 mL of buffer solution, and 1 mL of saturated bromine water into a 50 mL colorimetric tube. Shake well and let stand for 15 min. Then add 5 mL of 1% sodium formate and react for 5 min. Add 5 mL of starch-cadmium iodide solution, shake well, and let stand for 15 min. Measure the absorbance at 590 nm using a 1 cm cuvette. Calculate the concentration of the Russian polymer in the sample using the standard curve equation.

[0111] Method for determining COD content: HJ / T 399-2007 Rapid digestion spectrophotometric method

[0112] Add 1 mL of potassium dichromate (0.5 mol / L), 0.5 mL of mercuric sulfate (0.24 g / mL), 6 mL of silver sulfate-sulfuric acid (10 g / L), and 2 mL of the sample to be tested (diluted according to the selected range, the determination range is 100-1000 mg / L) to a colorimetric tube. Shake well and digest at 165℃±2℃ for 5 min. After cooling, measure the absorbance value at a wavelength of 600 nm. The detailed procedure for determining this index using a multi-parameter water quality analyzer is not described here; please refer to HJ / T 399-2007 Rapid Digestion Spectrophotometric Method for details.

[0113] Analysis of experimental results:

[0114] The degradation efficiency of polymers by different microbial agents, such as Figure 4 and Figure 5 As shown, Figure 4 The polymer degradation rate of different bacterial agents, Figure 5 The COD degradation rate of different microbial agents.

[0115] In the single-strain ecological model, P1, P2, and P3 showed lower polymer degradation capabilities than P4. P3 exhibited better degradation ability, while P1 and P2 showed increased polymer concentrations. Clearly, the P1:P2:P3 ecological model demonstrated higher polymer degradation capabilities than the dual-strain and single-strain models, but still lower than P4. The mixed P1:P2:P3:P4 ecological model showed higher polymer degradation capabilities than the single-strain P4 model and the mixed P1:P2:P3 model, increasing by 1.66% and 16.32% respectively, but the improvement in polymer degradation efficiency was relatively small.

[0116] The increased polymer content observed in the P1 and P2 single-cell models suggests that the degradation mechanism of polymers by P1 and P2 microorganisms may involve breaking down large polymer molecules, exposing the amide groups within them. During this process, the microorganisms use the generated small molecules as a carbon source for growth, and the exposed amide groups lead to the increase in polymer content.

[0117] In the single-strain mode, P3 bacteria exhibited the highest COD degradation rate at 29.47%, while P4 showed a higher degradation rate than P3 at 41.254%. In the two-strain mode, the P1:P2 combination showed a higher degradation rate than its single-strain counterpart, but the P1:P3 and P2:P3 combinations showed higher degradation rates than the P1 and P2 single-strain modes, though both were lower than the P3 single-strain mode. The P1:P2:P3 mode had the highest degradation rate, but all were lower than the P3 single-strain mode.

[0118] However, during COD determination, the added sulfuric acid and digestion process cause the bacterial cells in the test liquid to precipitate. Furthermore, the biomass produced by bacteria at different times varies significantly. P3, a strain with a large biomass, saw most of its cells settle to the bottom after a period of settling, but the culture medium remained turbid. In contrast, P1 and P2 showed less sedimentation after settling. Although the precipitate is removed during measurement, the presence of bacterial cells in the sample still affects the COD result, leading to either a higher or lower reading. Therefore, the degradation efficiency of the bacterial agent is determined by the polymer degradation rate.

[0119] In summary, P4 showed the best polymer degradation effect in single-strain cultures, while P3 showed the best polymer degradation effect among bacteria in single-strain cultures. The mixed culture culture of P1:P2:P3:P4 showed the best polymer degradation effect among all bacterial agents.

[0120] To maximize the biomass of the strain, the effects of adding trace elements and inorganic salts on strain proliferation were investigated using LB medium. Specific implementation method four:

[0122] The difference between this embodiment and one of the specific embodiments one to three is that the seed culture medium formula in step sixteen is as follows:

[0123] The selected single bacterial strains were inoculated into seed culture medium to increase the biomass of the strains. Seed culture medium: based on basic LB medium, 0.25% sodium acetate was added, and the culture was prepared into dry powder inoculum by vacuum freeze drying.

[0124] The other steps and parameters are the same as in any of the specific implementation methods one to three. Specific implementation method five:

[0126] The difference between this embodiment and one of the specific embodiments one to four is that the seed culture medium formula in step sixteen is as follows:

[0127] The selected single bacterial strains were inoculated into seed culture medium to increase the biomass of the strains. Seed culture medium: based on basic LB medium, 0.25% dipotassium hydrogen phosphate was added, and the culture was prepared into dry powder inoculum by vacuum freeze drying.

[0128] The other steps and parameters are the same as in any of the specific implementation methods one to four. Specific implementation method six:

[0130] The difference between this embodiment and one of specific embodiments one to five is that the seed culture medium formula in step sixteen is as follows:

[0131] The selected single bacterial strains were inoculated into seed culture medium to increase the biomass of the strains. Seed culture medium: based on basic LB medium, 0.05% magnesium sulfate was added, and the culture was prepared into dry powder inoculum by vacuum freeze drying.

[0132] The other steps and parameters are the same as those in any of the specific implementation methods one to five. Specific implementation method seven:

[0134] The difference between this embodiment and one of the specific embodiments one to six is ​​that the seed culture medium formula in step sixteen is as follows:

[0135] The selected single bacterial strains were inoculated into seed culture medium to increase the biomass of the strains. Seed culture medium: based on the basic LB medium, 0.05% manganese sulfate was added, and the dry powder bacterial agent was prepared by vacuum freeze drying.

[0136] The other steps and parameters are the same as those in any of the specific implementation methods one to six. Detailed implementation method eight:

[0138] The difference between this embodiment and one of the specific embodiments one to seven is that the seed culture medium formula in step sixteen is as follows:

[0139] The selected single bacterial strains were inoculated into seed culture medium to increase the biomass of the strains. Seed culture medium: based on basic LB medium, 0.05% ferric sulfate was added, and the culture was prepared into dry powder inoculum by vacuum freeze drying.

[0140] The other steps and parameters are the same as those in any of the specific implementation methods one to seven. Specific implementation method nine:

[0142] The difference between this embodiment and one of the specific embodiments one to seven is that the seed culture medium formula in step sixteen is as follows:

[0143] The selected single bacterial strains were inoculated into seed culture medium to increase the biomass of the strains. Seed culture medium: based on the basic LB medium, 0.25% triammonium citrate was added, and the dry powder bacterial agent was prepared by vacuum freeze drying.

[0144] The other steps and parameters are the same as those in one of the specific implementation methods one to eight.

[0145] Comparative implementation methods,

[0146] A blank control was set up, and the selected single bacterial strains were inoculated into seed culture medium to increase the biomass of the strains. Seed culture medium: basal LB medium, and the strains were prepared into dry powder bacterial agents by vacuum freeze drying.

[0147] The effects of inorganic salts and trace elements on bacterial proliferation were investigated by adjusting and optimizing the LB medium. Inorganic salts included dipotassium hydrogen phosphate, sodium acetate, and triammonium citrate, added at 0.25%; trace elements included magnesium, manganese, and iron ions, all added to the LB medium as sulfate hydrates at 0.05%. Biomass was represented by the absorbance value at 600 nm using a spectrophotometer.

[0148] Analysis of Experimental Results

[0149] Figure 6 The effect of trace elements on bacterial biomass. Figure 7 The effect of inorganic salts on the biomass of the strain.

[0150] Reference Figure 6 and Figure 7 In the figure, CN represents the blank control group. For strain P1, the addition of the three trace elements significantly affected its proliferation. Magnesium and manganese ions had significant proliferation effects, while iron ions had a negative effect on the cell growth of P1, with magnesium ions showing the highest proliferation effect. The addition of the three trace elements significantly affected the proliferation of strain P2, with all three showing a proliferation effect, among which manganese ions showed the best proliferation effect. For strain P3, there was no significant difference in biomass between strains with and without the addition of iron and manganese ions, while the addition of magnesium ions had a better proliferation effect. The addition of the three inorganic salts significantly affected the proliferation of strain P1, with triammonium citrate showing the best effect. For P2, the addition of triammonium citrate and potassium dihydrogen phosphate showed the best proliferation effect, with no significant difference in proliferation effect between the two. However, unlike P1 and P2, the addition of inorganic salts had no significant proliferation effect on P3. The biomass of strains with added triammonium citrate and potassium dihydrogen phosphate showed no significant difference between the biomass of the control group, and sodium acetate showed an inhibitory effect.

[0151] Therefore, the optimized seed culture medium is: yeast extract 0.5-2.0%, peptone 1-2%, triammonium citrate 0.25-0.35%, dipotassium hydrogen phosphate 0.05-0.15%, sodium chloride 0.1-0.5%, magnesium sulfate 0.01-0.05%, and distilled water 1L.

[0152] The effects of electrochemical and electrochemical-biological coupling treatment processes on the surface morphology of polymeric and oily sludge will be further explored below.

[0153] Figure 8 This is a scanning electron microscope image of the original aggregated oily sludge. Figure 9 This is a scanning electron microscope image of the electrochemically treated oily sludge containing polymers. Figure 10 This is a scanning electron microscope image of polymeric oily sludge after treatment by an electrochemical-biological coupling process.

[0154] Before treating aggregated oily sludge, to understand the sludge's morphology and composition characteristics, the sludge's morphology was observed under a scanning electron microscope, such as... Figure 8 As shown, at 500x magnification, the sludge particles are uneven in size; at 1000x magnification, the sludge surface is smooth; and at 3000x magnification, the sludge surface is clearly rounded. This is because the surface is coated with petroleum-based substances, and irregular polymer clumps are visible. After electrochemical treatment ( Figure 9 The sludge surface exhibits more pronounced layers and a stronger three-dimensional appearance, no longer appearing flat. This indicates that the electrochemical process alters the morphological characteristics of the sludge, disrupting its emulsified state. After treatment using the electrochemical-biological coupling process ( Figure 10 The surface has almost no flat areas, proving that the electrochemical-biological coupling process is more effective than simple electrochemical treatment.

[0155] exist Figure 10 In the image, the surface is not smooth; instead, there are slender, rod-shaped particles and some small, round particles. A distinct sense of layering is visible, along with some oval-shaped, smooth-edged spheres, which may be the degrading strains being scanned.

[0156] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an electrostatically resistant polymer-degrading bacterial agent for treating polymeric oily sludge, characterized in that, include: In an electrochemical-biological coupling treatment device, homogenized aggregated oily sludge is laid. The electrochemical-biological coupling treatment device includes electrode plates on both sides, and the electrode plates on both sides generate voltage to act on the aggregated oily sludge. After the electrochemical-biological coupling treatment device is powered on, it performs voltage acclimatization on the original microorganisms in the aggregated oily sludge. The voltage-acclimated oily sludge was inoculated into a culture medium for cultivation, separation, and purification to obtain at least one target strain; Ecological construction is carried out based on the obtained at least one target strain to obtain the polymer-degrading bacterial agent.

2. The preparation method according to claim 1, characterized in that, The process involves inoculating the voltage-acclimated, polymeric, oily sludge into a culture medium for cultivation, isolation, and purification to obtain at least one target strain, including: The voltage-acclimated oily sludge was inoculated into an enrichment medium and cultured under the following conditions: temperature 20-35℃, shaking speed 120-150rpm, culture time 24-48 hours. The bacterial solution in the enrichment medium is screened out by dilution and spreading on a solid separation medium to identify single colonies that grow independently. The solid separation medium includes a salt-resistant polymer. Single colonies are picked out from the solid separation medium and inoculated into liquid LB medium for culture, wherein the selection principle of single colonies is based on colony phenotype; The single-colony bacterial suspension in the liquid LB medium was purified by streak plating to obtain at least one of the target strains.

3. The preparation method according to claim 1, characterized in that, The voltage acclimatization period is 7-10 days. During the voltage acclimatization process, nutrient salt solution is sprayed at preset intervals, stirred evenly, and spread out.

4. The preparation method according to claim 1, characterized in that, The voltage for voltage conditioning is less than 30V.

5. The preparation method according to any one of claims 1 to 4, characterized in that, The target strains include one or more of Alcaligenes faecalis, Microbacterium oxysporum, Bacillus cereus, and Aspergillus sclerotiorum.

6. The preparation method according to claim 5, characterized in that, The ecological construction includes: mixing and culturing multiple target strains in a preset ratio.

7. The preparation method according to any one of claims 1 to 4, characterized in that, The preparation method further includes: The biomass of the polymer-degrading microbial agent is increased by using a seed culture medium, wherein the seed culture medium, in addition to the liquid-based LB medium, also includes inorganic salts and trace elements, wherein the inorganic salts are selected from at least one of dipotassium hydrogen phosphate, sodium acetate, and triammonium citrate, and the trace elements are selected from at least one of magnesium ions, manganese ions, and iron ions.

8. The preparation method according to claim 7, characterized in that, The seed culture medium comprises: 0.5-2.0% yeast extract, 1-2% peptone, 0.25-0.35% triammonium citrate, 0.05-0.15% dipotassium hydrogen phosphate, 0.5-1.0% sodium chloride, 0.01-0.05% magnesium sulfate, 0.1-0.5% sodium thiosulfate, and 1L distilled water.

9. A polymer-degrading bacterial agent, characterized in that, The polymer-degrading microbial agent is prepared by the method for preparing an electrostatic polymer-degrading microbial agent for treating polymeric oily sludge as described in any one of claims 1-8.

10. A method for applying a polymer-degrading microbial agent, characterized in that, The polymer-degrading bacterial agent as described in claim 9 is added to polymer-containing oily sludge and degraded by electricity in an electrochemical-biological coupling treatment device.

Images