Method for bioremediation of chlorinated hydrocarbons in groundwater by using bifunctional microspheres

Abstract

The application provides a method for reinforcing bioremediation of chlorinated hydrocarbon groundwater by using bifunctional microspheres, and a high-efficiency microorganism is screened independently, the method has the advantages of simple instrument and equipment used, mild conditions, eco-friendly preparation process, no secondary pollution, easy recycling, and wide market prospect of batch production.

Description

Technical Field

[0001] This invention belongs to the field of groundwater treatment, and in particular relates to a method for enhancing the bioremediation of chlorinated hydrocarbon groundwater with bifunctional microspheres. Background Technology

[0002] Chlorinated hydrocarbons are mostly carcinogenic, mutagenic, and teratogenic. They are generally denser than water and have low solubility in water. They are often used as raw materials, intermediate products, and organic solvents in chemical production. The widespread use of chlorinated hydrocarbons inevitably leads to their entry into the groundwater environment, causing a continuous decline in groundwater quality and seriously threatening human health and water environment safety.

[0003] Currently, remediation projects for groundwater contaminated with chlorinated hydrocarbons (CHPs) often employ iron-based materials for reduction dechlorination (such as injecting EHC reagents or constructing permeable reactive barriers made of zero-valent iron / iron-containing minerals) or bioremediation. Nano-zero-valent iron (NZVI) exhibits outstanding reactivity in effectively removing various pollutants from wastewater, including both inorganic and organic pollutants. However, due to interparticle interactions, NZVI particles tend to aggregate into larger particles, and their strong reducing power makes them easily oxidized in air, leading to decreased activity and stability. This significantly hinders their large-scale application under real-world working conditions. Furthermore, in-situ bioremediation of CHP contamination faces challenges such as incomplete degradation of CHPs due to a lack of exogenous organic carbon and electron donors, resulting in the accumulation of toxic and harmful intermediate products. Additionally, the addition of conventional water-soluble organic carbon sources can lead to increased site COD and secondary pollution. Summary of the Invention

[0004] In view of this, the present invention aims to propose a method for enhancing the bioremediation of chlorinated hydrocarbon groundwater with bifunctional microspheres, and independently screens a highly efficient microorganism. The method has the advantages of simple equipment, mild conditions, eco-friendly preparation process, no secondary pollution, easy recycling, and broad market prospects for mass production.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0006] A bifunctional microsphere-enhanced bioremediation agent for chlorinated hydrocarbon-remediated groundwater includes a Pseudomonas aeruginosa inoculum and bifunctional modified zero-valent iron microspheres (CS@ZVI microspheres), wherein the bifunctional modified zero-valent iron microspheres are zero-valent iron coated with chitosan on their surface.

[0007] Furthermore, the aforementioned Pseudomonas aeruginosa is deposited at the China General Microbiological Culture Collection Center, named HK-EPT-3, with accession number CGMCC NO.24871.

[0008] Furthermore, the mass ratio of the bifunctional modified zero-valent iron microspheres to the Pseudomonas aeruginosa inoculum is 0.2:1~3.

[0009] Furthermore, the preparation method of the bifunctional modified zero-valent iron microspheres includes the following steps:

[0010] 1) Prepare chitosan acetic acid solution: Dissolve an appropriate amount of chitosan in acetic acid solution and stir at 35℃~65℃ until the chitosan is completely dissolved;

[0011] 2) Preparation of mixed reaction solution: Take an appropriate amount of chitosan acetate solution, add zero-valent iron, and control the molar ratio of chitosan to zero-valent iron to be 1~3:1, and stir;

[0012] 3) Preparation of composite functional repair materials containing zero-valent iron and organic slow-release carbon source: Add the mixed liquid droplets to a 0.05mol / L~0.2mol / L NaOH solution;

[0013] 4) Preparation of chitosan-encapsulated zero-valent iron modified composite microspheres: The self-assembled microspheres obtained above were washed with deionized water until neutral, stored in an oxygen-free ethanol brown bottle, and refrigerated at 4°C for later use.

[0014] Chitosan dissolves under acidic conditions and precipitates under alkaline conditions. With increasing NaOH concentration, chitosan microspheres rapidly form, readily exhibiting uniform thickness, good morphology, and good toughness. Therefore, by controlling the NaOH solution to an appropriate concentration, chitosan completely precipitates into spheres with rounded shapes, good strength, and relatively uniform surface thickness, forming a good outer shell structure that encapsulates zero-valent iron to create a core-shell composite material. At this point, the microsphere surface is supported, revealing the loose, porous structure inherent in chitosan. However, at a concentration of 20%, sphere formation fails because the high concentration causes the microspheres to clump together before spheric formation, forming a flocculent mass. The core-shell thickness of the encapsulated chitosan is approximately 1-3 mm.

[0015] Furthermore, in step 1), the diameter of zero-valent iron is 100nm~1000nm, the mass of chitosan is 0.5g~5g, the mass fraction of acetic acid solution is 1%, and the volume of acetic acid solution is 100ml~2000ml.

[0016] Furthermore, in step 2), the stirring time is 3h~6h.

[0017] Furthermore, in step 4), the above mixed reaction solution is collected using an automatic sampler, and the dropper speed is controlled at 0.2 drops / s to 100 drops / min.

[0018] This invention also provides a method for enhancing the bioremediation of chlorinated hydrocarbon groundwater with bifunctional microspheres. A certain mass of gel-like bifunctional modified zero-valent iron microspheres is weighed, and an appropriate amount of the aforementioned Pseudomonas aeruginosa inoculum is added. The mass ratio of the bifunctional modified zero-valent iron microspheres to the Pseudomonas aeruginosa inoculum is maintained at 0.2:1~3. The mixture is allowed to stand under natural conditions for 20 min~3 h. Preferably, as the standing reaction time increases, a certain proportion of bifunctional modified zero-valent iron microspheres and the nutrient supplement glucose (or it can be omitted) need to be added periodically. The degradation effect of groundwater is tested after 14 days.

[0019] The chitosan shell of the CS@ZVI microspheres can provide nutrients for the screened HK-EPT-3 bacteria, supplementing the carbon source required for microbial growth. As the reaction proceeds under natural conditions, the microorganisms degrade the chitosan shell, causing the CS@ZVI microspheres to gradually release the ZVI encapsulated inside, promoting the degradation of chlorinated hydrocarbons by zero-valent iron, and realizing the dual-cycle chemical-biological coupling remediation and treatment of chlorinated hydrocarbon groundwater.

[0020] The CS@ZVI microspheres release zero-valent iron to reduce chlorinated hydrocarbons, and the Fe after losing electrons... 2+ Fe 3+ This further enhances bioavailability, especially for Fe. 3+ It is relatively easy to dissolve and can be absorbed by Fe naturally occurring in the environment. 3+ The rapid bioreduction by reducing bacteria (FeRB) is typically achieved through direct contact reduction of Fe(III) (hydroxyl) oxides with FeRB, which generates ligands and electron shuttles. This further enhances the indirect bioreduction process of HK-EPT-3 bacteria that degrade chlorinated hydrocarbons, thereby improving the efficiency of chlorinated hydrocarbon bioreduction.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. This invention solves the problems of easy deactivation of zero-valent iron and complicated and difficult preparation process of modified materials. It provides an efficient method for preparing modified zero-valent iron by self-assembling chitosan. The method uses simple instruments and equipment, has mild conditions, is eco-friendly in preparation process, and is easy to recycle zero-valent iron.

[0023] 2. This invention provides a method for enhancing the bioremediation of chlorinated hydrocarbon groundwater with bifunctional microspheres. It utilizes chitosan (CS), zero-valent iron (ZVI), and HK-EPT-3 to degrade chlorine in a synergistic manner, solving the problems of cost and secondary pollution associated with the additional carbon source in bioremediation. At the same time, the biodegradation achieves slow and controlled release of zero-valent iron. During the reduction of organic matter by zero-valent iron, the gain and loss of electrons and the reaction ligands stimulate microbial growth, achieving full utilization of materials. It has the advantages of being green and environmentally friendly and not generating secondary pollution.

[0024] 3. This invention fully combines zero-valent iron and the reducing dechlorination mechanism of microorganisms to specifically identify chlorinated hydrocarbon pollutants, significantly enhancing the targeted capture and degradation of chlorinated hydrocarbon pollutants; in addition, the dual-cycle reducing dechlorination mechanism provides more possibilities for the remediation of groundwater pollution and improves economic efficiency, thus demonstrating the superiority of the method of this invention.

[0025] Biological Preservation Instructions:

[0026] HK-EPT-3, Pseudomonas sp., was deposited on May 9, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.24871, and is in viable condition. Attached Figure Description

[0027] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0028] Figure 1 This is a schematic diagram of the preparation process of bifunctional modified zero-valent iron microspheres (CS@ZVI), wherein A is a schematic diagram of the preparation of chitosan acetate solution; B is a schematic diagram of the preparation of mixed reaction solution; C is a schematic diagram of the preparation of composite functional repair material containing ZVI and organic slow-release carbon source; and D is a schematic diagram of the preparation of the obtained chitosan-encapsulated zero-valent iron modified composite microspheres.

[0029] Figure 2 The results of streaking an HK-EPT-3 degrading bacterium (BL5) onto LB medium;

[0030] Figure 3 The growth curve of a single colony of HK-EPT-3 degrading bacteria (BL5) is shown. Detailed Implementation

[0031] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. The test reagents used in the following embodiments include anoxic ethanol, chitosan, sodium hydroxide aqueous ethanol solution, zero-valent iron, etc. The chitosan is C916461 chitosan with a degree of deacetylation of 90% and MW=200000, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; the zero-valent iron is reduced iron powder, 80 mesh, purchased from Sinopharm Chemical Reagent Co., Ltd. Unless otherwise specified, all are conventional biochemical reagents; the experimental methods described are conventional methods unless otherwise specified.

[0032] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0033] Example 1

[0034] The specific steps for preparing chitosan-encapsulated zero-valent iron modified bifunctional microspheres (CS@ZVI) proposed in this invention are as follows:

[0035] 1. Preparation of chitosan acetic acid solution: Dissolve 2g of chitosan (CS) in 200ml of acetic acid solution (the mass fraction of the acetic acid solution is 1%), stir at 50℃ for 3h with a magnetic stirrer until completely dissolved, and the mass fraction of the obtained chitosan acetic acid solution is 2.0g / 100mL.

[0036] 2. Preparation of mixed reaction solution: Take 500 ml of chitosan acetate solution and add 0.1 g of zero-valent iron (ZVI). The zero-valent iron used is purchased industrial-grade nano-zero-valent iron. Control the ratio of chitosan to zero-valent iron so that chitosan:iron = 3:2. Stir with a magnetic stirrer at 200 r / min.

[0037] 3. Preparation of composite functional repair material containing zero-valent iron and organic slow-release carbon source: Collect 500ml of the above mixed reaction solution using an automatic sampler, control the dropper speed at 0.5 drops / s, and add the mixed solution to 1000ml of 0.1mol / L NaOH aqueous ethanol solution, where V(0.1mol / L NaOH):V(ethanol)=4:1.

[0038] 4. Preparation of chitosan-encapsulated zero-valent iron modified composite microspheres: The self-assembled microspheres obtained above were washed with deionized water until neutral, stored in brown bottles, and refrigerated at 4°C for later use.

[0039] Example 2

[0040] The specific steps for screening the HK-EPT-3 strain described in this invention are as follows:

[0041] 1) Sampling: Samples of contaminated soil and wastewater from the pesticide factory were collected at the Yingli Company site in Tianjin. Three samples were taken from each of the contaminated soil and wastewater samples, for a total of six samples. Approximately 500g of soil sample was collected and sealed in a bag for storage. Approximately 500ml of water sample was collected and sealed in a plastic bottle for storage. After removing debris and stones, the samples were placed in sample bags, clearly labeled, and stored at 4℃ for later use.

[0042] 2) Domestication: Microbial domestication generally refers to the method of gradually adapting microorganisms to a certain condition through artificial means, thereby selectively breeding microorganisms. Domestication can yield strains with higher tolerance and activity. It is often used in the selection of highly efficient strains with high degradation capabilities for certain pollutants. In this experiment, vinyl chloride, dichloroethane, and DDVP were used as carbon sources, and the collected samples were domesticated and cultured in 250mL shake flasks at 30℃ and 220r / min.

[0043] 3) Transplantation: After acclimation for about one week in media containing 100 mg / L vinyl chloride, dichloroethane, and DDVP, respectively, obvious bacterial growth was observed in the shake flasks. 5% acclimation solution (microbial suspension after one week of acclimation) was added to media containing 200 mg / L contaminant, and cultured in 250 mL shake flasks at 30℃ and 220 r / min for about one week. Finally, the second acclimation solution was inoculated at a 5% inoculation ratio into media containing 300 mg / L vinyl chloride, dichloroethane, and DDVP, respectively, and acclimated for about one week.

[0044] 4) Dilution: Two days before the end of the third acclimatization cycle (7 days), select one bottle from the experimental group, take 1 mL of bacterial culture into a 1.5 mL centrifuge tube, and dilute it tenfold to 10. -4 10 -5 10 -6 10 -7 10 -8 10 -9 These six bacterial solutions were prepared at different concentration gradients. 0.1 mL of each concentration was spread onto different solid culture media plates ((LB) liquid medium (10 g peptone, 5 g yeast extract, 5 g NaCl) or beef extract agar medium (3 g / L beef extract, 10 g / L peptone, 5 g / L sodium chloride, pH 7.2-7.6)). The plates were incubated at 30°C for two days, and the microbial activity was monitored regularly. Three suitable dilutions were ultimately selected for the next plating step.

[0045] 5) Pouring Plates: Heat the solid culture medium used in the experiment in a water bath until melted. Once the medium has cooled to approximately 50°C, pour it into plates using aseptic technique. Place the plates horizontally and allow them to solidify. Hold the test tube or Erlenmeyer flask containing the culture medium in your right hand near a flame. Gently remove the stopper with your left hand, keeping the mouth of the tube or flask facing the flame. Then, hold the stopper between the edge of your right palm or your little and ring fingers (or place the stopper between the edge of your left hand or your little and ring fingers. If the culture medium in the test tube or Erlenmeyer flask is used up at once, the stopper does not need to be held). Hold the petri dish and open the lid slightly near the flame. Quickly pour in approximately 15 mL of culture medium. After capping, gently shake the petri dish to distribute the culture medium evenly at the bottom. Then, place it horizontally on a table and allow it to solidify. This is your petri dish.

[0046] 6) Plate spreading: Melt the sterilized culture medium and pour it hot into sterile plates. After solidification, number the plates. Then, use a pipette to take 0.1 ml of bacterial suspension and inoculate it into different dilutions, numbering them accordingly (microbial inoculation densities are 10-1). -6 10 -7 10 -8 10 -9Spread the bacterial culture onto agar plates (three replicates per number). Then, use a spreader to evenly spread the culture onto the plate until it dries. Use one spreader per dilution. When changing dilutions, sterilize the spreader by exposing it to the outer flame of an alcohol lamp. When spreading from a low concentration to a high concentration, the spreader may not need to be changed. Place the spread plate flat on a table for 20-30 minutes to allow the bacterial culture to penetrate the medium. Then, invert the plate and incubate overnight at 30°C.

[0047] 7) Streaking: The streak method yields single colonies. Divide the entire plate into sections, with the angle between sections approximately 120° (about 60° when the plate is rotated). This ensures full utilization of the plate's surface area and prevents the lines from touching. Use a smooth, flat inoculation loop and aseptically pick up a small amount of inoculum. Invert the plate next to an alcohol lamp, holding the bottom of the plate with your left hand, ensuring the plate is perpendicular to the table with the culture medium facing the lamp. After each streak, immediately burn off any remaining bacteria on the loop to prevent excessive bacteria from affecting the isolation of subsequent sections. When burning the loop, hold the bottom of the plate with your left hand and cover it above the lid (do not place it inside the lid) to prevent contamination. Repeat this process 4-5 times to better separate single colonies.

[0048] 8) Incubation at constant temperature: Invert the streak plate and incubate it in a 30℃ incubator. After about 24 hours, take it out and observe it to determine whether a single colony has been isolated. Observe the morphology of the strain and save the image.

[0049] 9) Preservation of bacteria: Preserve 2 tubes of glycerol.

[0050] 10) Glycerol-containing bacteria: Perform the procedure in a UV-sterilized laminar flow hood. Holding the conical flask containing the bacterial stock solution, shake well, remove the sealing film, and flask the mouth with the outer flame of an alcohol lamp. Using a 1000μL pipette, add 1mL of the bacterial solution to a high-pressure sterilized cryovial, then add 1mL of freshly prepared 50% glycerol, seal, and label for storage. Store two glycerol tubes for each bacterial strain, one at -80℃ and the other at -20℃.

[0051] Experimental Example 1

[0052] 1. The selected HK-EPT-3 strain was inoculated into shaker tubes containing 5 mL of suitable culture medium. The shaker was set to 220 rpm and cultured overnight at 30°C. After 12 hours, the strain was transferred to Erlenmeyer flasks and cultured under the same environmental conditions until it entered the logarithmic growth phase.

[0053] 2. Take the bacterial suspension obtained in the previous step and centrifuge it in a centrifuge tube at 8000 rpm for 10 minutes. After centrifugation, discard the supernatant under sterile conditions and resuspend the bacterial cells. Repeat the above steps three times to remove all nutrients. After discarding the supernatant for the last time, add an appropriate amount of sterile MSM medium to make the OD600 value of the bacterial suspension approximately 1. Resuspend the suspension and store it at -4°C to obtain 1000 ml of HK-EPT-3 bacterial agent for later use.

[0054] 3. Weigh 150g of the prepared bifunctional modified zero-valent iron microspheres (CS@ZVI) and place them in 1000L of chlorinated hydrocarbon-contaminated groundwater extracted from a contaminated site in Wuqing District, Tianjin. After thorough mixing, add 1000ml of the above-prepared HK-EPT-3 bacterial agent. As the reaction time increases, add 50g of the prepared bifunctional modified zero-valent iron microspheres and 100g of glucose solution every 3 days until the reaction has lasted for 14 days. Water samples were collected periodically for testing. The degradation effect on chlorinated hydrocarbons is shown in Table 1 below.

[0055] Table 1. Test results of chlorinated hydrocarbon degradation and remediation in groundwater at a contaminated site in Wuqing District, Tianjin.

[0056]

[0057] Comparative Example 1

[0058] 150g of zero-valent iron microspheres were weighed and placed in 1000L of groundwater contaminated with chlorinated hydrocarbons extracted from a contaminated site in Wuqing District, Tianjin. After thorough mixing, 50g of zero-valent iron microspheres were added every 3 days until the reaction lasted for 14 days. The degradation effect on chlorinated hydrocarbons is shown in Table 2 below.

[0059] Table 2. Test results of chlorinated hydrocarbon degradation and remediation in groundwater at a contaminated site in Wuqing District, Tianjin.

[0060]

[0061] Table 1 shows that after adding the remediation agent to groundwater samples collected from a contaminated site in Wuqing District, Tianjin, chloroform, 1,2-dichloroethane, and vinyl chloride achieved a 100% degradation rate, being completely degraded after seven days. 1,3,5-trimethylbenzene showed good degradation within seven days, with a degradation rate of 75%. For the degradation of chlorobenzenes, after 14 days, the degradation rates for chlorobenzene, 1,2-dichlorobenzene, 1,4-dichlorobenzene, and 1,3,5-trimethylbenzene were 48.94%, 91.35%, 74.24%, and 84.34%, respectively. Tetrahydrofuran did not show a significant reduction, further confirming that the remediation agent provided in this invention can specifically remediate and degrade environmental chlorinated hydrocarbon pollutants, thus demonstrating the superiority of this remediation method.

[0062] As shown in Table 2, the degradation efficiency of chlorinated hydrocarbons using zero-valent iron alone is low, and the degradation efficiency for chlorobenzene, 1,2-dichlorobenzene, 1,4-dichlorobenzene, and 1,3,5-trimethylbenzene is poor. As time goes on, the degradation effect of zero-valent iron alone shows signs of fatigue. Comparison with the degradation effect in Table 1 further verifies that the remediation agent provided by this method has significant degradation performance for chlorinated hydrocarbons and excellent degradation performance.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A bifunctional microsphere-enhanced bioremediation agent for chlorinated hydrocarbon-contaminated groundwater, characterized in that: The invention includes Pseudomonas sp. and bifunctional modified zero-valent iron microspheres. The bifunctional modified zero-valent iron microspheres are zero-valent iron coated with chitosan, and the molar ratio of chitosan to zero-valent iron is controlled to be 1~3:

1. The Pseudomonas sp. is deposited in the China General Microbiological Culture Collection Center, named HK-EPT-3, with accession number CGMCCNO.24871.

2. The repair agent according to claim 1, characterized in that: The mass ratio of the bifunctional modified zero-valent iron microspheres to the Pseudomonas agent is 0.2:1~3.

3. The repair agent according to claim 1, characterized in that: The preparation method of the bifunctional modified zero-valent iron microspheres includes the following steps: 1) Prepare chitosan acetic acid solution: Dissolve an appropriate amount of chitosan in acetic acid solution and stir at 35℃~65℃ until the chitosan is completely dissolved; 2) Preparation of mixed reaction solution: Take an appropriate amount of chitosan acetate solution, add zero-valent iron, and stir; 3) Preparation of composite functional repair materials containing zero-valent iron and organic slow-release carbon source: Add the mixed reaction solution dropwise to a 0.05 mol / L~0.2 mol / L NaOH solution; 4) Preparation of bifunctional modified zero-valent iron microspheres: The above-obtained composite functional repair material was washed with deionized water until neutral, stored in an oxygen-free ethanol brown bottle, and refrigerated at 4°C for later use.

4. The repair agent according to claim 3, characterized in that: In step 1), the diameter of zero-valent iron is 100nm~1000nm, the mass of chitosan is 0.5g~5g, the mass fraction of acetic acid solution is 1%, and the volume of acetic acid solution is 100ml~2000ml.

5. The repair agent according to claim 3, characterized in that: In step 2), the stirring time is 3h~6h.

6. The repair agent according to claim 3, characterized in that: In step 4), the above mixed reaction solution is collected using an automatic sampler, and the dropper speed is controlled at 0.2 drops / s to 100 drops / min.

7. A method for enhancing the bioremediation of chlorinated hydrocarbon groundwater with bifunctional microspheres, characterized in that: Weigh a certain mass of the bifunctional modified zero-valent iron microspheres according to any one of claims 1-6, add an appropriate amount of Pseudomonas sp. according to any one of claims 1-6, maintain the mass ratio of bifunctional modified zero-valent iron microspheres to Pseudomonas sp. at 0.2:1~3, let it stand under natural conditions for 20min~3h, and test the degradation effect on groundwater after 14 days.

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