Method for preserving anaerobic dehalogenating microorganisms and use thereof

Abstract

The application belongs to water pollution treatment technology, and particularly relates to an anaerobic dehalogenation microbial preservation method and application. The dehalogenation microorganism is resuspended in a embedding agent solution, and then the resuspension is added dropwise into a crosslinking agent, and then is reacted in an ice bath under anaerobic conditions for 10-20 min; after the reaction, the crosslinking is continued for 24-36 h under anaerobic conditions at 4-6 DEG C, and the preservation of the anaerobic dehalogenation microorganism is realized after the crosslinking. The anaerobic dehalogenation microorganism is fixed, and through centrifugation, resuspension and crosslinking into a ball under strict anaerobic conditions, the immobilized bacterial agent is obtained while the bacterial solution is concentrated and the cells are wrapped, the tolerance of the dehalogenation microorganism to oxygen is significantly improved, the problem that the dehalogenation microorganism is easy to be exposed to air and lose the degradation activity in the form of culture solution is overcome, and great convenience is provided for the implementation of in-situ bioremediation engineering of a contaminated site.

Description

Technical Field

[0001] This invention pertains to water pollution control technology, specifically a method for preserving anaerobic dehalogenated microorganisms and its application. Background Technology

[0002] The widespread use of organochlorines as dry cleaning agents and pesticides globally has made them a major category of pollutants commonly found in groundwater. Organochlorines have low solubility and strong migration capacity in soil, easily penetrating and contaminating deep groundwater, forming dense non-aqueous phase liquids (DNAPLs). As persistent organic pollutants, once organochlorines enter aquifers, they pose a long-term threat to underground ecosystems and drinking water sources. Since the 1980s, research has revealed that chlorinated alkenes such as tetrachloroethylene have high toxicity and carcinogenic, teratogenic, and mutagenic effects on humans, with trichloroethylene and monochloroethylene identified as human carcinogens. Currently, many organochlorines, including tetrachloroethylene and trichloroethylene, have been listed as "priority pollutants" by various countries.

[0003] The reductive dechlorination process mediated by specific microorganisms is the primary pathway for the biodegradation of organochlorides in anaerobic environments. In this process, microorganisms use organochlorides as electron acceptors to progressively reduce and dechlorinate them, producing hydrochloric acid and dechlorination products with low or no chlorine substituents. These microorganisms, which use organochlorides as electron acceptors to catalyze the reductive dechlorination process, are collectively referred to as dehalogenating microorganisms. Dehalococcoides are among the most studied dehalogenating microorganisms. The first Dehalococcoides strain, 195, was discovered and isolated in 1997; it was capable of completely dechlorinating tetrachloroethylene to non-toxic ethylene. Other microorganisms involved in tetrachloroethylene degradation include Sulfurospirillium, Geobacter, Dehalobacter, and Dehalogenimonas, but the crucial reduction process from dichloroethylene to non-toxic ethylene can only be mediated by a few microorganisms, represented by Dehalococcoides.

[0004] Dehalogenated microorganisms, such as dehalogenated cocci, are absolute anaerobic microorganisms that are extremely sensitive to oxygen; brief exposure to air can completely destroy their degradation activity. In in-situ remediation, large quantities of bacterial cultures cultivated in the laboratory need to be transported to the remediation site and injected into the underground environment. However, this transportation process is costly and it is difficult to maintain strictly anaerobic conditions, often resulting in a significant reduction in the degradation activity of the dehalogenated microorganisms. On the other hand, organochlorines above 0.5 mM often have a certain toxic inhibitory effect on dehalogenated microorganisms; however, in actual contaminated sites, concentrations far exceeding this, and even non-dissolved organochlorine contamination, are very common, greatly reducing the efficiency of in-situ bioremediation. Therefore, the transportation and storage of remediation agents, as well as their tolerance and degradation of high concentrations of organochlorine pollutants, are urgent problems to be solved in in-situ remediation projects. Summary of the Invention

[0005] In view of the problems in in-situ bioremediation of organochlorine pollutants, such as the reduced degradation activity of dehalogenated microorganisms after exposure to air and the high transportation cost of liquid microbial agents, this invention aims to provide a method for preserving anaerobic dehalogenated microorganisms and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for preserving anaerobic dehalogenated microorganisms involves resuspending the dehalogenated microorganisms in an encapsulating agent solution, then adding the resuspended liquid droplets to a crosslinking agent, and then reacting the mixture in an ice bath under anaerobic conditions for 10-20 minutes. After the reaction, the mixture is further crosslinked under anaerobic conditions at 4-6℃ for 24-36 hours, thus achieving the preservation of the anaerobic dehalogenated microorganisms.

[0008] The encapsulating agent is an aqueous solution containing 1-3 wt% sodium alginate, 2-4 wt% polyvinyl alcohol, and 1-1.5 wt% activated carbon, with the pH adjusted to 7.15-7.3 using sodium hydroxide; the crosslinking agent is an aqueous solution containing 1.5-4 wt% calcium chloride and 1.5-2 wt% boric acid, with the pH adjusted to 7.15-7.3 using sodium hydroxide.

[0009] The dehalogenated microbial strain was inoculated at a 3% (v / v) rate into an inorganic salt culture medium containing a carbon source, electron donor, and electron acceptor. A mixture of N2 and CO2 gas was maintained in the headspace of the closed system, and the culture was carried out under anaerobic conditions at 30°C until the electron acceptor in the system was completely consumed. The bacterial cells were then collected by centrifugation under anaerobic conditions for later use.

[0010] The concentration of the carbon source in the culture system is 5-10 mM, and the concentration of the electron acceptor liquid phase is 0.2-0.5 mM; wherein, the carbon source is acetate, lactate or pyruvate; the electron donor is hydrogen, formate or lactate; and the electron acceptor is an organochloride.

[0011] The electron acceptor was added to the culture system in batches.

[0012] The inorganic salt culture medium consists of the following components: NaCl 1.0 g / L, MgCl₂·6H₂O 0.5 g / L, KH₂PO₄ 0.2 g / L, NH₄Cl 0.3 g / L, KCl 0.3 g / L, CaCl₂·2H₂O 0.015 g / L, FeCl₂·4H₂O 1.5 mg / L, CoCl₂·6H₂O 190 μg / L, MnCl₂·4H₂O 100 μg / L, ZnCl₂ 70 μg / L, H₃BO₃ 6 μg / L, Na₂MoO₄·2H₂O 36 μg / L, NiCl₂·6H₂O 24 μg / L, CuCl₂·2H₂O 2 μg / L, Na₂SeO₃·5H₂O 6 μg / L, and Na₂WO₄·2H₂O. Prepare a solution containing 8 μg / L of resazurin indicator (0.025% w / v), L-cysteine ​​(24 mg / L, 0.2 mM), Na₂S·9H₂O (48 mg / L, 0.2 mM), dithiothreitol (77 mg / L, 0.5 mM), and NaHCO₃ (2.52 g / L, 30 mM). Adjust the pH to 7.2-7.3 and bring the volume to 1 L with water. Sterilize and add multivitamins. Set aside for use.

[0013] The inorganic salt culture medium was supplemented with a complex vitamin, and the final concentrations of the vitamins in the system were: biotin 20 μg / L, folic acid 20 μg / L, pyridoxine hydrochloride 100 μg / L, riboflavin 50 μg / L, thiamine 50 μg / L, pantothenic acid 50 μg / L, nicotinic acid 50 μg / L, and vitamin B12. 12 50 μg / L, p-aminobenzoic acid 50 μg / L, lipoic acid 50 μg / L.

[0014] The preferred preparation method is as follows:

[0015] S1. Anaerobic Culture of Dehalogenating Microorganisms: This invention is applicable to all anaerobic dehalogenating microorganisms. The dehalogenating microorganisms are cultured in a 160 mL sealed serum bottle containing 100 mL of inorganic salt medium, with a headspace ratio of N2 / CO2 (80 / 20, v / v). 5 mM sodium acetate or 5 mM sodium lactate is used as the carbon source, and 10 mL of hydrogen or 5 mM sodium formate is used as the electron donor. 8 μL (0.5 mM) of tetrachloroethylene or 6 μL (0.5 mM) of trichloroethylene, or other organochlorides, is added via a microsyringe as the electron acceptor. The concentration of the organochloride in the liquid phase generally does not exceed 0.5 mM to avoid toxicity to the dehalogenating microorganisms.

[0016] S2. Collection of dehalogenated microbial cells: In order to obtain more bacterial cells, after the dehalogenated microorganisms have completely depleted the electron acceptor, the same amount of electron acceptor is added twice. Then, in the anaerobic chamber, the culture medium is transferred to 50mL centrifuge tubes that have been placed in the anaerobic chamber for more than 48 hours, and the centrifuge tube caps are sealed with vacuum tape. The cells are then collected by centrifuging in a refrigerated high-speed centrifuge outside the anaerobic chamber.

[0017] S3. Preparation of bacterial suspension: In an anaerobic chamber, the cells obtained in step S2 are mixed evenly with the embedding gel to obtain a bacterial suspension concentrated 80-200 times. The embedding gel contains 1-3 wt% sodium alginate, 2-4 wt% polyvinyl alcohol, and 1-1.5 wt% 200-mesh activated carbon, with a pH of 7.15-7.3.

[0018] S4. Immobilization Process: In the anaerobic chamber, the bacterial suspension obtained in S3 is added dropwise to the cross-linking agent using a pipette to carry out the cross-linking reaction. The cross-linking agent contains 1.5-4 wt% calcium chloride and 1.5-2 wt% boric acid. The reaction is carried out in an ice bath. After 10-20 minutes, the formed immobilized microspheres and cross-linking agent are transferred into centrifuge tubes, sealed with vacuum tape to isolate oxygen, and removed from the anaerobic chamber. The tubes are then placed in a refrigerator at 4-6℃ for 24-30 hours to continue cross-linking. After the reaction is complete, the centrifuge tubes containing the immobilized microspheres are transferred into the anaerobic chamber. The residual cross-linking agent is poured out, and the immobilized microspheres are quickly rinsed three times with physiological saline to obtain the final immobilized bacterial agent with a network-like interwoven structure layer encapsulating anaerobic dehalogenated microorganisms. Its appearance is grayish-black granules with a particle size of 3-4 mm, which have a certain elasticity and mechanical strength, are not crushed by hand, and can rebound.

[0019] In step S2, the centrifugation parameters for collecting cells are: centrifugation speed of 20,000-25,000 x g, centrifugation time of 10-20 min, and centrifugation temperature of 20℃.

[0020] In step S2, after each centrifugation, the centrifuge tube is transferred to an anaerobic chamber to remove the supernatant and add fresh culture medium, avoiding contact with air throughout the process.

[0021] An immobilized microbial agent for dehalogenation is characterized by: obtaining a gray-black immobilized microbial agent with a particle size of 3-4 mm by the method described above; it has a certain elasticity and mechanical strength, and can be rebounded when squeezed by hand.

[0022] An application of microorganisms preserved by the method, wherein the microorganisms preserved by the method are encapsulated in a mesh-like interwoven structure, for the degradation of organochlorine pollutants in groundwater environments.

[0023] The preserved microorganisms are used in the degradation of 0.1-2.5 mM (or non-dissolved) organochlorine pollutants in groundwater environments.

[0024] The present invention has the following beneficial effects:

[0025] This invention preserves dehalogenated microorganisms by immobilizing them. The method involves separating the bacterial solution into solid and liquid phases under anaerobic conditions to obtain bacterial cells, which are then mixed with an encapsulating agent and subjected to a cross-linking reaction to form spheres. This process concentrates the bacterial solution and encapsulates the cells within a structure of sufficient mechanical strength, allowing for long-term preservation. Furthermore, this preservation method significantly improves the degradation efficiency of dehalogenated microorganisms against high concentrations of organochlorines, their tolerance to undissolved organochlorines, and their tolerance to oxygen exposure. It also reduces the transportation costs of remediation agents, greatly facilitating in-situ bioremediation of contaminated sites. Specifically:

[0026] 1. The preservation method of the present invention preserves dehalogenated microorganisms by means of activated carbon adsorption, gel embedding and cross-linking immobilization process. At the same time, all the above processes are carried out under anaerobic conditions, so as to maintain the degradation activity of the microorganisms to the greatest extent.

[0027] 2. The preservation method of the present invention enables dehalogenated microorganisms in an immobilized state to degrade organochlorine pollutants more efficiently.

[0028] 3. The oxygen tolerance of the immobilized dehalogenated microorganisms in the preservation method of the present invention is significantly enhanced, which solves the problem of reduced activity of dehalogenated microorganisms caused by exposure to air during bacterial liquid transportation and in-situ injection.

[0029] 4. The method of the present invention significantly enhances the tolerance and degradation capacity of dehalogenating microorganisms in the immobilized state to high concentrations of organochlorine pollutants, which solves the problem of high concentrations of organochlorine inhibiting dehalogenating microorganisms in actual remediation processes. Attached Figure Description

[0030] Figure 1 The degradation curve of trichloroethylene by the bacterial suspension provided in Example 1 of this invention is shown.

[0031] Figure 2 The degradation curve of trichloroethylene by immobilized Dhc 195 bacteria provided in Example 1 of the present invention.

[0032] Figure 3 The degradation curve of trichloroethylene by the Dhc 195 bacterial suspension provided in Example 1 of the present invention after being exposed to air for 12 hours.

[0033] Figure 4 The degradation curve of trichloroethylene by the Dhc 195 bacterial suspension provided in Example 1 of the present invention after being exposed to air for 24 hours.

[0034] Figure 5The degradation curve of trichloroethylene by the Dhc 195 bacterial suspension provided in Example 1 of the present invention after being exposed to air for 48 hours.

[0035] Figure 6 The degradation curve of trichloroethylene by the Dhc 195 bacterial suspension provided in Example 1 of the present invention after being exposed to air for 72 hours.

[0036] Figure 7 The degradation curve of trichloroethylene by immobilized Dhc 195 bacteria after exposure to air for 12 hours, as provided in Example 1 of this invention.

[0037] Figure 8 The degradation curve of trichloroethylene by immobilized Dhc 195 bacteria after exposure to air for 24 hours, as provided in Example 1 of this invention.

[0038] Figure 9 The degradation curve of trichloroethylene by immobilized Dhc 195 bacteria after exposure to air for 48 hours, as provided in Example 1 of this invention.

[0039] Figure 10 The degradation curve of trichloroethylene by immobilized Dhc 195 bacteria after exposure to air for 72 hours, as provided in Example 1 of this invention.

[0040] Figure 11 The degradation curve of high concentration of trichloroethylene by Dhc 195 bacterial suspension provided in Example 1 of the present invention.

[0041] Figure 12 The degradation curve of high concentration of trichloroethylene by immobilized Dhc 195 bacteria provided in Example 1 of the present invention. Detailed Implementation

[0042] The technical solution of the present invention will be further described below with reference to specific embodiments, but it should not be construed as limiting the present invention.

[0043] This invention involves uniformly mixing microbial cells with an adsorbent and an encapsulating gel. During the cross-linking reaction with a cross-linking agent, the microbial cells are adsorbed and encapsulated in a porous network structure. Sodium alginate and polyvinyl alcohol are used as the effective components of the encapsulating agent, which are mild and non-toxic to microorganisms, have high mechanical strength, and are biodegradable. The entire operation is carried out under anaerobic conditions, especially the drop-addition and pelleting process, which is completed in an anaerobic chamber. This avoids the inhibitory effect of oxygen contact on the activity of the microbial cells before they are encapsulated. The entire immobilization process is applicable to all dehalogenated microorganisms, thus enabling the preservation of microorganisms.

[0044] Example 1

[0045] The type strain of *Dehalococcoides mccartyi* (strain preservation number ATCC BAA-2266) is used. T (hereinafter referred to as Dhc 195) is used as an example for fixation.

[0046] S1. Culture of dehalogenated cocci: Dhc 195 was inoculated at a 3% inoculum into a 160 mL sealed serum bottle containing 100 mL of inorganic salt medium and cultured with a headspace of N2 / CO2 (80 / 20, v / v). 5 mM sodium acetate was added to the inorganic salt medium as a carbon source and 10 mL of hydrogen was added as an electron donor. The culture was then incubated in the dark at 30 °C.

[0047] The inorganic salt culture medium consists of: NaCl 1.0 g / L, MgCl2·6H2O 0.5 g / L, KH2PO4 0.2 g / L, NH4Cl 0.3 g / L, KCl 0.3 g / L, CaCl2·2H2O 0.015 g / L, FeCl2·4H2O 1.5 mg / L, CoCl2·6H2O 190 μg / L, MnCl2·4H2O 100 μg / L, ZnCl2 70 μg / L, H3BO3 6 μg / L, Na2MoO4·2H2O 36 μg / L, NiCl2·6H2O 24 μg / L, CuCl2·2H2O 2 μg / L, Na2SeO3·5H2O 6 μg / L, and Na2WO4·2H2O. 8 μg / L, resazurin indicator 0.025% (w / v), L-cysteine ​​24 mg / L (0.2 mM), Na₂S·9H₂O 48 mg / L (0.2 mM), dithiothreitol 77 mg / L (0.5 mM), NaHCO₃ 2.52 g / L (30 mM), sodium acetate 0.41 g / L (5 mM), pH adjusted to 7.2-7.3 with CO₂. After sterilization, a compound vitamin was added, and the final vitamin content in the culture medium was as follows: biotin 20 μg / L, folic acid 20 μg / L, pyridoxine hydrochloride 100 μg / L, riboflavin 50 μg / L, thiamine 50 μg / L, pantothenic acid 50 μg / L, nicotinic acid 50 μg / L, vitamin B 12 50 μg / L, p-aminobenzoic acid 50 μg / L, lipoic acid 50 μg / L.

[0048] S2. Collection of dehalogenated cocci cells: Add 6 μL of trichloroethylene as an electron acceptor to the culture three times. After all three additions of substrate have been degraded into a mixture of monochloroethylene and ethylene, purge the headspace of the culture system with nitrogen for 20 min to remove residual organochlorides. Transfer the serum bottles containing the culture medium to an anaerobic chamber. Remove the stoppers in the anaerobic chamber and pour the culture medium into 50 mL centrifuge tubes that have been placed in the anaerobic chamber for more than 48 h. Tighten the caps and immediately seal the centrifuge tube caps with vacuum tape after removing the anaerobic chamber to prevent air from entering.

[0049] Centrifuge the centrifuge tubes containing the culture medium in a floor-standing high-speed refrigerated centrifuge with the following parameters: 24,000 x g, 20 min, 20 °C. After centrifugation, slowly and steadily transfer the centrifuge tubes to an anaerobic chamber, pour off the supernatant, add the remaining culture medium, remove the tubes from the anaerobic chamber, and centrifuge again. Repeat this process until all the culture medium has been centrifuged. Care must be taken to ensure that no air is introduced into the culture medium during centrifugation.

[0050] S3. Preparation of bacterial suspension: In an anaerobic chamber, the obtained cells were resuspended in an appropriate volume of embedding agent aqueous solution to obtain a 100-fold concentrated Dhc 195 bacterial suspension. The embedding agent aqueous solution was prepared by adding activated carbon (200 mesh), sodium alginate, and polyvinyl alcohol to ultrapure water, and then adjusting the pH of the solution to 7.2 with sodium hydroxide. The concentrations of activated carbon, sodium alginate, and polyvinyl alcohol in the embedding agent aqueous solution were 1 wt%, 1.5 wt%, and 4 wt%.

[0051] S4. Immobilization Process: In an anaerobic chamber, the embedded cell suspension was pipetted dropwise (approximately 30 μL) into the crosslinking agent aqueous solution. The embedding agent and crosslinking agent were allowed to react fully under ice bath and magnetic stirring conditions. After 10 minutes in the ice bath, microspheres formed. The microspheres, along with the embedding agent and crosslinking agent, were transferred to 50 mL centrifuge tubes, and the tube caps were sealed with vacuum tape. After removing the tubes from the anaerobic chamber, they were placed in a 4°C refrigerator to continue the reaction for 24 hours. After the crosslinking reaction was complete, any remaining crosslinking agent was removed in the anaerobic chamber. The immobilized microspheres were then rapidly washed three times with sterile, deoxygenated physiological saline to solidify them. They were then stored at 4°C for later use. The crosslinking agent aqueous solution contained 1.5 wt% calcium chloride and 1.5 wt% boric acid, and the pH of the solution was adjusted to 7.2 using sodium hydroxide.

[0052] Each immobilized microsphere contains 2.02 ± 0.03 × 10⁻⁶ microspheres. 9 One Dhc 195 cells.

[0053] The dehalogenated cocci cell pellet obtained from S2 was partially embedded and partially resuspended in culture medium, and stored in 50 mL centrifuge tubes, which were then sealed with vacuum tape. After being removed from the anaerobic chamber, it was stored together with the immobilized microspheres in a refrigerator at 4°C for 24 hours.

[0054] The above method is applicable to *Dehalococcoides mccartyi* BAV1 strain (strain preservation number ATCC BAA-2100), *Geobacter lovleyi* SZ strain (strain preservation number ATCC BAA-1151), and *Sulfurospirillum multivorans* DSM 12446 strain (strain preservation number JCM 15788). T Preservation of various anaerobic dehalogenation microbial strains, including Dehalobacter restrictus PER-K23 strain (strain preservation number DSM 9455).

[0055] The immobilized Dhc 195 strain obtained in the above embodiments and the bacterial suspension obtained by resuspending in culture medium in step S3 were used for the degradation of trichloroethylene, respectively.

[0056] The immobilized Dhc 195 strain and the bacterial suspension obtained in step S3 were inoculated into the above-mentioned culture medium containing 0.5 mM trichloroethylene in an anaerobic chamber. The carbon source and electron donor were the same as above. The 16S rRNA gene of *Dehalococcus dehalogenates* was quantified by qPCR. The results showed that the biomass of the six immobilized microspheres was consistent, all containing 1.21 ± 0.02 × 10⁻⁶ microspheres. 10 Each 300 μL bacterial suspension contained 1.34 ± 0.11 × 10⁵ Dhc 195 cells; the biomass of each suspension was consistent, containing 1.34 ± 0.11 × 10⁵ cells. 10 One Dhc 195 cells.

[0057] The samples were incubated in the dark at 30°C for 15 days. Qualitative and quantitative determination of trichloroethylene and its degradation products in the liquid phase was performed using gas chromatography with a tandem flame ionization detector (GC-FID).

[0058] First, use Henry's Law to convert between the liquid phase concentration and the amount of substance:

[0059] M = C a ×(V a +V g ×H cc )

[0060] Where M represents the amount of substance, C a V represents the liquid phase concentration. aV represents the volume of the liquid phase. g H represents the volume of the gas phase. cc It represents the dimensionless Henry's constant (i.e., the ratio of gas phase concentration to liquid phase concentration).

[0061] Then, the degradation rate is calculated according to the following formula:

[0062] E = (M VC ×2+M ETH ×3) / (Va×d)

[0063] Where E represents the degradation rate (expressed as the rate of chloride ion release), M VC M represents the amount of vinyl chloride. ETH d represents the amount of ethylene, and d represents the number of days of cultivation required for trichloroethylene to be completely degraded into monochloroethylene and for ethylene to be produced.

[0064] The comparative experiments above show that the degradation rates of trichloroethylene by the Dhc 195 strain in both suspension and immobilized forms are 84.7 μM·d⁻¹. -1 and 129.9 μM·d -1 The degradation rate of the immobilized Dhc 195 strain increased by 53.3% compared to the bacterial suspension.

[0065] Example 2

[0066] The method described in Example 1 was used to immobilize Dhc 195 bacteria, except that activated carbon was not added in step S3, thereby achieving the corresponding immobilization of Dhc 195 bacteria.

[0067] The immobilized microspheres without activated carbon obtained in this embodiment and the Dhc 195 bacteria immobilized microspheres obtained in Example 1 were used for the degradation of trichloroethylene. The culture conditions, degradation product detection, and degradation rate calculation were the same as in Example 1.

[0068] Testing showed that the degradation rates of trichloroethylene by immobilized microspheres without activated carbon and immobilized microspheres with activated carbon were 95.3 μM·d⁻¹. -1 and 129.9 μM·d -1 Adding activated carbon can increase the degradation rate by 36.3%. This indicates that the adsorption effect of activated carbon on microorganisms avoids the toxic effects of boric acid on microorganisms during the cross-linking process.

[0069] Furthermore, the immobilized microspheres without activated carbon have lower mechanical strength than those with activated carbon. After being added to the culture medium and shaken at 150 rpm for 6 hours, the microspheres with activated carbon showed a breakage rate of 50%, while the microspheres with activated carbon showed a breakage rate of only 10%. Therefore, the method of this invention not only improves the degradation ability of solid dehalogenated microorganisms but also enhances the mechanical strength of the immobilized bacterial agent.

[0070] Application Example 1

[0071] Using the immobilized Dhc 195 microspheres and Dhc 195 bacterial suspension obtained in Example 1 (resuspended in culture medium in step S3 of Example), a comparative experiment on the degradation of organochlorine after air exposure was conducted.

[0072] The specific experimental steps are as follows:

[0073] Immobilized microspheres (6 per group) and bacterial suspensions (300 μL per batch) were transferred to multiple 2 mL centrifuge tubes in an anaerobic chamber. The 16S rRNA gene of *Dehalococcus* was quantified by qPCR. The results showed that the biomass of the six immobilized microspheres was consistent, all containing 1.21 ± 0.02 × 10⁻⁶ microspheres. 10 Each 300 μL bacterial suspension contained 1.34 ± 0.11 × 10⁵ Dhc 195 cells; the biomass of each suspension was consistent, containing 1.34 ± 0.11 × 10⁵ cells. 10 Dhc 195 cells were cultured. The microspheres were exposed to air in a clean bench with the centrifuge tubes opened for 12 h, 24 h, 48 h, and 72 h. After the exposure time, the microspheres and bacterial suspension were transferred to an anaerobic chamber and inoculated into fresh culture medium. 6 μL of trichloroethylene was added as an electron acceptor, and 10 mL of hydrogen gas was added as an electron donor. Other culture conditions were as described in Example 1. The degradation of trichloroethylene was monitored periodically.

[0074] Depend on Figure 1 and Figure 2 It was found that both immobilized microspheres and bacterial suspensions, which were not exposed to air, were able to completely degrade trichloroethylene into monochloroethylene within 15 days, accompanied by the production of ethylene. Among them, immobilized microspheres showed higher degradation efficiency for trichloroethylene, and could completely degrade trichloroethylene into monochloroethylene and ethylene within 9 days of cultivation.

[0075] Depend on Figure 3 , 4As shown in points 5 and 6, the activity of Dhc 195 bacterial suspensions significantly decreased after exposure to air for different durations. In the culture system inoculated with bacterial suspensions exposed to air for 12 hours, no trichloroethylene degradation products were detected within 10 days. The dechlorination reaction began around day 15 after inoculation and was completed in approximately day 25. The time required for dechlorination to be completed increased with the increase in the air exposure time of the inoculated bacterial suspension. In the culture system inoculated with bacterial suspensions exposed to air for 24 hours, the dechlorination reaction was completed in approximately day 35; in the culture systems inoculated with bacterial suspensions exposed to air for 48 hours and 72 hours, the dechlorination reaction was completed in approximately day 40.

[0076] Compared to bacterial suspension, immobilization significantly improved the oxygen tolerance of Dhc 195. Culture systems with immobilized microspheres exposed to air for 12 and 24 hours after inoculation completely degraded trichloroethylene into monochloroethylene and ethylene within 15 days; culture systems with immobilized microspheres exposed to air for 48 and 72 hours both completely degraded trichloroethylene into monochloroethylene and ethylene within 18 days.

[0077] After exposure to air for 12, 24, 48, and 72 hours, the average degradation rate of dehalogenated cocci in suspension decreased by 40.6%, 69.7%, 71.8%, and 80.3% over one month, respectively. In contrast, the degradation rate of immobilized dehalogenated cocci containing the same biomass decreased by 1.8%, 6.5%, 7.1%, and 14.6% after air exposure for 12, 24, 48, and 72 hours, respectively. This demonstrates that immobilized dehalogenated microorganisms are more tolerant of potential air exposure during long-term transportation than bacterial suspensions, which is beneficial for the effective implementation of in-situ remediation projects (Table 1).

[0078] Table 1. Degradation rate of trichloroethylene by microorganisms in different preservation forms after exposure to air for different periods (unit: chloride ion release per day, μM·d). -1 express)

[0079] bacterial suspension 84.7 50.3 25.7 23.9 16.7 Immobilization 129.9 127.6 121.4 120.7 110.9

[0080] Application Example 2

[0081] Using the immobilized Dhc 195 microspheres and Dhc 195 bacterial suspension obtained in Example 1 (same as in Application Example 1), a comparative experiment was conducted on the rate of degradation of high concentrations of trichloroethylene.

[0082] The specific experimental steps are as follows:

[0083] Immobilized microspheres (6 per group) and bacterial suspension (300 μL per batch) were inoculated into fresh culture medium in an anaerobic chamber, containing the biomass as described in Application Example 1. 20 μL (2.2 mM liquid concentration) of trichloroethylene was added as an electron acceptor and 10 mL of hydrogen gas as an electron donor; other culture conditions were as described in Example 1. The degradation of trichloroethylene was monitored periodically.

[0084] Depend on Figure 11 and Figure 12 It was found that high concentrations of trichloroethylene had a certain inhibitory effect on Dhc 195 bacterial suspension. During the first 20 days of culture, the degradation rate was very low, with approximately 30% of the trichloroethylene being degraded into cis-dichloroethylene and monochloroethylene. In contrast, during the first 20 days of culture, approximately 60% of the trichloroethylene in immobilized Dhc 195 bacteria was completely degraded into monochloroethylene. Both methods could degrade most of the trichloroethylene into monochloroethylene within about 40 days. However, possibly due to limitations in electron donors, both methods left small amounts of cis-dichloroethylene and trichloroethylene residues.

[0085] In summary, compared with bacterial suspension, immobilization treatment improved the degradation rate of trichloroethylene by Dhc 195 bacteria, especially significantly improved the degradation rate of high concentrations of trichloroethylene; more importantly, immobilization treatment can significantly improve the oxygen tolerance of Dhc 195 bacteria, which is of great significance for the in-situ remediation of organochlorine pollution.

[0086] The embodiments and beneficial effects of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the specific details in the embodiments.

Claims

1. A method for preserving anaerobic dehalogenated microorganisms, characterized in that: Dehalogenated cocci ( Dehalococcoides mccartyi The model strain 195 was resuspended in an encapsulating agent solution, and then the resuspended liquid was added dropwise to a cross-linking agent. The reaction was then carried out under anaerobic conditions in an ice bath for 10 min. After the reaction, the cross-linking was continued at 4℃ for 24 h under anaerobic conditions. After cross-linking, the anaerobic dehalogenation microorganism was preserved. The dehalogenated cocci ( Dehalococcoides mccartyi The model strain 195 was inoculated at a 3% (v / v) rate into an inorganic salt medium containing a carbon source, electron donor, and electron acceptor. A mixture of N2 and CO2 was maintained in the headspace of the closed system, and the cells were cultured under anaerobic conditions at 30°C until the electron acceptor in the system was completely consumed. The cells were then collected by centrifugation under anaerobic conditions for later use. The encapsulating agent is an aqueous solution containing 1.5 wt% sodium alginate, 4 wt% polyvinyl alcohol, and 1 wt% activated carbon, with the pH adjusted to 7.2 using sodium hydroxide; the crosslinking agent is an aqueous solution containing 1.5 wt% calcium chloride and 1.5 wt% boric acid, with the pH adjusted to 7.2 using sodium hydroxide.

2. An application of the microorganism preserved by the method of claim 1, characterized in that: Microorganisms preserved by the method described in claim 1 are encapsulated in a mesh-like interwoven structure and applied to the bioremediation of typical organochlorine pollutants in groundwater environments.

3. The application according to claim 2, characterized in that: The preserved microorganisms are used in the degradation of 0.1-2.5 mM or non-dissolved organochlorine pollutants in groundwater environments.

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