PH / ORP (Oxidation Reduction Potential) double-response type microbial remediation microsphere as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of energy conservation and environmental protection, and particularly relates to a pH / ORP (oxidation-reduction potential) double-response type microbial remediation microsphere as well as a preparation method and application thereof. The microbial remediation microspheres disclosed by the invention are prepared by mixing a mixed bacteria suspension and a composite gel solution, and carrying out ionic crosslinking and oxidative crosslinking on the mixed bacteria suspension and the composite gel solution. Wherein the mixed bacteria suspension comprises desulfurization vibrio and citrobacter; the effective components of the composite gel solution comprise xanthan gum, low-fat pectin and rice hull ash. According to the microbial remediation microsphere, the remediation process is automatically and rapidly triggered through the pH and ORP double-response slow release function design, heavy metal is fixed through the biochemical process of functional bacteria, and long-term stabilization and harmlessness of the heavy metal are achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of energy saving and environmental protection, and particularly relates to a pH / ORP dual-response microbial remediation microsphere as well as a preparation method and application thereof. BACKGROUND

[0002] With the continuous development of industrialization in China, the problem of groundwater heavy metal pollution in industrial and mining enterprises is becoming increasingly serious. Such sites not only have long-term chronic pollution, but also have the risk of sudden leakage, and the pollutants will quickly migrate downstream with rainwater or leachate, forming a dynamically changing pollution plume, which poses an immediate and serious threat to the surrounding ecological environment.

[0003] Existing remediation technologies are mostly focused on long-term chronic pollution treatment, and have deficiencies in dealing with sudden leakage risks. Patent CN120328675A designs an inner-outer double-layer slow release, which realizes long-term slow release of remediation agents. However, the slow-release shell lacks specific signal response, and in the case of sudden pollution impact, the best interception time may be missed. Patent CN112374596A encapsulates nano zero-valent iron with paraffin, and repairs groundwater by slowly releasing nano zero-valent iron. Sudden leakage may be accompanied by oxidizing substances, causing the surface passivation of nano iron to be deactivated before it can be used. Patent CN114249508A uses a biologically active composite material + quartz sand to fill a reaction wall, and an external electrode to drive the migration of pollutants. This system uses solar power to reduce operating costs, but the construction of the reaction wall takes a long time, and if it is not deployed in advance, it cannot respond to sudden leaks. Patent CN114259993A and patent CN111151225A respectively prepare two kinds of metal composite gel adsorbents, which can be added for adsorbing heavy metals in the case of sudden pollution. However, the adsorption capacity of pure physical and chemical adsorbents is limited and they have no sustainable pollutant interception capability. The above remediation technologies have good removal effect on groundwater heavy metal pollution, but they have problems such as mismatch between material response rate and pollution diffusion rate and instantaneous inactivation at high concentrations in dealing with sudden leakage risks.

[0004] Therefore, there is an urgent need for an intelligent response material that meets the requirements of rapid triggering, high efficiency and environmental adaptability to deal with sudden leakage risks. SUMMARY

[0005] The purpose of the present application is to provide a pH / ORP dual-response microbial remediation microsphere as well as a preparation method and application thereof, which can be triggered by natural chemical signals in groundwater to solve the problem of targeted delivery of bacterial agents and reduce heavy metal pollution.

[0006] To achieve the above purpose, the present application provides the following technical solutions: The present application provides a kind of pH / ORP dual-response microbial remediation microspheres, the microbial remediation microspheres are prepared by the mixed solution of composite gel solution and bacteria suspension liquid by ionic crosslinking and oxidation crosslinking; The composition of the composite gel solution includes xanthan gum, low-fat pectin and rice husk ash; The bacteria suspension liquid contains desulfurication vibrio and citric acid bacillus.

[0007] Preferably, the particle size of the microbial remediation microspheres is 2-3 mm.

[0008] The present application also provides a preparation method of pH / ORP dual-response microbial remediation microspheres, including the following steps: S1, mix xanthan gum and low-fat pectin, add deionized water to the mixed solution, stir uniformly, then add 20% of the total dry weight of xanthan gum and low-fat pectin gel to the rice husk ash, stir again to obtain a composite gel solution; S2, mix the freeze-dried powder of desulfurication vibrio and citric acid bacillus, then add 0.1% (w / v) L-cysteine to 0.85% sodium chloride to obtain a bacteria suspension liquid; S3, mix the bacteria suspension liquid and the composite gel solution uniformly, then perform ionic crosslinking and oxidation crosslinking to obtain wet microspheres, and freeze-dry to obtain pH / ORP dual-response microbial remediation microspheres.

[0009] Preferably, in step S1, the xanthan gum and low-fat pectin are mixed in a mass ratio of (6-8):(3-5); the solid-liquid ratio of the mixed solution to deionized water is 1:(18-25); and the stirring conditions of the mixed solution and deionized water are 45-55°C and 2h of stirring until thick.

[0010] Preferably, in step S2, the mixed mass ratio of the freeze-dried powder of desulfurication vibrio and citric acid bacillus is 1:1; and the addition amount of 0.85% sodium chloride is 1-1.5 times the total mass of the bacteria powder of desulfurication vibrio and citric acid bacillus.

[0011] Preferably, in step S3, the mixing ratio of the bacteria suspension liquid to the composite gel solution is 1:9.

[0012] Preferably, in step S3, the ionic crosslinking process is: drop the mixed solution of the bacteria suspension liquid and the composite gel solution into a 2% (w / v) calcium chloride solution for solidification for 30 min to obtain gel balls; and the oxidation crosslinking process is: place the gel balls in a 0.5% (w / v) hydrogen peroxide solution for stirring reaction for 20 min.

[0013] Preferably, in step S3, the freeze-drying process is: freeze-drying at -40~-60°C and less than 10 Pa for 24 h.

[0014] This invention also provides the application of the above-mentioned microbial remediation microspheres or the microbial remediation microspheres prepared by the above-mentioned preparation method in the remediation of groundwater contaminated with heavy metals.

[0015] This invention also provides the application of the above-mentioned microbial remediation microspheres or the microbial remediation microspheres prepared by the above-mentioned preparation method in the removal of heavy metal ions in polluted water, wherein the heavy metal ions are cadmium, zinc, lead and copper.

[0016] The beneficial effects of this invention are: This invention provides a pH / ORP dual-response microbial remediation microsphere and its application, which has the advantages of simple preparation process and easy industrial production. Through the design of pH and ORP dual-response slow release function, the remediation process is automatically and quickly triggered. The biochemical process of functional bacteria is used to fix heavy metals, thereby achieving long-term stabilization and harmlessness of heavy metals. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a comparison chart of the heavy metal removal rates of Examples 1-5 and Comparative Examples 1-3. Detailed Implementation

[0019] This invention provides a pH / ORP dual-responsive microbial remediation microsphere, which is prepared by mixing a composite gel solution and a bacterial suspension, followed by ionic cross-linking and oxidative cross-linking. The composite gel solution is prepared from xanthan gum, low-fat pectin, and rice husk ash; the bacterial suspension contains *Desulfovibrio* and *Citrobacter*.

[0020] In the preparation process of the microbial remediation microspheres described in this invention, the ionic crosslinking involves adding a bacterial suspension to a composite gel solution (volume ratio 1:9) that has been cooled to room temperature, mixing thoroughly, and then adding the solution dropwise into a 2% (w / v) calcium chloride solution. The gel spheres are then solidified in the calcium chloride solution for 30 minutes to complete the ionic crosslinking. The principle is that the carboxyl groups on the low-fat pectin molecular chain bridge with calcium ions to form a three-dimensional network structure, encapsulating water, bacterial agent, and rice husk ash within it, and solidifying it. The gel spheres are then removed and placed in a 0.5% (w / v) hydrogen peroxide solution, and gently stirred for 20 minutes to complete the oxidative crosslinking. The thiol groups on the xanthan gum molecular chain form disulfide bonds under the action of the oxidant. The microspheres are then removed and washed with deionized water to obtain wet microspheres.

[0021] The microorganism repairing microspheres of the present application, when pH<5, the carboxyl ionization triggers swelling, when the environmental pH decreases, H + The concentration increases, competes with calcium ions for carboxyl sites, forms uncharged carboxylic acid, three-dimensional network structure swells and collapses, releases the core bactericide; ORP<-200 mV, disulfide bond breaks, when the reducing substances in the environment increase, the reducing agent attacks and breaks the disulfide bond, destroys the cross-linked network, the shell structure collapses, and releases the core bactericide.

[0022] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they cannot be understood as limiting the protection scope of the present application.

[0023] The production process, experimental method or detection method involved in the embodiments of the present application, if not specially mentioned, are all conventional methods in the prior art, and the name and / or abbreviation thereof all belong to the conventional name in the field, which is very clear and definite in the related application field, and the person skilled in the art can understand the conventional process steps and apply the corresponding equipment according to the name, and implement it according to the conventional conditions or the conditions recommended by the manufacturer.

[0024] The various instruments, equipment, raw materials or reagents used in the embodiments of the present application do not have special restrictions on the source, and are all conventional products that can be purchased through normal commercial channels, or can be prepared according to the conventional method well known to the person skilled in the art.

[0025] Example 1 pH and ORP dual response (1) Take xanthan gum and low-fat pectin according to the mass ratio of 7:3, add deionized water according to the solid-liquid ratio of 1:20, and stir at 50°C for 2 hours to obtain a uniform solution; take the previously ground rice husk ash to 200 mesh according to 20% of the dry weight of the gel, add the uniform solution, and continue to stir for 1 hour to obtain a composite gel; take the freeze-dried powder of desulfovibrio and citrobacter according to the mass ratio of 1:1, add sterile normal saline (0.85% sodium chloride) containing 0.1% (w / v) L-cysteine according to the solid-liquid ratio of 1:1.25, and mix gently to obtain a bacterial suspension; blend the bacterial suspension and the composite gel according to the volume ratio of 1:9, mix evenly, and obtain a mixed solution.

[0026] (2) Extract the mixed solution with a syringe (outlet diameter 2 mm), drop it into a 2% (w / v) calcium chloride solution at a constant speed, and solidify for 30 minutes to obtain solidified microspheres; take out the solidified microspheres and put them into a 0.5% (w / v) hydrogen peroxide solution, stir gently for 20 minutes, then take them out again, and wash them with deionized water for 3 times to obtain wet microspheres. Place the wet microspheres in a freeze dryer, freeze dry at -50°C and <10 Pa for 24 hours to obtain dry porous gel microspheres, screen and select microspheres with a particle size of 2-3 mm, seal and package, and store under dry and dark conditions at 4°C.

[0027] (3) Accurately weigh 1 g of freeze-dried microspheres, add 100 mL of sterile normal saline, and sonicate for 5 minutes at a power of 400 W until the spherical structure is completely destroyed. Take another portion of the bacterial suspension, and the encapsulation rate = the number of microspheres (CFU / g) / the number of bacterial colonies (CFU / g) in the bacterial suspension x 100%. Accurately weigh 1 g of freeze-dried microspheres, add 100 mL of phosphate buffer (pH 4.0, 0.1 M), and soak for 4 hours before measuring the number of bacterial colonies in the solution. The pH-responsive release rate = the number of released bacterial colonies (CFU / g) / the number of encapsulated bacterial colonies (CFU / g) x 100%. Measure 100 mL of phosphate buffer (pH 7.0, 0.1 M), slowly add sodium hydrosulfite powder, control the ORP at -250 mV, add accurately weighed 1 g of freeze-dried microspheres, and soak for 6 hours before measuring the number of bacterial colonies in the solution. The ORP-responsive release rate = the number of released bacterial colonies (CFU / g) / the number of encapsulated bacterial colonies (CFU / g) x 100%.

[0028] (4) Take a circular column with a diameter of 10 cm and a length of 50 cm, fill it with 2 kg of dry spheres, and simulate the inflow of Cd 2+ = 15 mg / L, Zn 2+ = 5 mg / L, Pb 2+ = 4 mg / L, Cu 2+ = 2 mg / L, SO4 2- = 50 mg / L, pH 4.5, ORP -250 mV, flow rate 350 mL / min, time 30 days.

[0029] Table 1: Sphere data of Example 1

[0030] Example 2: pH response The same as steps 1-3 of Example 1.

[0031] Take a circular column with a diameter of 10 cm and a length of 50 cm, fill it with 2 kg of dry spheres, and simulate the inflow of Cd 2+ = 15 mg / L, Zn 2+ = 5 mg / L, Pb 2+ = 4 mg / L, Cu 2+ = 2 mg / L, SO4 2- = 50 mg / L, pH 4.5, ORP +200 mV, flow rate 350 mL / min, time 30 days.

[0032] Example 3: ORP response The same as steps 1-3 of Example 1.

[0033] A 10 cm diameter by 50 cm long cylindrical column was packed with 2 kg of dry balls and simulated influent Cd 2+ = 15 mg / L, Zn 2+ = 5 mg / L, Pb 2+ = 4 mg / L, Cu 2+ = 2 mg / L, SO4 2- = 50 mg / L, pH 7.0, ORP -250 mV, flow rate 350 mL / min, time 30 days.

[0034] Example 4 Nitrate Competition Steps 1-3 were the same as Example 1.

[0035] A 10 cm diameter by 50 cm long cylindrical column was packed with 2 kg of dry balls and simulated influent Cd 2+ = 15 mg / L, Zn 2+ = 5 mg / L, Pb 2+ = 4 mg / L, Cu 2+ = 2 mg / L, SO4 2- = 50 mg / L, NO3 - = 50 mg / L, pH 4.5, ORP -250 mV, flow rate 350 mL / min, time 30 days.

[0036] Example 5 High Hydraulic Loading Steps 1-3 were the same as Example 1.

[0037] A 10 cm diameter by 50 cm long cylindrical column was packed with 2 kg of dry balls and simulated influent Cd 2+ = 15 mg / L, Zn 2+ = 5 mg / L, Pb 2+ = 4 mg / L, Cu 2+ = 2 mg / L, SO4 2- = 50 mg / L, pH 4.5, ORP -250 mV, flow rate 700 mL / min, time 30 days.

[0038] Comparative Example 1 Sodium alginate and deionized water were weighed according to a solid-liquid ratio of 1:20, and stirred at a constant temperature of 50°C for 2 hours to obtain a uniform solution; rice husk ash that was previously ground to 200 mesh was weighed according to 20% of the dry weight of the gel, added to the uniform solution, and stirred for another 1 hour to obtain a composite gel; freeze-dried powders of Desulfovibrio and Citrobacter were weighed according to a mass ratio of 1:1, added to sterile normal saline (0.85% sodium chloride) containing 0.1% (w / v) L-cysteine according to a solid-liquid ratio of 1:1.25, and gently mixed to obtain a bacterial suspension; the bacterial suspension and the composite gel were blended according to a volume ratio of 1:9, and mixed uniformly to obtain a mixed solution.

[0039] Steps 2-4 of Example 1 were followed.

[0040] Comparative Example 2 Comparative Example 2 is an oxidation-free crosslinking control of Example 1.

[0041] (1) Xanthan gum and low-fat pectin were weighed according to a mass ratio of 7:3, deionized water was added according to a solid-liquid ratio of 1:20, and stirred at a constant temperature of 50°C for 2 hours to obtain a uniform solution; rice husk ash that was previously ground to 200 mesh was weighed according to 20% of the dry weight of the gel, added to the uniform solution, and stirred for another 1 hour to obtain a composite gel; freeze-dried powders of Desulfovibrio and Citrobacter were weighed according to a mass ratio of 1:1, added to sterile normal saline (0.85% sodium chloride) containing 0.1% (w / v) L-cysteine according to a solid-liquid ratio of 1:1.25, and gently mixed to obtain a bacterial suspension; the bacterial suspension and the composite gel were blended according to a volume ratio of 1:9, and mixed uniformly to obtain a mixed solution.

[0042] (2) The mixed solution was drawn into a syringe (outlet diameter 2 mm) and dripped into a 2% (w / v) calcium chloride solution at a constant speed, and the solidification time was 30 minutes to obtain solidified microspheres; the wet microspheres were washed with deionized water for 3 times to obtain wet microspheres. The wet microspheres were placed in a freeze dryer and freeze-dried at -50°C and <10 Pa for 24 hours to obtain dry porous gel microspheres, and microspheres with a particle size of 2-3 mm were selected by sieving, sealed and packaged, and stored at 4°C under dry and dark conditions.

[0043] (3) Accurately weigh 1 g of the freeze-dried microspheres, add 100 mL of sterile normal saline, and sonicate at 400 W power for 5 minutes until the spherical structure is completely destroyed. Take another portion of the bacterial suspension. The encapsulation efficiency = the number of microspheres (CFU / g) / the number of bacteria in the bacterial suspension (CFU / g) x 100%. Accurately weigh 1 g of the freeze-dried microspheres, add 100 mL of phosphate buffer (pH 4.0, 0.1 M), and soak for 4 hours before measuring the number of bacteria in the solution. The pH-responsive release rate = the number of released bacteria (CFU / g) / the number of encapsulated bacteria (CFU / g) x 100%. Measure 100 mL of phosphate buffer (pH 7.0, 0.1 M), slowly add sodium hydrosulfite powder, control the ORP at -250 mV, add accurately weighed 1 g of freeze-dried microspheres, and soak for 6 hours before measuring the number of bacteria in the solution. The ORP-responsive release rate = the number of released bacteria (CFU / g) / the number of encapsulated bacteria (CFU / g) x 100%.

[0044] (4) Take a circular column with a diameter of 10 cm and a length of 50 cm, fill it with 2 kg of dry balls, and simulate the water inlet Cd 2+ = 15 mg / L, Zn 2+ = 5 mg / L, Pb 2+ = 4 mg / L, Cu 2+ = 2 mg / L, SO4 2- = 50 mg / L, pH 4.5, ORP -250 mV, flow rate 350 mL / min, time 30 days.

[0045] Comparative Example 3 Comparative Example 3 is a control without the addition of rice husk ash of Example 1.

[0046] (1) Weigh xanthan gum and low-fat pectin at a mass ratio of 7:3, add deionized water at a solid-liquid ratio of 1:20, and stir at 50°C for 2 hours to obtain a uniform solution. Weigh the freeze-dried powder of Desulfovibrio and Citrobacter at a mass ratio of 1:1, add sterile normal saline (0.85% sodium chloride) containing 0.1% (w / v) L-cysteine at a solid-liquid ratio of 1:1.25, and mix gently to obtain a bacterial suspension. Blend the bacterial suspension and the composite gel at a volume ratio of 1:9, mix well, and obtain a mixed solution.

[0047] (2) Using a syringe (2 mm outlet diameter), extract the mixture and drip it into a 2% (w / v) calcium chloride solution at a uniform rate. The curing time is 30 minutes to obtain cured microspheres. Remove the cured microspheres and place them in a 0.5% (w / v) hydrogen peroxide solution. Stir gently for 20 minutes, then remove them again and wash them three times with deionized water to obtain wet microspheres. Place the wet microspheres in a freeze dryer and freeze-dry at -50℃ and <10 Pa for 24 hours to obtain dry, porous gel microspheres. Select microspheres with a particle size of 2-3 mm by sieving, seal them in a package, and store them at 4℃ under dry, light-protected conditions.

[0048] (3) Accurately weigh 1 g of freeze-dried microspheres, add them to 100 mL of sterile physiological saline, and sonicate them at 400 W for 5 minutes until the microsphere structure is completely destroyed. Take another portion of the bacterial suspension. The encapsulation rate = microsphere colony count (CFU / g) / bacterial suspension colony count (CFU / g) × 100%. Accurately weigh 1 g of freeze-dried microspheres, add them to 100 mL of phosphate buffer (pH 4.0, 0.1 M), soak them for 4 hours, and then measure the colony count in the solution. The pH response release rate = released colony count (CFU / g) / encapsulated colony count (CFU / g) × 100%. Measure 100 mL of phosphate buffer (pH 7.0, 0.1 M), slowly add sodium dithionite powder, control the ORP to be stable at -250 mV, and add 1 g of accurately weighed microspheres. After soaking the freeze-dried microspheres for 6 hours, the number of colonies in the solution was measured. The ORP response release rate = number of released colonies (CFU / g) / number of encapsulated colonies (CFU / g) × 100%.

[0049] (4) Take a circular column with a diameter of 10 cm and a length of 50 cm, fill it with 2 kg of dry bulbs, and simulate the ingress of Cd water. 2+ =15 mg / L, Zn 2+ =5 mg / L, Pb 2+ =4 mg / L, Cu 2+ =2 mg / L, SO4 2- =50 mg / L, pH 4.5, ORP -250 mV, flow rate 350 mL / min, time 30 days.

[0050] The ion removal performance of the microbial remediation response microspheres prepared in Examples 1-5 and Comparative Examples 1-3 was compared, and the results are shown in Table 2.

[0051] The results are as follows Figure 1As shown, Example 1, triggered by both pH and ORP, verifies the upper limit of the technology and demonstrates the optimal remediation effect achievable when the environmental signal is perfectly matched. Example 2, triggered only by pH, demonstrates that the system can be activated through a pH response mechanism in the absence of strong reducing conditions. Example 3, triggered only by ORP, demonstrates that the remediation process can be initiated as long as an environmental field is formed. Example 4, involving nitrate competition, demonstrates that after being inhibited by other microbial processes, the system can complete the remediation task through continuous release and microbial system recovery. Example 5, involving high hydraulic loading, demonstrates that the microspheres can still effectively immobilize the bacterial agent and maintain a high removal rate under high flow rate flushing.

[0052] Further evidence indicates that the microbial remediation microspheres exhibit swelling triggered by carboxyl ionization at pH < 5, and that H+ ionization occurs when the ambient pH decreases. + As the concentration increases, it competes with calcium ions for carboxyl sites, forming uncharged carboxylic acids. The three-dimensional network structure swells and disintegrates, releasing the core bacterial agent. When ORP < -200 mV, disulfide bonds break. When the reducing substances in the environment increase, the reducing agent attacks and breaks the disulfide bonds, destroying the cross-linked network. The spherical shell structure disintegrates, releasing the core bacterial agent.

[0053] Compared with the comparative example, the microbial remediation microspheres prepared in this invention, through the design of pH and ORP dual-response sustained-release function, can effectively remove ions such as cadmium, zinc, lead and copper, and further achieve long-term stabilization and harmlessness of heavy metals.

[0054] Table 2 Ion removal performance of the examples and comparative examples

[0055] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A pH / ORP dual-responsive microbial remediation microsphere, characterized in that, The microbial repair microspheres are prepared by ion crosslinking and oxidative crosslinking of a mixture of composite gel solution and bacterial suspension; The composite gel solution comprises xanthan gum, low-fat pectin, and rice husk ash. The bacterial suspension contains desulfovibrio and citrate bacillus.

2. The microbial remediation microspheres according to claim 1, characterized in that, The microbial repair microspheres have a particle size of 2-3 mm.

3. A method for preparing pH / ORP dual-responsive microbial remediation microspheres, characterized in that, The preparation method includes the following steps: S1. Mix xanthan gum and low-fat pectin, add deionized water to the mixture, stir well, then add rice husk ash, which is 20% of the total dry weight of xanthan gum and low-fat pectin gel, and stir again to obtain a composite gel solution. S2. After mixing the freeze-dried powders of Desulfovibrio and Citrobacter, add 0.85% sodium chloride containing 0.1% (w / v) L-cysteine ​​and mix well to obtain a bacterial suspension. S3. After the bacterial suspension and the composite gel solution are mixed evenly, wet microspheres are obtained by ionic cross-linking and oxidative cross-linking. After freeze-drying, pH / ORP dual-responsive microbial repair microspheres are obtained.

4. The preparation method according to claim 3, characterized in that, In step S1, xanthan gum and low-fat pectin are mixed at a mass ratio of (6-8):(3-5); the solid-liquid ratio of the mixture to deionized water is 1:(18-25); the stirring conditions for the mixture and deionized water are: 45-55℃, stirring for 2 h until viscous.

5. The preparation method according to claim 3, characterized in that, In step S2, the mass ratio of the freeze-dried powders of *Vibrio desulfurans* and *Citrobacter citric acid* is 1:1; the amount of 0.85% sodium chloride added is 1-1.5 times the total mass of the *Vibrio desulfurans* and *Citrobacter citric acid* powders.

6. The preparation method according to claim 3, characterized in that, The mixing ratio of the bacterial suspension and the composite gel solution in step S3 is 1:

9.

7. The preparation method according to claim 3, characterized in that, The ionic crosslinking process in step S3 is as follows: the mixed solution of bacterial suspension and composite gel solution is dropped into a 2% (w / v) calcium chloride solution and solidified for 30 min to obtain gel balls; the oxidative crosslinking process is as follows: the gel balls are placed in a 0.5% (w / v) hydrogen peroxide solution and stirred for 20 min.

8. The preparation method according to claim 3, characterized in that, The freeze-drying process in step S3 is as follows: freeze-drying at -40~-60℃ for 24 h at a pressure less than 10 Pa.

9. The application of the microbial remediation microspheres of claim 1 or the microbial remediation microspheres prepared by the preparation method of claim 3 in the remediation of groundwater contaminated with heavy metals.

10. The application of the microbial remediation microspheres of claim 1 or the microbial remediation microspheres prepared by the preparation method of claim 3 in the removal of heavy metal ions from polluted water, characterized in that, The heavy metal ions are cadmium, zinc, lead, and copper.

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