A method for treating heavy metals in soil based on microorganisms

Abstract

The application provides a method for treating heavy metals in soil based on microorganisms. The method for treating heavy metals in soil based on microorganisms comprises the following steps: adding styrene sulfonic acid to perform a modification reaction; adjusting PH and performing alcohol precipitation; activating and pretreating Bacillus amyloliquefaciens; adding sodium alginate solution and performing a gelation reaction; adding modified chitosan to perform coating; applying an electric field and removing heavy metals in soil. Under the action of the electric field, the film-coated microspheres move from top to bottom into deep soil, can fully attract positively charged heavy metal ions in the heavy metal soil, and then the heavy metal ions are adsorbed and removed by the Bacillus amyloliquefaciens in the film. The film-coated bacterial solution microspheres adsorbed with heavy metals move from bottom to top under the action of the reverse electric field and are suspended on the water surface, so that the heavy metals are separated and removed from the soil, the soil quality is improved, and the heavy metal removal rate is relatively high.

Description

Technical Field

[0001] The present invention relates to the field of soil microbial remediation, and in particular to a method for treating heavy metals in soil based on microorganisms. Background Art

[0002] Heavy metal soil refers to soil in which the content of heavy metal elements is significantly higher than its natural background value. Heavy metal soil in mining areas is usually caused by the indiscriminate discharge of waste, wastewater and exhaust gas. Heavy metal contaminated soil fertility decreases, making it difficult for plants to grow, which leads to environmental pollution and waste of land resources. Therefore, heavy metal soil needs to be treated to reduce its heavy metal content and reduce pollution.

[0003] Microorganisms have a short growth cycle, low remediation costs and will not cause secondary pollution, which gives them great advantages in heavy metal soil treatment. At present, the existing microbial treatment methods mostly directly put microorganisms into heavy metal soil, using the microorganisms' biological adsorption and accumulation capabilities to adsorb heavy metals, thereby reducing the toxic effects of heavy metals. However, due to the slow movement of microorganisms, this method is slow to take effect and lasts for a long time, resulting in a relatively low heavy metal removal rate. In addition, the treated heavy metals are still in the soil and cannot be separated and removed from the soil. Long-term accumulation will lead to a decrease in soil quality.

[0004] Therefore, we proposed a fast-acting microbial-based method for treating heavy metals in soil, which can separate and remove heavy metals from the soil to improve soil quality. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a method for treating heavy metals in soil based on microorganisms.

[0006] A method for treating heavy metals in soil based on microorganisms comprises the following steps:

[0007] S1: Add styrene sulfonic acid for modification reaction

[0008] The chitosan and acetic acid solution are fully mixed and dissolved, and then nitrogen is introduced to exhaust the air, and then ammonium persulfate is added for initiation, and finally styrene sulfonic acid is added to carry out a modification reaction to obtain a modified chitosan mixture;

[0009] S2: Adjust pH and precipitate with alcohol

[0010] Adding sodium hydroxide solution to the modified chitosan mixture until the pH of the modified chitosan mixture is adjusted to 6-8, then adding ethanol to carry out precipitation reaction, filtering, washing and drying to obtain modified chitosan;

[0011] S3: Activation and pretreatment of Bacillus amyloliquefaciens

[0012] The Bacillus amyloliquefaciens is inoculated into a seed culture medium for activation culture, 10% of which is inoculated into a transformation culture medium for shaking culture, then washed with physiological saline to obtain washed bacterial seeds;

[0013] S4: adding sodium alginate solution and performing gelation reaction

[0014] The washed bacterial seeds and the sodium alginate solution are stirred and mixed, then nano-sized calcium carbonate, sorbitan trioleate and liquid paraffin are added, mixed, then glacial acetic acid is added, and gelation reaction is performed, and after washing and sieving, bacterial gel balls are obtained;

[0015] S5: adding modified chitosan for coating

[0016] The modified chitosan is dissolved in methanol, then oscillation reaction is performed with the bacterial gel balls to perform coating, then washing is performed, then a sodium citrate solution is added, and the sol is oscillated to obtain membrane-coated bacterial liquid microspheres;

[0017] S6: applying an electric field and removing heavy metals in soil

[0018] The heavy metal soil is irrigated, then the membrane-coated bacterial liquid microspheres are uniformly scattered, then an electric field is applied for 16-18 days, then a reverse electric field is applied for 20-24 days, then the membrane-coated bacterial liquid microspheres suspended on the surface of the soil are collected to obtain heavy metal-removed soil.

[0019] Further, step S1 of adding styrene sulfonic acid for modification reaction specifically includes the following steps:

[0020] S1.1: chitosan and acetic acid solution are added into a reactor at a solid-liquid ratio of 1g:8-12mL, until the gravity sensor in the reactor detects that the gravity in the reactor no longer increases, and the gravity sensor sends a signal to the controller;

[0021] S1.2: after the controller receives the signal sent by the gravity sensor, the stirrer in the reactor is controlled at a stirring rate of 700-800r / min for 1-2h for sufficient dissolution;

[0022] S1.3: the controller controls the air pump to pump nitrogen into the reactor, until the nitrogen concentration detector in the reactor detects that the nitrogen concentration is 90-98%, and the nitrogen concentration detector sends a signal to the controller;

[0023] S1.4: after the controller receives the signal sent by the nitrogen concentration detector, the stirring rate of the stirrer is adjusted to 200-300r / min, and the controller hopper adds ammonium persulfate into the reactor for 20-30min;

[0024] S1.5: The controller controls the heater in the reactor to heat at a rate of 5-10°C / min until the temperature sensor in the reactor detects that the temperature has risen to 60-70°C, and the temperature sensor sends a signal to the controller;

[0025] S1.6: After receiving the signal from the temperature sensor, the controller controls the hydraulic pump to pump styrene sulfonic acid into the reactor, and heats the reaction for 4-6 hours to perform modification and obtain a modified chitosan mixture.

[0026] Furthermore, the activation and pretreatment of Bacillus amyloliquefaciens in step S3 specifically includes the following steps:

[0027] S3.1: Inoculate the seed culture medium with Bacillus amyloliquefaciens, then place the seed culture medium in an incubator and activate the culture at 30-34°C and 160-180 rpm for 12-16 hours to obtain a seed solution;

[0028] S3.2: Inoculate 10% of the above seed solution into the transformation medium, then place the transformation medium in an incubator, maintain the temperature and shaking rate constant, and continue shaking culture for 8-10 hours to obtain an activated bacterial solution;

[0029] S3.3: Place the activated bacterial solution in a centrifuge and centrifuge for 20-30 minutes. Discard the first supernatant to obtain the activated bacterial strain.

[0030] S3.4: Add physiological saline to the activated bacterial strain, centrifuge again for 10-20 minutes, wash, and discharge the second supernatant to obtain the washed bacterial strain.

[0031] Furthermore, step S4 of adding sodium alginate solution and performing gelation reaction specifically includes the following steps:

[0032] S4.1: Add the washed bacterial strain prepared in step S3.4 and the sodium alginate solution into a blender and stir at a rate of 500-600 rpm for 20-30 min;

[0033] S4.2: Add nano-calcium carbonate to a blender at a solid-liquid ratio of 1 g:90-100 mL. Adjust the stirring rate to 300-400 rpm and continue stirring for 30-40 minutes to thoroughly mix to obtain component A.

[0034] S4.3: Add sorbitan trioleate and liquid paraffin in a volume ratio of 2-3:1 to a mixing tank and stir thoroughly with a stirrer to obtain component B.

[0035] S4.4: Add component B to component A, adjust the stirring rate of the mixer to 150-250 rpm, and continue stirring for 1-2 hours to obtain a Bacillus amyloliquefaciens emulsion;

[0036] S4.5: Add glacial acetic acid to the above-mentioned Bacillus amyloliquefaciens emulsion, adjust the stirring rate to 100-150 r / min, and stir for 40-50 min to carry out gelation reaction to obtain gel spheres;

[0037] S4.6: Add the mixed solution to the above gel balls, adjust the stirring rate to 350-500 r / min, stir for 10-20 minutes, wash, and pass through a 60-80 mesh sieve to obtain bacteria-containing gel balls.

[0038] Furthermore, step S5 of adding modified chitosan for coating specifically includes the following steps:

[0039] S5.1: Add the modified chitosan prepared in step S2 and methanol into a batching machine, stir and mix thoroughly to dissolve, and prepare a 5-10 g / L modified chitosan solution;

[0040] S5.2: Add the bacteria-containing rubber balls prepared in step S4.6 and the modified chitosan solution described above to an oscillating reaction tank at a solid-liquid ratio of 1:9-12, and oscillate the reaction tank at a rate of 100-200 rpm for 3-5 hours to obtain membrane-coated rubber balls;

[0041] S5.3: Wash the membrane-coated microspheres 2-3 times with sodium chloride solution, then add 50-60 mmol / L sodium citrate solution to the washed membrane-coated microspheres at a solid-to-liquid ratio of 1:10-12;

[0042] S5.4: Adjust the oscillation rate of the oscillating reaction tank to 200-300 r / min and continue oscillating for 40-50 min to perform sol formation to obtain membrane-coated bacterial liquid microspheres.

[0043] Furthermore, step S6 of applying an electric field and removing heavy metals in the soil specifically includes the following steps:

[0044] S6.1: Irrigate the heavy metal soil until the water covers the heavy metal soil to obtain waterlogged soil;

[0045] S6.2: Evenly spread the membrane-coated bacterial solution microspheres prepared in step S5.4 into the water-soaked soil at a concentration of 50-60 g / m². An electric field is then applied to the water-soaked soil. Heavy metals migrate upward under the action of the electric field, while the membrane-coated bacterial solution microspheres migrate downward under the action of the electric field, adsorbing heavy metals in the soil.

[0046] S6.3: After applying the electric field for 16-18 days, apply a reverse electric field to the heavy metal-removing soil. After applying the electric field for 20-24 days, collect the membrane-coated bacterial liquid microspheres suspended on the soil surface to obtain the heavy metal-removing soil.

[0047] Furthermore, the solid-liquid ratio of chitosan to styrene sulfonic acid is 1 g:5-7 mL.

[0048] Furthermore, the seed culture medium is prepared from glucose, peptone, yeast extract, L-phenylalanine, potassium hydrogen phosphate trihydrate, magnesium sulfate heptahydrate and purified water.

[0049] Furthermore, the sodium alginate solution is prepared by mixing sodium alginate and physiological saline at a solid-liquid ratio of 1g:60-70mL.

[0050] Furthermore, the mixed solution is prepared by mixing 1-3% Tween-80 and 1.1-1.5% calcium chloride.

[0051] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0052] 1. The present invention modifies chitosan by using styrene sulfonic acid so that the surface of the prepared film-coated microspheres is negatively charged. Then, under the action of the electric field, the film-coated microspheres move from top to bottom and enter deep into the soil, which can fully attract positively charged heavy metal ions in the heavy metal soil. The heavy metal ions can enter the interior of the membrane-coated bacterial liquid microspheres through the modified chitosan membrane, and then the amylobacillus in the membrane adsorbs and removes the heavy metal ions. The membrane-coated bacterial liquid microspheres that have adsorbed heavy metals move from bottom to top under the action of the reverse electric field until they are suspended on the water surface, thereby achieving the effect of separating and removing heavy metals from the soil, improving soil quality, and having a relatively high heavy metal removal rate.

[0053] 2. The present invention adopts Mn 2+ 、Fe 3+ and Cr 6+ Bacillus amyloliquefaciens, which has good tolerance to heavy metals such as chloramphenicol, is used to treat heavy metal-contaminated soil in coal mining areas in a targeted manner, which can improve the efficiency of heavy metal treatment and reduce the types of bacteria used.

[0054] 3. The present invention can relatively reduce the impact of environmental factors on Bacillus amyloliquefaciens by coating Bacillus amyloliquefaciens with a modified chitosan film, thereby improving the stability and adsorption efficiency of Bacillus amyloliquefaciens.

[0055] 4. The present invention applies an electric field after spreading the membrane-coated bacterial liquid microspheres, causing the membrane-coated microspheres with negative charges on the surface to move downward and the positively charged heavy metal ions to migrate upward. This can reduce the encounter time between the membrane-coated microspheres and the heavy metal ions, thereby reducing the treatment time of microorganisms and achieving rapid results. At the same time, since the membrane-coated microspheres and the heavy metal ions move toward each other, from a macroscopic perspective, the contact area between the two can be increased, allowing the two to mix more fully, thereby further improving the adsorption efficiency of Bacillus amyloliquefaciens for heavy metals. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a flow chart of the method for treating heavy metals in soil based on microorganisms adopted in an embodiment of the present invention.

[0057] Figure 2 This is a summary table of the calculation results of heavy metal removal rates of Examples 1, 2 and 3 of the present invention.

[0058] Figure 3 The table below summarizes the calculation results of heavy metal removal rates for Example 1 and Comparative Example 1 of the present invention.

[0059] Figure 4 The table below summarizes the calculation results of heavy metal removal rates for Example 1 and Comparative Example 2 of the present invention.

[0060] Figure 5 The table below summarizes the calculation results of heavy metal removal rates for Example 1 and Comparative Example 3 of the present invention.

[0061] Figure 6 The table below summarizes the calculation results of heavy metal removal rates for Example 1 and Comparative Example 4 of the present invention. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below with reference to specific embodiments.

[0063] Example 1

[0064] A microbial-based method for treating heavy metals in soil, such as Figure 1 and Figure 2 As shown, the following steps are included:

[0065] S1: Add styrene sulfonic acid for modification reaction

[0066] Chitosan and acetic acid solution are added to the reactor at a solid-liquid ratio of 1g:8mL until the gravity sensor in the reactor detects that the gravity in the reactor no longer increases. The gravity sensor sends a signal to the controller. After receiving the signal sent by the gravity sensor, the controller controls the stirrer in the reactor to stir at a rate of 700r / min for 1h to fully dissolve. Subsequently, the controller controls the air pump to pump nitrogen into the reactor until the nitrogen concentration detector in the reactor detects that the nitrogen concentration is 90%. The nitrogen concentration detector sends a signal to the controller. The controller receives the signal sent by the nitrogen concentration detector. After the signal is received, the stirring rate of the stirrer is controlled to be adjusted to 200r / min, and the controller feed hopper is used to add ammonium persulfate into the reactor. After 20 minutes of initiation, the controller controls the heater in the reactor to heat at a rate of 5°C / min until the temperature sensor in the reactor detects that the temperature rises to 60°C. The temperature sensor sends a signal to the controller. After receiving the signal sent by the temperature sensor, the controller controls the hydraulic pump to pump styrene sulfonic acid into the reactor at a solid-liquid ratio of chitosan to styrene sulfonic acid of 1g:5mL, and heats the reaction for 4h to carry out modification to obtain a modified chitosan mixture;

[0067] S2: Adjust pH and precipitate with alcohol

[0068] Adding sodium hydroxide solution to the modified chitosan mixture until the pH of the modified chitosan mixture is adjusted to 6, then adding ethanol to carry out precipitation reaction, filtering, washing and drying to obtain modified chitosan;

[0069] S3: Activation and pretreatment of Bacillus amyloliquefaciens

[0070] Bacillus amyloliquefaciens is inoculated into a seed culture medium prepared from glucose, peptone, yeast extract, L-phenylalanine, potassium hydrogen phosphate trihydrate, magnesium sulfate heptahydrate, and purified water; the seed culture medium is then placed in an incubator and activated and cultured at 30° C. and 160 rpm for 12 hours to obtain a seed solution; 10% of the seed solution is then inoculated into a transformation culture medium; the transformation culture medium is then placed in an incubator, the temperature and the shaking rate are maintained constant, and the shaking culture is continued for 8 hours to obtain an activated bacterial solution; the activated bacterial solution is then placed in a centrifuge and centrifuged for 20 minutes, a first supernatant is discharged, and an activated bacterial strain is obtained; finally, physiological saline is added to the activated bacterial strain, the culture is centrifuged again for 10 minutes, and the culture is washed; and a second supernatant is discharged to obtain a washed bacterial strain;

[0071] S4: Add sodium alginate solution and perform gelation reaction

[0072] The washing bacteria and a sodium alginate solution prepared by mixing sodium alginate and normal saline at a solid-liquid ratio of 1 g:60 mL are added into a blender, stirred at a speed of 500 r / min for 20 min, then nano-sized calcium carbonate is added into the blender at a solid-liquid ratio of 1 g:90 mL, the stirring speed is adjusted to 300 r / min, and the stirring is continued for 30 min, so as to be fully mixed, thereby obtaining component A; sorbitan trioleate and liquid paraffin are added into a mixing tank at a volume ratio of 2:1, and fully stirred and mixed by a stirrer, thereby obtaining component B; then component B is added into component A, the stirring speed of the blender is adjusted to 150 r / min, and the stirring is continued for 1 h, thereby obtaining a bacillus amyloliquefaciens emulsion; glacial acetic acid is added into the bacillus amyloliquefaciens emulsion, and the stirring speed is adjusted to 100 r / min, and the stirring is continued for 40 min, so as to perform a gelation reaction, thereby obtaining gel balls; finally, a mixed solution prepared by mixing 1% Tween-80 and 1.1% calcium chloride is added into the gel balls, the stirring speed is adjusted to 350 r / min, and the stirring is continued for 10 min, so as to perform a washing, and the gel balls are screened through a 60-80 mesh sieve, thereby obtaining the gel balls containing bacteria;

[0073] S5: adding modified chitosan for coating

[0074] The modified chitosan prepared in step S2 and methanol are added into a batching machine, fully stirred and mixed, and dissolved, so as to prepare a 5 g / L modified chitosan solution; the gel balls containing bacteria and the modified chitosan solution are added into a shaking reaction tank at a solid-liquid ratio of 1:9, and shaken at a speed of 100 r / min for 3 h, so as to perform a coating, thereby obtaining membrane-coated gel balls; the membrane-coated gel balls are washed twice with a sodium chloride solution; 50 mmol / L sodium citrate solution is added into the washed membrane-coated gel balls at a solid-liquid ratio of 1:10, the shaking speed of the shaking reaction tank is adjusted to 200 r / min, and the shaking is continued for 40 min, so as to perform a sol, thereby obtaining membrane-coated bacterial liquid microspheres;

[0075] S6: applying an electric field and removing heavy metals in soil

[0076] The heavy metal soil with Mn, Fe and Cr contents of about 2235 mg / kg, 2476 mg / kg and 485 mg / kg respectively is irrigated until the heavy metal soil is submerged in water, thereby obtaining a waterlogged soil; then 50 g / m 2 The membrane-coated bacterial liquid microspheres are uniformly scattered into the waterlogged soil, and then an electric field is applied to the waterlogged soil; under the action of the electric field, the heavy metals migrate upward, and the membrane-coated bacterial liquid microspheres migrate downward under the action of the electric field, thereby adsorbing the heavy metals in the soil; after the electric field is applied for 16 d, a reverse electric field is applied to the heavy metal-removed soil, and after the reverse electric field is applied for 20 d, the membrane-coated bacterial liquid microspheres suspended on the surface of the soil are collected, thereby obtaining the heavy metal-removed soil.

[0077] Then, the content of heavy metal elements in the heavy metal-removed soil was tested. The results showed that the contents of Mn, Fe and Cr in the heavy metal-removed soil were approximately 378 mg / kg, 433 mg / kg and 96 mg / kg, respectively, that is, the removal rates of Mn, Fe and Cr were approximately 83.09%, 82.51% and 80.2%, respectively.

[0078] Example 2

[0079] A microbial-based method for treating heavy metals in soil, such as Figure 1 and Figure 2 As shown, the following steps are included:

[0080] S1: Add styrene sulfonic acid for modification reaction

[0081] Chitosan and acetic acid solution are added to the reactor at a solid-liquid ratio of 1g:10mL until the gravity sensor in the reactor detects that the gravity in the reactor no longer increases. The gravity sensor sends a signal to the controller. After receiving the signal sent by the gravity sensor, the controller controls the stirrer in the reactor to stir at a rate of 750r / min for 1.5h to fully dissolve. Subsequently, the controller controls the air pump to pump nitrogen into the reactor until the nitrogen concentration detector in the reactor detects that the nitrogen concentration is 94%. The nitrogen concentration detector sends a signal to the controller. The controller receives the signal sent by the nitrogen concentration detector. After the signal is received, the stirring rate of the stirrer is controlled to be adjusted to 250r / min, and the controller feed hopper is used to add ammonium persulfate into the reactor. After 25 minutes of initiation, the controller controls the heater in the reactor to heat at a rate of 7.5°C / min until the temperature sensor in the reactor detects that the temperature rises to 65°C. The temperature sensor sends a signal to the controller. After receiving the signal sent by the temperature sensor, the controller controls the hydraulic pump to pump styrene sulfonic acid into the reactor at a solid-liquid ratio of chitosan to styrene sulfonic acid of 1g:6mL, and heats the reaction for 5 hours to carry out modification to obtain a modified chitosan mixture;

[0082] S2: Adjust pH and precipitate with alcohol

[0083] Adding sodium hydroxide solution to the modified chitosan mixture until the pH of the modified chitosan mixture is adjusted to 7, then adding ethanol to carry out precipitation reaction, filtering, washing and drying to obtain modified chitosan;

[0084] S3: Activation and pretreatment of Bacillus amyloliquefaciens

[0085] Bacillus amyloliquefaciens is inoculated into a seed culture medium prepared from glucose, peptone, yeast extract, L-phenylalanine, potassium hydrogen phosphate trihydrate, magnesium sulfate heptahydrate, and purified water; the seed culture medium is then placed in an incubator and activated and cultured at 32° C. and 170 rpm for 14 hours to obtain a seed solution; 10% of the seed solution is then inoculated into a transformation culture medium; the transformation culture medium is then placed in an incubator, the temperature and the shaking rate are maintained constant, and the shaking culture is continued for 9 hours to obtain an activated bacterial solution; the activated bacterial solution is then placed in a centrifuge and centrifuged for 25 minutes, a first supernatant is discharged, and an activated bacterial strain is obtained; finally, physiological saline is added to the activated bacterial strain, the culture is centrifuged again for 15 minutes, and the culture is washed; a second supernatant is discharged, and a washed bacterial strain is obtained;

[0086] S4: Add sodium alginate solution and perform gelation reaction

[0087] The washed bacterial strain and a sodium alginate solution prepared by mixing sodium alginate and physiological saline at a solid-liquid ratio of 1g:65mL were added to a blender, stirred at a rate of 550r / min for 25min, and then nano-scale calcium carbonate was added to the blender at a solid-liquid ratio of 1g:95mL. The stirring rate was adjusted to 350r / min, and the stirring was continued for 35min. The mixture was thoroughly mixed to obtain component A. Sorbitan trioleate and liquid paraffin were then added to a mixing tank at a volume ratio of 2.5:1. The mixture was thoroughly stirred with a blender to obtain component B. Component B was added to component A, the stirring rate of the stirrer was adjusted to 200 r / min, and stirring was continued for 1.5 hours to obtain a Bacillus amyloliquefaciens emulsion. Glacial acetic acid was then added to the Bacillus amyloliquefaciens emulsion, and the stirring rate was adjusted to 175 r / min. The mixture was stirred for 45 minutes to perform a gelation reaction to obtain gel balls. Finally, a mixed solution prepared by mixing 2% Tween-80 and 1.3% calcium chloride was added to the gel balls. The mixture was stirred at a stirring rate of 425 r / min and stirred for 15 minutes. The mixture was washed and passed through a 70-mesh sieve to obtain bacteria-containing gel balls.

[0088] S5: Add modified chitosan for coating

[0089] The modified chitosan prepared in step S2 and methanol were added to a batching machine, and the mixture was thoroughly stirred and dissolved to prepare a 7.5 g / L modified chitosan solution. The bacteria-containing rubber balls and the modified chitosan solution were then added to an oscillation reaction tank at a solid-liquid ratio of 1:10. The reaction was oscillated at a rate of 150 r / min for 4 h to obtain membrane-coated rubber balls. The membrane-coated rubber balls were then washed twice with a sodium chloride solution. 55 mmol / L sodium citrate solution was then added to the washed membrane-coated microspheres at a solid-liquid ratio of 1:11. The oscillation rate of the oscillation reaction tank was adjusted to 250 r / min, and the oscillation was continued for 45 min to obtain a sol, thereby obtaining membrane-coated bacterial liquid microspheres.

[0090] S6: Applying an electric field and removing heavy metals from soil

[0091] The heavy metal soil with Mn, Fe and Cr contents of approximately 2235 mg / kg, 2476 mg / kg and 485 mg / kg, respectively, was irrigated until the water covered the heavy metal soil to obtain waterlogged soil, and then the soil was heated at 55 g / m 2 The membrane-coated bacterial liquid microspheres are evenly sprinkled into the soaked soil, and then an electric field is applied to the soaked soil. The heavy metals migrate upward under the action of the electric field, and the membrane-coated bacterial liquid microspheres migrate downward under the action of the electric field, adsorbing the heavy metals in the soil. After applying the electric field for 17 days, a reverse electric field is applied to the heavy metal-removing soil. After 22 days of application, the membrane-coated bacterial liquid microspheres suspended on the soil surface are collected to obtain the heavy metal-removing soil.

[0092] Then, the content of heavy metal elements in the heavy metal-free soil was tested. The results showed that the content of Mn, Fe and Cr in the heavy metal-free soil was approximately 383 mg / kg, 427 mg / kg and 94 mg / kg, respectively. 2+ 、Fe 3+ and Cr 6+ The removal rates were approximately 82.86%, 82.75% and 80.62%, respectively.

[0093] Example 3

[0094] A microbial-based method for treating heavy metals in soil, such as Figure 1 and Figure 2 As shown, the following steps are included:

[0095] S1: Add styrene sulfonic acid for modification reaction

[0096] Chitosan and acetic acid solution are added to the reactor at a solid-liquid ratio of 1g:12mL until the gravity sensor in the reactor detects that the gravity in the reactor no longer increases. The gravity sensor sends a signal to the controller. After receiving the signal sent by the gravity sensor, the controller controls the stirrer in the reactor to stir at a rate of 800r / min for 2h to fully dissolve. Subsequently, the controller controls the air pump to pump nitrogen into the reactor until the nitrogen concentration detector in the reactor detects that the nitrogen concentration is 98%. The nitrogen concentration detector sends a signal to the controller. The controller receives the signal sent by the nitrogen concentration detector. After the signal is received, the stirring rate of the stirrer is controlled to be adjusted to 300r / min, and the controller feed hopper is used to add ammonium persulfate into the reactor. After 30 minutes of initiation, the controller controls the heater in the reactor to heat at a rate of 10°C / min until the temperature sensor in the reactor detects that the temperature rises to 70°C. The temperature sensor sends a signal to the controller. After receiving the signal sent by the temperature sensor, the controller controls the hydraulic pump to pump styrene sulfonic acid into the reactor at a solid-liquid ratio of chitosan to styrene sulfonic acid of 1g:7mL, and heats the reaction for 6 hours to carry out modification to obtain a modified chitosan mixture;

[0097] S2: Adjust pH and precipitate with alcohol

[0098] Adding sodium hydroxide solution to the modified chitosan mixture until the pH of the modified chitosan mixture is adjusted to 8, then adding ethanol to carry out precipitation reaction, filtering, washing and drying to obtain modified chitosan;

[0099] S3: Activation and pretreatment of Bacillus amyloliquefaciens

[0100] Bacillus amyloliquefaciens is inoculated into a seed culture medium prepared from glucose, peptone, yeast extract, L-phenylalanine, potassium hydrogen phosphate trihydrate, magnesium sulfate heptahydrate, and purified water; the seed culture medium is then placed in an incubator and activated and cultured at 34° C. and 180 rpm for 16 hours to obtain a seed solution; 10% of the seed solution is then inoculated into a transformation culture medium; the transformation culture medium is then placed in an incubator, the temperature and the shaking rate are maintained constant, and the shaking culture is continued for 10 hours to obtain an activated bacterial solution; the activated bacterial solution is then placed in a centrifuge and centrifuged for 30 minutes, a first supernatant is discharged, and an activated bacterial strain is obtained; finally, physiological saline is added to the activated bacterial strain, the culture is centrifuged again for 20 minutes, and the washed bacterial strain is obtained after a second supernatant is discharged;

[0101] S4: Add sodium alginate solution and perform gelation reaction

[0102] The washed bacterial strain and a sodium alginate solution prepared by mixing sodium alginate and physiological saline at a solid-liquid ratio of 1g:70mL were added to a blender, stirred at a rate of 600r / min for 30min, and then nano-scale calcium carbonate was added to the blender at a solid-liquid ratio of 1g:100mL. The stirring rate was adjusted to 400r / min, and the stirring was continued for 40min. The mixture was thoroughly mixed to obtain component A. Sorbitan trioleate and liquid paraffin were then added to a mixing tank at a volume ratio of 3:1. The mixture was thoroughly stirred with a blender to obtain component B. Component B is added to component A, the stirring rate of the stirrer is adjusted to 250 r / min, and stirring is continued for 2 hours to obtain a Bacillus amyloliquefaciens emulsion. Glacial acetic acid is then added to the Bacillus amyloliquefaciens emulsion, and the stirring rate is adjusted to 150 r / min. Stirring is carried out for 50 minutes to perform a gelation reaction to obtain gel balls. Finally, a mixed solution prepared by mixing 3% Tween-80 and 1.5% calcium chloride is added to the gel balls. The stirring rate is adjusted to 500 r / min, stirring is carried out for 20 minutes, and the gel balls are washed and passed through an 80-mesh sieve to obtain bacteria-containing gel balls.

[0103] S5: Add modified chitosan for coating

[0104] The modified chitosan prepared in step S2 and methanol were added to a batching machine, and the mixture was thoroughly stirred and dissolved to prepare a 10 g / L modified chitosan solution. The bacteria-containing rubber balls and the modified chitosan solution were then added to an oscillation reaction tank at a solid-liquid ratio of 1:12. The reaction was oscillated at a rate of 200 r / min for 5 h to obtain membrane-coated rubber balls. The membrane-coated rubber balls were then washed three times with a sodium chloride solution. 60 mmol / L sodium citrate solution was then added to the washed membrane-coated microspheres at a solid-liquid ratio of 1:12. The oscillation rate of the oscillation reaction tank was adjusted to 300 r / min, and the oscillation was continued for 50 min to obtain a sol to obtain membrane-coated bacterial liquid microspheres.

[0105] S6: Applying an electric field and removing heavy metals from soil

[0106] The heavy metal soil with Mn, Fe and Cr contents of approximately 2235 mg / kg, 2476 mg / kg and 485 mg / kg, respectively, was irrigated until the water covered the heavy metal soil to obtain waterlogged soil, and then the soil was heated at 60 g / m 2 The membrane-coated bacterial liquid microspheres are evenly sprinkled into the soaked soil, and then an electric field is applied to the soaked soil. The heavy metals migrate upward under the action of the electric field, and the membrane-coated bacterial liquid microspheres migrate downward under the action of the electric field, adsorbing the heavy metals in the soil. After applying the electric field for 18 days, a reverse electric field is applied to the heavy metal-removing soil. After 24 days of application, the membrane-coated bacterial liquid microspheres suspended on the soil surface are collected to obtain the heavy metal-removing soil.

[0107] Then, the content of heavy metal elements in the heavy metal-removed soil was tested. The results showed that the contents of Mn, Fe and Cr in the heavy metal-removed soil were approximately 375 mg / kg, 429 mg / kg and 98 mg / kg, respectively, that is, the removal rates of Mn, Fe and Cr were approximately 83.22%, 82.67% and 79.79%, respectively.

[0108] Comparative Example 1

[0109] Refer to the preparation steps of Example 1, other conditions remain unchanged, only step S3 is retained, and then the washed bacteria are heated at 50-60g / m 2 Evenly add heavy metal soil containing Mn, Fe and Cr of about 2236mg / kg, 2473mg / kg and 483mg / kg, respectively. After 36 days, the Mn, Fe and Cr contents in the soil were detected to be about 2235mg / kg, 2473mg / kg and 483mg / kg, respectively. 2+ 、Fe 3+ and Cr 6+ The contents are approximately 1182 mg / kg, 1237 mg / kg and 242 mg / kg, respectively, that is, the removal rates of Mn, Fe and Cr are approximately 47.14%, 49.98% and 49.9%, respectively.

[0110] like Figure 3 As shown, by comparing the test results of Example 1, it can be seen that by modifying chitosan with styrenesulfonic acid, the surface of the prepared film-coated microspheres is negatively charged, and then under the action of the electric field, the film-coated microspheres move from top to bottom and enter deep into the soil, which can fully attract positively charged heavy metal ions in the heavy metal soil. The heavy metal ions can enter the interior of the membrane-coated bacterial liquid microspheres through the modified chitosan membrane, and then the amylobacillus in the membrane adsorbs and removes the heavy metal ions. The membrane-coated bacterial liquid microspheres that adsorb heavy metals move from bottom to top under the action of the reverse electric field until they are suspended on the water surface, thereby achieving the effect of separating and removing heavy metals from the soil, thereby improving soil quality, and the heavy metal removal rate is relatively high.

[0111] Comparative Example 2

[0112] Referring to the preparation steps of Example 1, other conditions remained unchanged, only the Bacillus amyloliquefaciens in step S3 was replaced with an equal amount of Bacillus subtilis, and then the heavy metal element content in the heavy metal removal soil was detected. The results showed that the Mn, Fe and Cr contents in the heavy metal removal soil were approximately 834 mg / kg, 1042 mg / kg and 229 mg / kg, respectively, that is, the removal rates of Mn, Fe and Cr were approximately 62.7%, 57.92% and 52.78%, respectively.

[0113] like Figure 4As shown, by comparing the test results of Example 1, it can be seen that by using 2+ 、Fe 3+ and Cr 6+ Bacillus amyloliquefaciens, which has good tolerance to heavy metals such as chloramphenicol, is used to treat heavy metal-contaminated soil in coal mining areas in a targeted manner, which can improve the efficiency of heavy metal treatment and reduce the types of bacteria used.

[0114] Comparative Example 3

[0115] Referring to the preparation steps of Example 1, other conditions remain unchanged, only steps S1 and S2 are removed, and the modified chitosan in step S5 is replaced with an equal amount of physiological saline. Then, the content of heavy metal elements in the heavy metal-removed soil is detected. The results show that the contents of Mn, Fe and Cr in the heavy metal-removed soil are approximately 2235 mg / kg, 2475 mg / kg and 484 mg / kg, respectively, and the free Mn 2+ 、Fe 3+ and Cr 6+ The contents are approximately 986 mg / kg, 1124 mg / kg and 221 mg / kg, respectively, that is, the removal rates of Mn, Fe and Cr are approximately 55.88%, 54.6% and 54.43%, respectively.

[0116] like Figure 5 As shown, by comparing the test results of Example 1, it can be seen that coating Bacillus amyloliquefaciens with a modified chitosan film can relatively reduce the impact of environmental factors on Bacillus amyloliquefaciens and improve its stability and adsorption efficiency.

[0117] Comparative Example 4

[0118] Referring to the preparation steps of Example 1, other conditions remain unchanged, only the electric field in step S6 is removed, and then the heavy metal element content in the heavy metal-removed soil is detected. The results show that the content of Mn, Fe and Cr in the heavy metal-removed soil is approximately 2235 mg / kg, 2476 mg / kg and 483 mg / kg, respectively, and the free Mn 2+ 、Fe 3+ and Cr 6+ The contents are approximately 597 mg / kg, 683 mg / kg and 148 mg / kg, respectively, that is, the removal rates of Mn, Fe and Cr are approximately 73.29%, 72.42% and 69.48%, respectively.

[0119] like Figure 6As shown, by comparing the test results of Example 1, it can be seen that by applying an electric field after sprinkling the membrane-coated bacterial liquid microspheres, the membrane-coated microspheres with negative surface charge move downward and the positively charged heavy metal ions migrate upward, which can reduce the encounter time between the membrane-coated microspheres and the heavy metal ions, thereby reducing the treatment time of the microorganisms and achieving a quick effect. At the same time, since the membrane-coated microspheres and the heavy metal ions move toward each other, from a macroscopic perspective, the contact area between the two can be increased, so that the two are mixed more fully, thereby further improving the adsorption efficiency of Bacillus amyloliquefaciens for heavy metals and improving the heavy metal removal rate.

[0120] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for treating heavy metals in soil based on microorganisms, characterized in that: The steps include: S1: Add styrene sulfonic acid for modification reaction The chitosan and acetic acid solution are fully mixed and dissolved, and then nitrogen is introduced to exhaust the air, and then ammonium persulfate is added for initiation, and finally styrene sulfonic acid is added to carry out a modification reaction to obtain a modified chitosan mixture; S2: Adjust pH and precipitate with alcohol Adding sodium hydroxide solution to the modified chitosan mixture until the pH of the modified chitosan mixture is adjusted to 6-8, then adding ethanol to carry out precipitation reaction, filtering, washing and drying to obtain modified chitosan; S3: Activation and pretreatment of Bacillus amyloliquefaciens The amyloliquefaciens was inoculated into a seed culture medium for activation culture, 10% of the amyloliquefaciens was inoculated into a transformation culture medium for shaking culture, and then washed with physiological saline to obtain a washed strain; S4: Add sodium alginate solution and perform gelation reaction The washed bacterial strain and sodium alginate solution are stirred and mixed, and then nano-scale calcium carbonate, sorbitan trioleate and liquid paraffin are added. After mixing, glacial acetic acid is added to carry out gelation reaction, and after washing and sieving, bacteria-containing gel balls are obtained; S5: Add modified chitosan for coating The modified chitosan is dissolved in methanol, and then reacted with the bacteria-containing gel balls by oscillation to coat them. After washing, sodium citrate solution is added and the sol is shaken to obtain membrane-coated bacteria liquid microspheres. S6: Applying an electric field and removing heavy metals from soil Irrigating the heavy metal soil, then evenly spreading the membrane-coated bacterial solution microspheres, then applying an electric field for 16-18 days, and then applying a reverse electric field for 20-24 days, collecting the membrane-coated bacterial solution microspheres suspended on the soil surface to obtain heavy metal-removed soil; The modification reaction of adding styrenesulfonic acid in step S1 specifically comprises the following steps: S1.1: Chitosan and acetic acid solution are added to the reactor at a solid-liquid ratio of 1 g:8-12 mL. The reaction is continued until the gravity sensor in the reactor detects that the gravity in the reactor no longer increases and sends a signal to the controller. S1.2: After receiving the signal from the gravity sensor, the controller controls the stirrer in the reactor to stir at a rate of 700-800 rpm for 1-2 hours to fully dissolve the solution. S1.3: The controller controls the air pump to pump nitrogen into the reactor until the nitrogen concentration detector in the reactor detects that the nitrogen concentration is 90-98%, and the nitrogen concentration detector sends a signal to the controller; S1.4: After receiving the signal from the nitrogen concentration detector, the controller controls the stirring speed of the stirrer to 200-300 r / min, and controls the feed hopper to add ammonium persulfate into the reactor for 20-30 minutes; S1.5: The controller controls the heater in the reactor to heat at a rate of 5-10°C / min until the temperature sensor in the reactor detects that the temperature has risen to 60-70°C, and the temperature sensor sends a signal to the controller; S1.6: After receiving the signal from the temperature sensor, the controller controls the hydraulic pump to pump styrene sulfonic acid into the reactor and heat the reaction for 4-6 hours to modify the chitosan to obtain a modified chitosan mixture. Step S5 of adding modified chitosan for coating specifically includes the following steps: S5.1: Add the modified chitosan prepared in step S2 and methanol into a batching machine, stir and mix thoroughly to dissolve, and prepare a 5-10 g / L modified chitosan solution; S5.2: Add the bacteria-containing rubber balls prepared in step S4.6 and the modified chitosan solution described above to an oscillating reaction tank at a solid-liquid ratio of 1:9-12, and oscillate the reaction tank at a rate of 100-200 rpm for 3-5 hours to obtain membrane-coated rubber balls; S5.3: Wash the membrane-coated microspheres 2-3 times with sodium chloride solution, then add 50-60 mmol / L sodium citrate solution to the washed membrane-coated microspheres at a solid-to-liquid ratio of 1:10-12; S5.4: Adjust the oscillation rate of the oscillating reaction tank to 200-300 r / min and continue oscillating for 40-50 min to perform sol formation to obtain membrane-coated bacterial liquid microspheres.

2. The method for treating heavy metals in soil based on microorganisms according to claim 1, characterized in that: The activation and pretreatment of Bacillus amyloliquefaciens in step S3 specifically comprises the following steps: S3.1: Inoculate the seed culture medium with Bacillus amyloliquefaciens, then place the seed culture medium in an incubator and activate the culture at 30-34°C and 160-180 rpm for 12-16 hours to obtain a seed solution; S3.2: Inoculate 10% of the above seed solution into the transformation medium, then place the transformation medium in an incubator, maintain the temperature and shaking rate constant, and continue shaking for 8-10 hours to obtain an activated bacterial solution; S3.3: Place the activated bacterial solution in a centrifuge and centrifuge for 20-30 minutes. Discard the first supernatant to obtain the activated bacterial strain. S3.4: Add physiological saline to the activated bacterial strain, centrifuge again for 10-20 minutes, wash, and discharge the second supernatant to obtain the washed bacterial strain.

3. The method for treating heavy metals in soil based on microorganisms according to claim 2, characterized in that: Step S4 of adding sodium alginate solution and performing gelation reaction specifically includes the following steps: S4.1: Add the washed bacterial strain prepared in step S3.4 and the sodium alginate solution into a blender and stir at a rate of 500-600 rpm for 20-30 min; S4.2: Add nano-calcium carbonate to a blender at a solid-liquid ratio of 1 g:90-100 mL. Adjust the stirring rate to 300-400 rpm and continue stirring for 30-40 minutes to thoroughly mix to obtain component A. S4.3: Add sorbitan trioleate and liquid paraffin in a volume ratio of 2-3:1 to a mixing tank and stir thoroughly with a stirrer to obtain component B. S4.4: Add component B to component A, adjust the stirring rate of the mixer to 150-250 rpm, and continue stirring for 1-2 hours to obtain a Bacillus amyloliquefaciens emulsion; S4.5: Add glacial acetic acid to the above-mentioned Bacillus amyloliquefaciens emulsion, adjust the stirring rate to 100-150 r / min, and stir for 40-50 min to carry out gelation reaction to obtain gel spheres; S4.6: Add the mixed solution to the above gel balls, adjust the stirring rate to 350-500 r / min, stir for 10-20 minutes, wash, and pass through a 60-80 mesh sieve to obtain bacteria-containing gel balls.

4. The method for treating heavy metals in soil based on microorganisms according to claim 3, characterized in that: Step S6 of applying an electric field and removing heavy metals from the soil specifically includes the following steps: S6.1: Irrigate the heavy metal soil until the water covers the heavy metal soil to obtain waterlogged soil; S6.2: Evenly spread the membrane-coated bacterial solution microspheres prepared in step S5.4 into the water-soaked soil at a concentration of 50-60 g / m². An electric field is then applied to the water-soaked soil. Heavy metals migrate upward under the action of the electric field, while the membrane-coated bacterial solution microspheres migrate downward under the action of the electric field, adsorbing heavy metals in the soil. S6.3: After applying the electric field for 16-18 days, apply a reverse electric field. After applying the electric field for 20-24 days, collect the membrane-coated bacterial liquid microspheres suspended on the soil surface to obtain heavy metal-removed soil.

5. The method for treating heavy metals in soil based on microorganisms according to claim 1, characterized in that: The solid-liquid ratio of chitosan to styrene sulfonic acid is 1g:5-7mL.

6. The method for treating heavy metals in soil based on microorganisms according to claim 2, characterized in that: The seed culture medium is prepared from glucose, peptone, yeast extract, L-phenylalanine, potassium hydrogen phosphate trihydrate, magnesium sulfate heptahydrate and purified water.

7. The method for treating heavy metals in soil based on microorganisms according to claim 3, characterized in that: The sodium alginate solution is prepared by mixing sodium alginate and physiological saline at a solid-liquid ratio of 1g:60-70mL.

8. The method for treating heavy metals in soil based on microorganisms according to claim 3, characterized in that: The mixed solution is prepared by mixing 1-3% Tween-80 and 1.1-1.5% calcium chloride.

Images