A method for in-situ restoration of polluted water by bacterial plant collaboration

Abstract

The present invention discloses a method for in-situ restoration of polluted water bodies by bacterial plant synergy. First, composite water purification bacterial agent capsules are prepared, and then the composite water purification bacterial agent capsules are mixed with sand and stone in a certain proportion as a plant root matrix, and the matrix is ​​wrapped in the root part of the water purification plant with a gauze net, and then it is placed in the water body that needs to be purified. The composite water purification bacterial agent capsules prepared by the method of the present invention can gradually disintegrate at the sedimentation site and release highly efficient water purification bacteria to the bottom mud and water body; the water purification plant in the present invention adopts an emergent plant, which has a well-developed root system, can not only absorb and adsorb pollutants in the water body, but also has an oxygen secretion effect, and can provide an aerobic microenvironment for the respiratory metabolism of water purification microorganisms, and the two work together to exert a long-term water purification effect.

Description

Technical Field

[0001] The present invention belongs to the field of bacterial agent immobilization and embedding technology and the field of polluted water body restoration, relates to the preparation and use of water purification bacterial agents, and particularly to a method for collaboratively restoring polluted water bodies using a composite water purification bacterial agent capsule and a water purification plant. Background Art

[0002] With the development of social economy and the expansion of urban population, the discharge of various industrial wastewater and domestic sewage has increased significantly. However, the sewage collection and treatment capacity of most cities is relatively backward, and a large amount of sewage is often discharged into urban rivers without treatment, resulting in serious pollution of rivers running through towns and even seasonal and perennial black and smelly water.

[0003] Water pollution control is a systematic project. It is necessary to control pollution at the source by relying on various source control and pollution interception projects, and to adopt a variety of means to control and ecologically restore existing polluted water bodies. Microbial water purification agents have significant advantages in the restoration of polluted water bodies, such as low cost, simple construction, quick results and no negative environmental impact. However, most of the water purification agents on the market are prepared by adding adsorbents and excipients to bacterial fermentation broth, and then drying and forming. Such water purification agents usually have shortcomings such as short shelf life and difficulty in evenly dispersing microorganisms in water bodies. There are also microcapsule agents prepared by embedding methods. This type of preparation method can significantly improve the shelf life and the tolerance of strains to the water environment, but most of the artificially added agents are lost in the river with the flow of water, and cannot continuously exert the water purification effect.

[0004] In addition, water purification plant restoration technology has also been widely used in water body restoration practice, among which plant floating beds are the most common. For example, the three-dimensional ecological floating bed designed by Li Xianning et al. uses aquatic plants, aquatic animals and artificial media to purify water, but its disadvantage is that the structure is complex and difficult to make; another example is the artificial method of planting aquatic plants on polystyrene foam boards or wood to make a purification floating bed frame, using the absorption and adsorption of plant roots to remove pollutants such as n and p in polluted water; there are also artificial fillers added to the floating bed to cooperate with biofilm to enhance the water purification effect. Plant floating bed technology is also unable to exert a sustained water purification effect due to seasonal factors.

[0005] In summary, due to the complex hydrology and water quality in various places, the existing single aquatic plant floating bed or microbial remediation technology is difficult to achieve the expected biological treatment effect when actually used in river sewage treatment, and the cost is relatively high. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for in-situ restoration of polluted water bodies by the synergistic use of bacteria and plants. First, a composite water-purifying bacterial agent capsule with a long-lasting water purification effect is prepared, and then the composite water-purifying bacterial agent capsule is synergistically applied with water-purifying plants. The method of the present invention organically integrates multiple water purification mechanisms such as adsorption, microbial degradation and plant absorption and utilization, thereby exerting a long-lasting water purification effect.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for in-situ restoration of polluted water by bacterial planting, comprising the following steps:

[0009] Step (1), Fe(NO3)3, attapulgite and water are mixed and stirred and then allowed to stand. After filtering, the filter cake is first dried and then roasted, and then ground and sieved to obtain Fe 3+ Modified attapulgite;

[0010] Step (2), the Fe obtained in step (1) 3+ The modified attapulgite and the unmodified attapulgite are mixed, water is added and stirred to prepare an attapulgite floc suspension;

[0011] Step (3), inoculating Bacillus pumilus and Bacillus cereus into liquid culture medium for enrichment culture respectively, discarding the supernatant after centrifugation, collecting the bacterial sludge precipitate at the lower layer, adjusting the concentration of the bacterial sludge precipitate with physiological saline, and preparing a concentrated bacterial solution;

[0012] Step (4), adding the concentrated bacterial solution of Bacillus pumilus and the concentrated bacterial solution of Bacillus cereus obtained in step (3) to the attapulgite floc suspension obtained in step (2), and oscillating and stirring to allow the strains to be stably fixed on the surface and gaps of the attapulgite flocs;

[0013] Step (5), controlling the water content of the mixed solution obtained in step (4) to 40% to 50% to obtain mud, adding sodium alginate solution to the mud, and stirring to obtain plastic mud;

[0014] Step (6), preparing the plastic mud obtained in step (5) into spherical particles in a mold, and immediately placing the particles in a calcium chloride solution, stirring to complete the embedding of the bacterial agent, separating the embedded spherical particles, washing the residual calcium chloride with clean water, and freeze-drying the particles to obtain a finished composite water purification bacterial agent capsule.

[0015] Step (7), mixing the composite water purification bacterial agent capsule obtained in step (6) with the sand and stone matrix in a certain proportion, then wrapping it around the root system of the water purification plant with a permeable material, and then putting it into the water body.

[0016] A further improvement of the present invention is:

[0017] The mass ratio of Fe(NO3)3, attapulgite and water in step (1) is 0.8~1.2:0.8~1.2:100, the standing time is 10~14h, the drying temperature is 100~110°C, the roasting temperature is 350~450°C, the roasting time is 2~4h, and the product is ground through a 200-mesh sieve.

[0018] Furthermore, in step (2), Fe 3+ The mass ratio of modified attapulgite to unmodified attapulgite is 1.8~2.2:1, and the mass fraction of solid components in the attapulgite floc suspension is 8~12%.

[0019] Furthermore, the liquid culture components in step (3) include 5 g beef extract, 10 g peptone, 5 g sodium chloride, and 1000 mL sterile water.

[0020] Furthermore, the culturing process in step (3) is as follows: culturing in a shaking incubator at 35-40°C for 22-26 hours; the centrifugation temperature is 2-6°C, the speed is 3500r / min-4500r / min, and the time is 10-15min; the mass percentage concentration of the physiological saline is 0.5-1.5%, and the concentration of the concentrated bacterial solution is 10 10 CFU / mL.

[0021] Furthermore, in step (4), the volume ratio of the concentrated Bacillus pumilus solution to the concentrated Bacillus cereus solution is 1:0.8-1.2, and 0.8-1.2 g of the attapulgite suspension is required to mix 1 mL of the mixed solution of the concentrated Bacillus pumilus solution and the concentrated Bacillus cereus solution, and the oscillating stirring temperature is 35-40° C. and the time is 22-26 h.

[0022] Furthermore, in step (5), the water content of the mixed solution is controlled by vacuum filtration, the mass of the sodium alginate solution is 3% to 5% of the mass of the mud, and the concentration of the sodium alginate solution is 20 to 40 mg / ml.

[0023] Furthermore, in step (6), spherical particles with a particle size of 2 to 5 mm are prepared in a mold, the concentration of the calcium chloride solution is 15 to 25 mg / ml, and the freeze-drying process is: first pre-freeze in a -22 to -18°C refrigerator for 1.5 to 2.5 hours, and then place in a -40 to -35°C, 5.5 to 6.5 Pa vacuum freezer for 22 to 26 hours.

[0024] Furthermore, the mass ratio of the bacterial agent capsule to the sand and stone matrix in step (7) is 2-5:100.

[0025] Furthermore, water-purifying plants include but are not limited to emergent plants such as reeds and calamus, and the height of the plants should be at least 5 to 10 cm above the water surface.

[0026] Furthermore, the water-permeable material wrapping the substrate includes but is not limited to nylon mesh and other soft fabrics.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] First, the Bacillus pumilus and Bacillus cereus used in the present invention are facultative oligotrophic water purification strains. After being embedded, the strains can play a water purification role under the concentration gradient of individual pollutants and can be used for purifying water bodies with different pollution degrees.

[0029] Second, the present invention adopts Fe modified attapulgite and original attapulgite to form a floc structure with a positive charge on the surface, which makes it easier for microorganisms to attach and form on the carrier surface; the floc structure has abundant void space, which can greatly increase the bacterial load.

[0030] Third, the present invention uses sodium alginate and calcium chloride to cross-link to form a wall material to encapsulate the bacterial agent, and the strain can be continuously and slowly released in the water body; after the capsule wall material is degraded, the embedded bacterial agent can continue to form a biofilm with concave soil and plant roots as carriers, and continue to be released into the water body.

[0031] Fourth, the concave soil used in the present invention is an excellent adsorbent, and has a strong adsorption capacity for organic matter / nitrogen, phosphorus and other nutrients in the water body, can achieve nutrient enrichment, and can provide continuous support for the growth of microorganisms and plants, thereby achieving microbial colonization.

[0032] Fifth, the water purification plants in the present invention are emergent plants with well-developed root systems, which can not only absorb and adsorb pollutants in the water, but also have an oxygen-secreting effect, which can provide an aerobic microenvironment for the respiratory metabolism of water purification microorganisms, thereby overcoming the inhibitory effect of low dissolved oxygen concentration in polluted water on microbial growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the root system of water purification plants embedded with sand and bacterial agent capsules of the present invention;

[0034] Figure 2 This is an appearance diagram of the composite water purification bacterial agent capsule prepared by the present invention;

[0035] Figure 2 The finished capsules are light yellow spherical particles. DETAILED DESCRIPTION

[0036] The following is a detailed description of the embodiments of the present invention.

[0037] Detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0038] In the present invention, Bacillus pumilus and Bacillus cereus used are two highly efficient facultative water-purifying oligotrophic bacteria purified and separated by the inventor's research group. The separation process is described in the literature "Research on Screening Oligotrophic Water-Purifying Microorganisms by Gradient Nutrient Dilution Method" (Hubei Agricultural Sciences, 2022, Vol. 61: 50-53.). Example 1

[0039] (1) Modified attapulgite: Fe(NO3)3, attapulgite and water were mixed in a mass ratio of 1:1:100, stirred and allowed to stand for 12 h, filtered and dried at 105 °C, calcined at about 400 °C for 3 h, and then ground through a 200-mesh sieve to obtain modified attapulgite.

[0040] (2) Preparing a suspension of attapulgite flocs: The modified attapulgite, unmodified attapulgite and water are mixed and stirred in a mass ratio of 2:1:27 to obtain a suspension of attapulgite flocs.

[0041] (3) Preparation of concentrated bacterial solution: Inoculate Bacillus pumilus and Bacillus cereus into liquid culture medium respectively, and culture in a shaking incubator at 37°C for 24 hours. Centrifuge at 4°C and 4000r / min for 12 minutes, save the lower layer of concentrated bacterial solution, and adjust the concentration of the concentrated bacterial solution to 10 with 0.9% saline. 10 CFU / mL.

[0042] (4) The concentrated bacterial solutions of Bacillus pumilus and Bacillus cereus were mixed in a volume ratio of 1:1, and then added to the modified attapulgite floc solution at a mass ratio of 1 mL (mixed concentrated bacterial solution) / g (attapulgite). The mixture was stirred and shaken at 37 °C for 24 h to allow the strains to stably adhere to the surface of the attapulgite flocs and the voids of the flocs, thereby obtaining a mixed solution of attapulgite and the strains.

[0043] (5) Preparation of plastic mud: The mixed solution is filtered under reduced pressure to obtain plastic mud with a water content of 40%, and 30 mg / ml sodium alginate solution with a mass ratio of 5% is added, and the mixture is stirred evenly to obtain plastic mud.

[0044] (6) Preparation of composite water purification bacterial capsules: The plastic clay was placed in a mold and pressed into spherical particles with a particle size of 3 mm, and immediately demolded and placed in a 20 mg / ml calcium chloride solution and stirred for about 30 min. After the embedded spherical particles were separated, the residual calcium chloride was washed with clean water, and the particles were pre-frozen in a -20°C refrigerator for 2 h, and then placed in a -40°C, 5.9 Pa vacuum freezer for 24 h to obtain freeze-dried composite water purification bacterial capsules.

[0045] (7) Water purification by bacterial plant synergy: Mix the bacterial capsules with the sand and gravel matrix in a mass ratio of 1:100, weigh 250 g, wrap it around the roots of the reed seedlings with a 60-mesh nylon mesh, and release it into the water. Example 2

[0046] (1) Modified attapulgite: Fe(NO3)3, attapulgite and water were mixed in a mass ratio of 0.8:0.8:100, stirred and allowed to stand for 12 h, filtered and dried at 100 °C, calcined at about 350 °C for 3 h, and then ground through a 200-mesh sieve to obtain modified attapulgite.

[0047] (2) Preparing a suspension of attapulgite flocs: The modified attapulgite, unmodified attapulgite and water are mixed and stirred in a mass ratio of 1.8:1:25 to obtain a suspension of attapulgite flocs.

[0048] (3) Preparation of concentrated bacterial solution: Inoculate Bacillus pumilus and Bacillus cereus into liquid culture medium respectively, and culture in a shaking incubator at 35°C for 22 hours. Centrifuge at 2°C and 3500r / min for 12 minutes, keep the lower layer of concentrated bacterial solution, and adjust the concentration of the concentrated bacterial solution to 10 with 0.9% saline. 10 CFU / mL.

[0049] (4) The concentrated bacterial solutions of Bacillus pumilus and Bacillus cereus were mixed at a volume ratio of 1:0.8, and then added to the modified attapulgite floc solution at a mass ratio of 0.8 mL (mixed concentrated bacterial solution) / g (attapulgite). The mixture was stirred and shaken at 35 °C for 22 h to allow the strains to stably adhere to the surface of the attapulgite flocs and the voids of the flocs, thereby obtaining a mixed solution of attapulgite and the strains.

[0050] (5) Preparation of plastic mud: The mixed solution is filtered under reduced pressure to obtain plastic mud with a water content of 40%, and 30 mg / ml sodium alginate solution with a mass ratio of 3% is added, and the mixture is stirred evenly to obtain plastic mud.

[0051] (6) Preparation of composite water purification bacterial capsules: The plastic clay was placed in a mold and pressed into spherical particles with a particle size of 2 mm, and immediately demolded and placed in a 15 mg / ml calcium chloride solution and stirred for about 30 min. After the embedded spherical particles were separated, the residual calcium chloride was washed with clean water, and the particles were placed in a -22°C refrigerator for pre-freezing for 1.5 h, and then placed in a -40°C, 5.5 Pa vacuum freezer for 22 h to obtain freeze-dried composite water purification bacterial capsules.

[0052] (7) Water purification by bacterial plant synergy: Mix the bacterial capsules with the sand and gravel matrix in a mass ratio of 2:100, weigh 300 g, wrap it around the roots of the calamus seedlings with a 60-mesh nylon mesh, and release it into the water. Example 3

[0053] (1) Modified attapulgite: Fe(NO3)3, attapulgite and water were mixed in a mass ratio of 1.2:1.2:100, stirred and allowed to stand for 14 h, filtered and dried at 110 °C, calcined at about 450 °C for 4 h, and then ground through a 200-mesh sieve to obtain modified attapulgite.

[0054] (2) Preparing a suspension of attapulgite flocs: The modified attapulgite, unmodified attapulgite and water are mixed and stirred in a mass ratio of 2.2:1:28 to obtain a suspension of attapulgite flocs.

[0055] (3) Preparation of concentrated bacterial solution: Inoculate Bacillus pumilus and Bacillus cereus into liquid culture medium respectively, and culture in a shaking incubator at 40°C for 22 hours. Centrifuge at 6°C and 4500r / min for 10 minutes, keep the lower layer of concentrated bacterial solution, and adjust the concentration of the concentrated bacterial solution to 10 with 0.9% saline. 10 CFU / mL.

[0056] (4) The concentrated bacterial solutions of Bacillus pumilus and Bacillus cereus were mixed at a volume ratio of 1:1.2, and then added to the modified attapulgite floc solution at a mass ratio of 1.2 mL (mixed concentrated bacterial solution) / g (attapulgite). The mixture was stirred and shaken at 37 °C for 24 h to allow the strains to stably adhere to the surface of the attapulgite flocs and the voids of the flocs, thereby obtaining a mixed solution of attapulgite and the strains.

[0057] (5) Preparation of plastic mud: The mixed solution is filtered under reduced pressure to obtain plastic mud with a water content of 50%, and 40 mg / ml sodium alginate solution with a mass ratio of 5% is added, and the mixture is stirred evenly to obtain plastic mud.

[0058] (6) Preparation of composite water purification bacterial capsules: The plastic clay was placed in a mold and pressed into spherical particles with a particle size of 5 mm, and immediately demolded and placed in a 20 mg / ml calcium chloride solution and stirred for about 40 min. After the embedded spherical particles were separated, the residual calcium chloride was washed with clean water, and the particles were pre-frozen in a -18°C refrigerator for 2 h, and then placed in a -35°C, 6.5 Pa vacuum freezer for 22 h to obtain freeze-dried composite water purification bacterial capsules.

[0059] (7) Water purification by bacterial planting: Mix the bacterial capsules with the sand and gravel matrix in a mass ratio of 5:100, weigh 200 g, wrap it around the roots of the canna seedlings with a 60-mesh nylon mesh, and place it in the water.

[0060] Example 4: Physical and chemical properties and water purification effect test

[0061] The composite water purification bacterial agent capsule prepared in Example 1 was used as the object to test its physical and chemical properties. The results are as follows:

[0062] 1. The particle size of the bacterial capsule is 3mm, the density is 1.87*103kg / m3, and the equilibrium sedimentation velocity in the water can reach 1.8m / s.

[0063] 2. Stability test of bacterial capsules: The unencapsulated and encapsulated strains were stored at a constant temperature of 57°C and a relative humidity of 60-75% for 7 days, which is equivalent to one year at room temperature, and the changes in the number of live bacteria were measured. The results showed that the unencapsulated strains had basically no live bacteria detected after 5 days. However, the number of live bacteria after encapsulation did not change significantly after 7 days.

[0064] 3. When bacterial capsules were added to the river device simulated in the laboratory, the water flow velocity ranged from 1m / s to 3m / s, and the bacterial capsules could settle rapidly in the river, and could maintain a stable position in the sandy and muddy bottom matrix; in the self-circulating flow device, the inlet COD concentration was set to 40mg / L, the flow rate was 2m3 / s, and the amount of bacterial capsules added was 50g. After continuous operation for 21 days, the effluent COD removal rate reached 70%; the spherical structure of the bacterial capsules had been destroyed, and Bacillus brevis and Bacillus cereus could be cultured in both the water and the bottom sediment, indicating that the water purification strains were successfully colonized.

[0065] 4. Reed seedlings with roots wrapped with bacterial agents were added to the black and smelly river. After 21 days, the reeds grew well. Water samples, bottom mud and plant roots were collected, and it was observed that the spherical structure of the bacterial agent capsules in the roots had been destroyed. Bacillus breve and Bacillus cereus could be cultured in the water, bottom mud and plant roots, indicating that the water purification strains were successfully colonized.

Claims

1. A method for in-situ restoration of polluted water by bacterial planting, characterized in that: The following steps are involved: Step (1), Fe(NO3)3, attapulgite and water are mixed and stirred and then allowed to stand. After filtering, the filter cake is first dried and then roasted, and then ground and sieved to obtain Fe 3+ Modified attapulgite; Step (2), the Fe obtained in step (1) 3+ The modified attapulgite and the unmodified attapulgite are mixed, water is added and stirred to prepare an attapulgite floc suspension; Step (3), inoculating Bacillus pumilus and Bacillus cereus into liquid culture medium for enrichment culture respectively, discarding the supernatant after centrifugation, collecting the bacterial sludge precipitate at the lower layer, adjusting the concentration of the bacterial sludge precipitate with physiological saline, and preparing a concentrated bacterial solution; Step (4), adding the concentrated bacterial solution of Bacillus pumilus and the concentrated bacterial solution of Bacillus cereus obtained in step (3) to the attapulgite floc suspension obtained in step (2), and oscillating and stirring to allow the strains to be stably fixed on the surface and gaps of the attapulgite flocs; Step (5), controlling the water content of the mixed solution obtained in step (4) to 40% to 50% to obtain mud, adding sodium alginate solution to the mud, and stirring to obtain plastic mud; Step (6), preparing the plastic mud obtained in step (5) into spherical particles in a mold, and immediately placing the particles in a calcium chloride solution, stirring to complete the embedding of the bacterial agent, separating the embedded spherical particles, washing the residual calcium chloride with clean water, and freeze-drying the particles to obtain composite water purification bacterial agent capsules; Step (7), mixing the composite water purification bacterial agent capsule obtained in step (6) with the sand and stone matrix in a certain proportion, then wrapping it around the root system of the water purification plant with a permeable material, and then putting it into the water body.

2. The method for in-situ remediation of polluted water by bacterial plant collaboration according to claim 1 is characterized by: The mass ratio of Fe(NO3)3, attapulgite and water in step (1) is 0.8~1.2:0.8~1.2:100, the standing time is 10~14h, the drying temperature is 100~110°C, the roasting temperature is 350~450°C, the roasting time is 2~4h, and the product is ground through a 200-mesh sieve.

3. The method for in-situ remediation of polluted water by bacterial plant collaboration according to claim 1 is characterized by: Step (2) Fe 3+ The mass ratio of modified attapulgite to unmodified attapulgite is 1.8~2.2:1, and the mass fraction of solid components in the attapulgite floc suspension is 8~12%.

4. The method for in-situ remediation of polluted water by bacterial plant collaboration according to claim 1 is characterized in that: The liquid culture medium in step (3) includes 5 g beef extract, 10 g peptone, 5 g sodium chloride, and 1000 mL sterile water.

5. The method for in-situ remediation of polluted water by bacterial plant collaboration according to claim 1 is characterized by: The culturing process in step (3) is as follows: culturing in a shaking incubator at 35-40°C for 22-26 hours; the centrifugation temperature is 2-6°C, the speed is 3500r / min-4500r / min, and the time is 10-15min; the mass percentage concentration of the physiological saline is 0.5-1.5%, and the concentration of the concentrated bacterial solution is 10 10 CFU / mL.

6. The method of in-situ remediation of polluted water by bacterial plant collaboration according to claim 1 is characterized by: The volume ratio of the concentrated bacterial solution of Bacillus pumilus to the concentrated bacterial solution of Bacillus cereus in step (4) is 1:0.8-1.2, and 0.8-1.2 g of the attapulgite suspension is required to mix 1 mL of the mixed bacterial solution of the concentrated bacterial solution of Bacillus pumilus and the concentrated bacterial solution of Bacillus cereus, and the oscillating stirring temperature is 35-40° C. and the time is 22-26 h.

7. The method for in-situ remediation of polluted water by bacterial plant collaboration according to claim 1 is characterized by: In step (5), the water content of the mixed solution is controlled by vacuum filtration, the mass of the sodium alginate solution is 3% to 5% of the mass of the mud, and the concentration of the sodium alginate solution is 20 to 40 mg / ml.

8. The method of in-situ remediation of polluted water by bacterial plant collaboration according to claim 1 is characterized by: In step (6), spherical particles with a particle size of 2 to 5 mm are prepared in a mold, the concentration of the calcium chloride solution is 15 to 25 mg / ml, and the freeze-drying process is: first pre-freeze in a -22 to -18°C refrigerator for 1.5 to 2.5 hours, and then place in a -40 to -35°C, 5.5 to 6.5 Pa vacuum freezer for 22 to 26 hours.

9. The method of in-situ remediation of polluted water by bacterial plant collaboration according to claim 1, characterized in that: The mass ratio of the bacterial agent capsule to the sand and stone matrix in step (7) is 2-5:

100.

10. The method for in-situ remediation of polluted water by bacterial plant collaboration as claimed in claim 1, characterized in that: In step (7), the water purification plant is reed or calamus, and the height of the plant should be at least 5 cm above the water surface; the permeable material is nylon mesh and other soft fabrics.

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