In-situ remediation process method for rural black and odorous water body

Abstract

The invention belongs to the technical field of water environment treatment, and relates to an in-situ remediation process method for rural black and odorous water, which is suitable for ecological treatment of black and odorous water bottom mud and low-temperature regions. The technology comprises the following steps: screening thiobacillus denitrificans, denitrifying bacteria and phosphorus-accumulating bacteria from the bottom mud of a target water area, preparing a special fungicide combination for three stages of deodorization, denitrification and phosphorus accumulation, and carrying out cross antagonism culture; drying-free agglomeration and wrapping granulation are adopted to prepare double-layer particles; injecting into a bottom mud anaerobic layer through a bottom mud injection device in three stages, and adding corresponding microbial agents according to the stages. Through cold-resistant flora directional reinforcement, double-layer particle controlled release and staged precise addition, the microbial inoculum activity and bottom mud permeability in a low-temperature environment are remarkably improved, the problems of microbial inoculum loss, agglomeration and insufficient pertinence are effectively solved, and efficient ecological management of black and odorous water is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of water environment treatment, more particularly, it relates to a rural black and odorous water in-situ remediation process method. BACKGROUND

[0002] Rural black and odorous water treatment is an important topic of water environment ecological restoration. The nitrogen, phosphorus and organic pollutants enriched in the sediment not only cause the intensification of black and odorous water, but also easily cause secondary pollution through water circulation, seriously affecting the rural ecological environment and the quality of life of residents. At present, biological remediation technology is widely used in black and odorous water treatment due to its ecological friendliness and controllable cost, and the addition of functional bacteria is one of the core means.

[0003] However, the existing technology still has many limitations in practical application. The environmental adaptability of the bacteria agent is insufficient, and the metabolic activity of the conventional normal temperature bacteria in low temperature sediment decreases sharply, which makes it difficult to effectively degrade pollutants. The competition between exogenous bacteria and indigenous microorganisms is weak, and it is easy to be inactivated due to environmental exclusion. Although there are individual cold-resistant strains, a single strain cannot cope with the complex coexistence system of pollutants in black and odorous water. At the same time, the traditional bacteria agent is in the form of powder, which is easy to form a covering layer on the surface of the sediment after being added, and cannot effectively penetrate into the anaerobic layer to fully contact with the deep pollutants. Under the action of water flow disturbance, the bacteria agent is also easy to be lost with the water body, further reducing the utilization efficiency. In addition, the existing bacteria adding method lacks pertinence, and mostly uses single or mixed adding mode, which cannot accurately act in stages according to the removal needs of different pollutants in the water body, resulting in insufficient synergy between functional bacteria groups, unbalanced removal effect of nitrogen, phosphorus and organic pollutants, and increasing the treatment cost, which makes it difficult to achieve efficient and long-term remediation of black and odorous water.

[0004] Therefore, the present application provides a rural black and odorous water in-situ remediation process method to solve the above technical problems. SUMMARY

[0005] The present application aims to provide a rural black and odorous water in-situ remediation process method, which is suitable for black and odorous water sediment ecological treatment and low temperature areas. Through directional strengthening of bacterial groups, double-layer particle controlled release and precise adding in stages, the activity of bacteria agents and the permeability of sediment in low temperature environment are significantly improved, the problems of bacteria loss, aggregation and lack of pertinence are effectively solved, and efficient ecological treatment of black and odorous water is achieved.

[0006] The present application provides a rural black and odorous water in-situ remediation process method, which comprises the following technical solutions:

[0007] S1, sampling from the sediment of the target water area, inoculating into the culture medium by gradient dilution coating method, and purifying the denitrifying bacteria, denitrifying bacteria and phosphorus accumulating bacteria single colonies by plate streaking method after 5-15 DEG C culture for 3-7 days;

[0008] S2. The S1 strain was designed with a three-stage bacterial agent combination. The deodorizing bacterial agent consisted of denitrifying bacteria, lactic acid bacteria and yeast. The denitrifying bacterial agent consisted of denitrifying bacteria, Bacillus subtilis and actinomycetes. The polyphosphate-accumulating bacterial agent consisted of polyphosphate-accumulating bacteria, Bacillus coagulans and Pseudomonas. All bacterial agents were cultured in a constant temperature shaker at 15-25℃ for 48-72h. The co-culture environment was alternated every 24h to enhance the antagonistic adaptation until the OD600 reached 0.8-1.2.

[0009] S3. Centrifuge and concentrate the liquid culture of the three types of bacterial agents in S2 to a viable count of 10^9-10^10 CFU / mL. Mix the deodorizing bacterial agent concentrate with magnesium sulfate monohydrate and calcium oxide and granulate. Form core particles with a particle size of 1-3 mm at 10-15℃. Separately, prepare a coating solution by taking the same batch of deodorizing bacterial agent concentrate, milk powder, glucose, chitosan, modified bentonite, and sterile water. Spray the solution evenly onto the surface of the core particles and harden at 25-30℃ for 24-48 hours to obtain bacterial agent particles. Denitrification and polyphosphate-accumulating bacterial agent particles are prepared independently using the same method.

[0010] S4. The compound bacterial agent is injected into the anaerobic layer of the bottom sediment in three stages through the bottom sediment injection device. In the first stage, deodorizing bacterial agent granules are added to control the dissolved oxygen in the water to 2-4 mg / L. After the chemical oxygen demand removal rate exceeds 85%, the second stage is entered to add denitrifying bacterial agent granules. After the ammonia nitrogen removal rate reaches more than 90%, the third stage is entered to add polyphosphate bacterial agent granules.

[0011] Preferably, in step S2, the deodorizing agent is composed of denitrifying bacteria, lactic acid bacteria and yeast in a weight ratio of (3-5):(2-3):(1-2), the denitrifying agent is composed of denitrifying bacteria, Bacillus subtilis and actinomycetes in a weight ratio of (4-6):(2-3):(1-2), and the polyphosphate-accumulating agent is composed of polyphosphate-accumulating bacteria, Bacillus coagulans and Pseudomonas in a weight ratio of (3-5):(2-3):(1-2).

[0012] Preferably, the preparation step of the modified bentonite in step S3 is as follows:

[0013] A1. Sodium-based bentonite is added to a 0.7-0.9 mol / L oxalic acid solution and reacted at 65-70℃ for 2-3 hours. After filtration, washing, and drying, acidified bentonite is obtained.

[0014] A2. Disperse acidified bentonite in anhydrous ethanol to form a suspension, add functional additives, and then add 20-25wt% ceryl acetylacetone solution. Sonicate at 45-50℃ for 50-60 min, then heat to 75-80℃ and stir for 2-4 h to obtain the reaction solution.

[0015] A3. Add glutaraldehyde to the reaction solution obtained in A2, adjust the pH to 4.8-5.2, stir the reaction at 50-60℃ for 1-3 hours, then filter, wash, and vacuum dry to obtain modified bentonite.

[0016] Preferably, the preparation step of the functional additive in step A2 is as follows:

[0017] (1) Dissolve 2-mercaptobenzothiazole, γ-chloropropyltrimethoxysilane and triethylamine in toluene, reflux at 80-90℃ for 5-7 h under nitrogen atmosphere, filter and wash, and then evaporate the filtrate by rotary evaporation to obtain the intermediate.

[0018] (2) Chitosan, L-lactic acid and p-toluenesulfonic acid are dispersed in dimethyl sulfoxide and stirred at 100-120℃ for 7-9 hours under nitrogen protection. After precipitation, filtration, washing and drying, the copolymer is obtained.

[0019] (3) Dissolve the intermediate in a mixed solution of ethanol and water with a volume ratio of (8-10):1, let it stand at room temperature for 25-35 min to hydrolyze, add the copolymer, adjust the pH to 4.8-5.2, stir and react at 40-50℃ for 3-5 h, and then precipitate, filter, wash and dry to obtain the functional additive.

[0020] Preferably, in step S3, the core particles consist of 14-18 parts by weight of bacterial agent concentrate, 30-35 parts by weight of magnesium sulfate monohydrate and 20-24 parts by weight of calcium oxide, and the coating layer consists of 35-40 parts by weight of bacterial agent concentrate, 5-10 parts by weight of milk powder, 3-8 parts by weight of glucose, 0.5-1.5 parts by weight of chitosan, 20-25 parts by weight of modified bentonite and sterile water to make up to 100 parts.

[0021] Preferably, step A1 comprises 8-12 parts by weight of sodium bentonite and 35-45 parts by weight of oxalic acid solution; step A2 comprises 8-12 parts by weight of acidified bentonite, 35-45 parts by weight of anhydrous ethanol, 12-18 parts by weight of functional additives and 5-7 parts by weight of ceryl acetylacetone solution; and step A3 comprises 0.6-0.9 parts by weight of glutaraldehyde.

[0022] Preferably, in step (1), the components by weight are 10-14 parts of 2-mercaptobenzothiazole, 19-25 parts of γ-chloropropyltrimethoxysilane, 4-7 parts of triethylamine and 50-55 parts of toluene.

[0023] Preferably, in step (2), the ingredients are 10-15 parts by weight of chitosan, 15-20 parts of L-lactic acid, 0.5-0.9 parts of p-toluenesulfonic acid, and 60-65 parts of dimethyl sulfoxide.

[0024] Preferably, in step (3), the components are 12-18 parts by weight of intermediate, 90-100 parts by weight of mixed solution and 8-12 parts by weight of copolymer.

[0025] In summary, the present invention has the following beneficial effects:

[0026] 1. This invention significantly improves adaptability to low-temperature environments. By screening for denitrifying bacteria, denitrifying bacteria, and polyphosphate-accumulating bacteria in the target water area, and using a cross-antagonistic co-culture process to enhance the synergy of the bacterial community, it effectively improves the metabolic activity of functional bacteria in low-temperature sediment environments. This solves the problem of the sudden drop in activity and low pollutant degradation efficiency of conventional bacterial agents at low temperatures, ensuring stable treatment effects in low-temperature areas.

[0027] 2. This invention optimizes the efficacy and long-lasting effect of the microbial agent. It employs a drying-free agglomeration-encapsulation granulation process to construct a double-layer particle structure. The core layer undergoes dehydration and solidification through hydration reaction and microbial metabolism, avoiding damage to the microbial community caused by high-temperature drying. The encapsulation layer consists of a composite controlled-release framework formed by acidified, cerium acetylacetone-grafted, and glutaraldehyde-crosslinked bentonite and chitosan. This modified bentonite possesses enhanced ion exchange capacity, adsorption sites, and structural stability, enabling precise control of particle disintegration and release kinetics in an anaerobic environment, achieving slow-release penetration of the microbial agent. Furthermore, it firmly adsorbs and immobilizes functional microorganisms, effectively preventing agent loss. Simultaneously, the modified bentonite, in conjunction with functional additives containing thiol compounds and chitosan copolymers, significantly enhances the chelation and passivation ability against sediment pollutants, creating a more favorable microenvironment for the colonization of functional microorganisms. This completely overcomes the defects of powdered microbial agents, such as surface agglomeration, water loss, insufficient penetration, and inhibition by environmental toxins.

[0028] 3. This invention achieves precise and efficient treatment by adopting a three-stage dosing strategy. Based on the removal requirements of different pollutants in the water, it adds a combination of deodorizing, denitrifying, and polyphosphate-accumulating microbial agents in stages, which specifically strengthens the treatment targets at each stage and improves the synergistic efficiency of functional microbial communities. This avoids the problems of insufficient synergy of microbial communities and uneven pollutant removal caused by single or mixed dosing modes, thereby improving the treatment effect while reducing the treatment cost. By combining targeted injection of microbial agent particles with static maintenance, the colonization and propagation of functional microbial communities in the anaerobic layer of the bottom sediment are ensured. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used, unless otherwise specified, were all purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the data are the average of the three replicates or the average ± standard deviation.

[0031] Lactic acid bacteria, purchased from Hubei Chengfeng Chemical Co., Ltd.;

[0032] Both the yeast and Bacillus subtilis were purchased from Jiangsu Caiwei Biotechnology Co., Ltd.

[0033] Actinomycetes, purchased from Wuhan Penglei Biotechnology Co., Ltd.;

[0034] Both Bacillus coagulans and Pseudomonas aeruginosa were purchased from Shanghai SenTai Biotechnology Co., Ltd.

[0035] Example 1

[0036] This embodiment provides a method for in-situ remediation of black and odorous water bodies in rural areas. The specific implementation steps are as follows:

[0037] S1. Collect 500g of bottom sediment samples from the target water area, place them in a sterile sampling bag, and transport them at 4℃. Take 10g of bottom sediment and add 90mL of sterile physiological saline. Stir at 200r / min for 30min to prepare a dilution solution. Use the gradient dilution plating method to inoculate the selective medium for denitrifying bacteria, denitrifying bacteria, and polyphosphate-accumulating bacteria, respectively. Place the inoculated plates in a constant temperature incubator at 5℃ and incubate for 7 days. Purify the colonies by the streak plate method and repeat 3 times to obtain single colonies of denitrifying bacteria, denitrifying bacteria, and polyphosphate-accumulating bacteria. Store the colonies on slant at 4℃.

[0038] S2. Prepare a deodorizing bacterial agent by weighing denitrifying bacteria, lactic acid bacteria, and yeast in a weight ratio of 3:2:1; prepare a denitrifying bacterial agent by weighing denitrifying bacteria, Bacillus subtilis, and actinomycetes in a weight ratio of 4:2:1; and prepare an polyphosphate-accumulating bacterial agent by weighing polyphosphate-accumulating bacteria, Bacillus coagulans, and Pseudomonas in a weight ratio of 3:2:1. Inoculate each bacterial agent into a liquid culture medium and culture in a constant temperature shaker at 15℃ with a shaking speed of 120r / min for 72h. Perform alternating co-culture environment enhancement every 24h, specifically by first culturing in an anaerobic environment for 12h and then in a micro-aerobic environment with dissolved oxygen of 1-2mg / L for 12h, until the OD600 of the bacterial solution reaches 0.8.

[0039] S3. Take 14 parts of the deodorizing agent concentrate to a viable count of 10^9 CFU / mL, add 30 parts of magnesium sulfate monohydrate and 20 parts of calcium oxide, place in a double planetary mixer and stir at 500 r / min for 15 min to mix evenly, transfer to a granulator and form core particles with a particle size of 1 mm at 10℃; separately take 35 parts of the same batch of agent concentrate, 5 parts of milk powder, 3 parts of glucose, 0.5 parts of chitosan, 20 parts of modified bentonite, and sterile water to make up to 100 parts, and ultrasonically treat with 300W power for 20 min to make a coating liquid; place the core particles in a fluidized bed coating machine, spray the coating liquid evenly at 10℃ and 1.5 m / s wind speed, and let stand at 25℃ for 48 h to harden to obtain deodorizing agent particles; denitrifying agent particles and polyphosphate agent particles are prepared independently using the same method.

[0040] S4. Using a bottom sediment injection device, the compound bacterial agent is injected into the anaerobic layer of the bottom sediment in three stages. In the first stage, 200g of deodorizing bacterial agent granules are added per square meter of bottom sediment area. After injection, the dissolved oxygen in the water is controlled at 4mg / L, and the mixture is left to stand for 14 days until the chemical oxygen demand removal rate exceeds 85%. In the second stage, 150g of denitrifying bacterial agent granules are added per square meter, and the mixture is left to stand for 14 days until the ammonia nitrogen removal rate reaches more than 90%. In the third stage, 100g of polyphosphate bacterial agent granules are added per square meter, and the mixture is left to stand for 14 days to complete the restoration.

[0041] The preparation steps of modified bentonite in step S3 are as follows:

[0042] A1. Add 8 parts of sodium-based bentonite to 35 parts of 0.9 mol / L oxalic acid solution, react at 65℃ and 320 rpm for 3 hours, then filter, wash and dry to obtain acidified bentonite.

[0043] A2. Disperse 8 parts of acidified bentonite in 35 parts of anhydrous ethanol to form a suspension, add 12 parts of functional additives, and then add 5 parts of 25wt% ceryl acetylacetone solution. Sonicate at 300W for 60 minutes at 45℃, and then heat to 75℃ and stir at 220rpm for 4 hours to obtain the reaction solution.

[0044] A3. Add 0.6 parts of glutaraldehyde to the reaction solution obtained in A2, adjust the pH to 4.8, stir at 180 rpm at 50℃ for 3 hours, filter, wash, and vacuum dry to obtain modified bentonite.

[0045] The preparation steps of the functional additives in step A2 are as follows:

[0046] (1) Dissolve 10 parts of 2-mercaptobenzothiazole, 19 parts of γ-chloropropyltrimethoxysilane and 4 parts of triethylamine in 50 parts of toluene, reflux at 80°C for 7 h under nitrogen atmosphere, filter and wash, and then evaporate the filtrate by rotary evaporation to obtain the intermediate.

[0047] (2) 10 parts chitosan, 15 parts L-lactic acid and 0.5 parts p-toluenesulfonic acid were dispersed in 60 parts dimethyl sulfoxide. The mixture was stirred at 300 rpm for 9 h at 100 °C under nitrogen protection. After precipitation, filtration, washing and drying, the copolymer was obtained.

[0048] (3) Take 12 parts of the intermediate and dissolve them in a mixed solution of 90 parts of ethanol and water with a volume ratio of 8:1. Let it stand at room temperature for 35 minutes to hydrolyze. Add 8 parts of the copolymer to adjust the pH to 4.8. Stir at 300 rpm at 40°C for 5 hours. After precipitation, filtration, washing and drying, the functional additive is obtained.

[0049] Example 2

[0050] This embodiment provides a method for in-situ remediation of black and odorous water bodies in rural areas. The specific implementation steps are as follows:

[0051] S1. Collect 500g of bottom sediment sample from the target water area, place it in a sterile sampling bag, and transport it at 4℃.

[0052] Take 10g of bottom sediment and add 90mL of sterile physiological saline. Stir magnetically at 250r / min for 25min to prepare a dilution. Use the gradient dilution plating method to inoculate the selective medium for denitrifying bacteria, denitrifying bacteria, and polyphosphate-accumulating bacteria, respectively. Place the inoculated plates in a constant temperature incubator at 10℃ and incubate for 5 days. Purify the colonies by streak plating and repeat 3 times to obtain single colonies of denitrifying bacteria, denitrifying bacteria, and polyphosphate-accumulating bacteria. Store on slant at 4℃.

[0053] S2. Prepare a deodorizing bacterial agent by weighing denitrifying bacteria, lactic acid bacteria, and yeast in a weight ratio of 4:2.5:1.5; prepare a denitrifying bacterial agent by weighing denitrifying bacteria, Bacillus subtilis, and actinomycetes in a weight ratio of 5:2.5:1.5; and prepare an polyphosphate-accumulating bacterial agent by weighing polyphosphate-accumulating bacteria, Bacillus coagulans, and Pseudomonas aeruginosa in a weight ratio of 4:2.5:1.5. Inoculate each bacterial agent into a liquid culture medium and culture in a constant temperature shaker at 20°C at a speed of 180 r / min for 60 h. Perform alternating co-culture environment enhancement every 24 h, specifically by first culturing in an anaerobic environment for 12 h and then in a micro-aerobic environment with dissolved oxygen of 1-2 mg / L for 12 h, until the OD600 of the bacterial solution reaches 1.0.

[0054] S3. Take 16 parts of the deodorizing agent concentrate to a viable count of 10^9.5 CFU / mL, add 32 parts of magnesium sulfate monohydrate and 22 parts of calcium oxide, place in a double planetary mixer and stir at 550 r / min for 12 min to mix evenly, transfer to a granulator and form core particles with a particle size of 2 mm at 12℃; separately take 37 parts of the same batch of agent concentrate, 7 parts of milk powder, 5 parts of glucose, 1.0 part of chitosan, 22 parts of modified bentonite, and sterile water to make up to 100 parts, and ultrasonically treat with 350W power for 15 min to make a coating liquid; place the core particles in a fluidized bed coating machine, spray the coating liquid evenly at 12℃ and 1.8 m / s wind speed, and let stand at 28℃ for 36 h to harden to obtain deodorizing agent particles; denitrifying agent particles and polyphosphate agent particles are prepared independently using the same method.

[0055] S4. Using a bottom sediment injection device, the compound bacterial agent is injected into the anaerobic layer of the bottom sediment in three stages. In the first stage, 300g of deodorizing bacterial agent granules are added per square meter of bottom sediment area. After injection, the dissolved oxygen in the water is controlled at 3mg / L, and the mixture is left to stand for 10 days until the chemical oxygen demand removal rate exceeds 87%. In the second stage, 225g of denitrifying bacterial agent granules are added per square meter, and the mixture is left to stand for 10 days until the ammonia nitrogen removal rate reaches more than 92%. In the third stage, 175g of polyphosphate bacterial agent granules are added per square meter, and the mixture is left to stand for 10 days to complete the restoration.

[0056] The preparation steps of modified bentonite in step S3 are as follows:

[0057] A1. Add 9 parts of sodium-based bentonite to 40 parts of 0.8 mol / L oxalic acid solution, react at 68℃ and 350 rpm for 2.8 h, then filter, wash and dry to obtain acidified bentonite;

[0058] A2. Disperse 10 parts of acidified bentonite in 38 parts of anhydrous ethanol to form a suspension, add 15 parts of functional additives, and then add 6 parts of 23wt% ceryl acetylacetone solution. Sonicate at 320W for 58 minutes at 47℃, and then heat to 78℃ and stir at 240rpm for 3.7 hours to obtain the reaction solution.

[0059] A3. Add 0.8 parts of glutaraldehyde to the reaction solution obtained in A2, adjust the pH to 5, stir at 200 rpm at 53℃ for 2.8 h, filter, wash, and vacuum dry to obtain modified bentonite.

[0060] The preparation steps of the functional additives in step A2 are as follows:

[0061] (1) Dissolve 13 parts of 2-mercaptobenzothiazole, 20 parts of γ-chloropropyltrimethoxysilane and 5 parts of triethylamine in 52 parts of toluene, reflux at 85°C for 6.8 h under a nitrogen atmosphere, filter and wash, and then evaporate the filtrate by rotary evaporation to obtain the intermediate.

[0062] (2) 13 parts chitosan, 17 parts L-lactic acid and 0.8 parts p-toluenesulfonic acid were dispersed in 63 parts dimethyl sulfoxide. The mixture was stirred at 320 rpm at 110 °C for 8 h under nitrogen protection. After precipitation, filtration, washing and drying, the copolymer was obtained.

[0063] (3) Take 15 parts of the intermediate and dissolve them in a mixed solution of 95 parts of ethanol and water in a volume ratio of 9:1. Let it stand at room temperature for 32 min to hydrolyze. Add 10 parts of the copolymer to it, adjust the pH to 5, stir at 360 rpm at 45℃ for 4.8 h, and then precipitate, filter, wash and dry to obtain the functional additive.

[0064] Example 3

[0065] This embodiment provides a method for in-situ remediation of black and odorous water bodies in rural areas. The specific implementation steps are as follows:

[0066] S1. Collect 500g of bottom sediment sample from the target water area, place it in a sterile sampling bag, and transport it at 4℃.

[0067] Take 10g of bottom sediment and add 90mL of sterile physiological saline. Stir magnetically at 300r / min for 20min to prepare a dilution. Use the gradient dilution plating method to inoculate the selective medium for denitrifying bacteria, denitrifying bacteria, and polyphosphate-accumulating bacteria, respectively. Place the inoculated plates in a constant temperature incubator at 15℃ and incubate for 3 days. Purify the colonies by streak plating and repeat 3 times to obtain single colonies of denitrifying bacteria, denitrifying bacteria, and polyphosphate-accumulating bacteria. Store on slant at 4℃.

[0068] S2. Prepare a deodorizing bacterial agent by weighing denitrifying bacteria, lactic acid bacteria, and yeast in a weight ratio of 5:3:2; prepare a denitrifying bacterial agent by weighing denitrifying bacteria, Bacillus subtilis, and actinomycetes in a weight ratio of 6:3:2; and prepare an polyphosphate-accumulating bacterial agent by weighing polyphosphate-accumulating bacteria, Bacillus coagulans, and Pseudomonas in a weight ratio of 5:3:2. Inoculate each bacterial agent into a liquid culture medium and culture in a constant temperature shaker at 25°C with a shaking speed of 220 r / min for 48 h. Perform alternating co-culture environment enhancement every 24 h, specifically by first culturing in an anaerobic environment for 12 h and then in a micro-aerobic environment with dissolved oxygen of 1-2 mg / L for 12 h, until the OD600 of the bacterial solution reaches 1.2.

[0069] S3. Take 18 parts of the deodorizing agent concentrate to a viable bacteria count of 10^10 CFU / mL, add 35 parts of magnesium sulfate monohydrate and 24 parts of calcium oxide, place in a double planetary mixer and stir at 600 r / min for 10 min to mix evenly, transfer to a granulator and form core particles with a particle size of 3 mm at 15℃; separately take 40 parts of the same batch of agent concentrate, 10 parts of milk powder, 8 parts of glucose, 1.5 parts of chitosan, 25 parts of modified bentonite, and sterile water to make up to 100 parts, and ultrasonically treat with 400W power for 10 min to make a coating liquid; place the core particles in a fluidized bed coating machine, spray the coating liquid evenly at 15℃ and 2.0 m / s wind speed, and let stand at 30℃ for 24 h to harden to obtain deodorizing agent particles; denitrifying agent particles and polyphosphate agent particles are prepared independently using the same method.

[0070] S4. Using a bottom sediment injection device, the compound bacterial agent is injected into the anaerobic layer of the bottom sediment in three stages. In the first stage, 400g of deodorizing bacterial agent granules are added per square meter of bottom sediment area. After injection, the dissolved oxygen in the water is controlled at 2mg / L, and the mixture is left to stand for 7 days until the chemical oxygen demand removal rate exceeds 90%. In the second stage, 300g of denitrifying bacterial agent granules are added per square meter, and the mixture is left to stand for 7 days until the ammonia nitrogen removal rate reaches more than 95%. In the third stage, 250g of polyphosphate bacterial agent granules are added per square meter, and the mixture is left to stand for 7 days to complete the restoration.

[0071] The preparation steps of modified bentonite in step S3 are as follows:

[0072] A1. Add 12 parts of sodium-based bentonite to 45 parts of 0.7 mol / L oxalic acid solution, react at 70℃ and 380 rpm for 2 hours, then filter, wash and dry to obtain acidified bentonite.

[0073] A2. Disperse 12 parts of acidified bentonite in 45 parts of anhydrous ethanol to form a suspension, add 18 parts of functional additives, and then add 7 parts of 20wt% ceryl acetylacetone solution. Sonicate at 50℃ for 50 min at 400W, then heat to 80℃ and stir at 280rpm for 2 h to obtain the reaction solution.

[0074] A3. Add 0.9 parts of glutaraldehyde to the reaction solution obtained in A2, adjust the pH to 5.2, stir at 220 rpm at 60℃ for 1 hour, filter, wash, and vacuum dry to obtain modified bentonite.

[0075] The preparation steps of the functional additives in step A2 are as follows:

[0076] (1) Dissolve 14 parts of 2-mercaptobenzothiazole, 25 parts of γ-chloropropyltrimethoxysilane and 7 parts of triethylamine in 55 parts of toluene, reflux at 90°C for 5 hours under nitrogen atmosphere, filter and wash, and then obtain the intermediate by rotary evaporation of the filtrate.

[0077] (2) 15 parts chitosan, 20 parts L-lactic acid and 0.9 parts p-toluenesulfonic acid were dispersed in 65 parts dimethyl sulfoxide. The mixture was stirred at 350 rpm for 7 h at 120 °C under nitrogen protection. After precipitation, filtration, washing and drying, the copolymer was obtained.

[0078] (3) Take 18 parts of the intermediate and dissolve them in a 100-part volume ratio of ethanol and water mixed solution. Let it stand at room temperature for 25 minutes to hydrolyze. Add 12 parts of the copolymer to it, adjust the pH to 5.2, stir at 400 rpm at 50℃ for 3 hours, and then precipitate, filter, wash and dry to obtain the functional additive.

[0079] Example 4

[0080] This embodiment provides an in-situ remediation process for black and odorous water bodies in rural areas. The specific implementation steps are as follows: S1. Collect 500g of bottom sediment samples from the target water area, place them in a sterile sampling bag, and transport them at 4℃. Take 10g of bottom sediment and add 90mL of sterile physiological saline. Stir magnetically at 280r / min for 22min to prepare a dilution solution. Use the gradient dilution plating method to inoculate the optimized selective medium for denitrifying bacteria, denitrifying bacteria, and polyphosphate-accumulating bacteria. Place the inoculated plates in a constant temperature incubator at 12℃ for 4 days. Purify the colonies by streak plating and repeat 3 times to obtain single colonies of denitrifying bacteria, denitrifying bacteria, and polyphosphate-accumulating bacteria. Store the colonies on slant at 4℃.

[0081] S2. Prepare a deodorizing bacterial agent by weighing denitrifying bacteria, lactic acid bacteria, and yeast in a weight ratio of 4:3:1.5; prepare a denitrifying bacterial agent by weighing denitrifying bacteria, Bacillus subtilis, and actinomycetes in a weight ratio of 5:2:1.5; and prepare an polyphosphate-accumulating bacterial agent by weighing polyphosphate-accumulating bacteria, Bacillus coagulans, and Pseudomonas in a weight ratio of 4:3:1.5. Inoculate each bacterial agent into a liquid culture medium and culture in a constant temperature shaker at 22℃ with a shaking speed of 200 r / min for 54 h. Perform alternating co-culture environment enhancement every 24 h, specifically by first culturing in an anaerobic environment for 12 h and then in a micro-aerobic environment with dissolved oxygen of 1-2 mg / L for 12 h, until the OD600 of the bacterial solution reaches 1.1.

[0082] S3. Take 17 parts of the deodorizing agent concentrate to a viable count of 10^9.8 CFU / mL, add 33 parts of magnesium sulfate monohydrate and 23 parts of calcium oxide, place in a double planetary mixer and stir at 550 r / min for 13 min to mix evenly, transfer to a granulator and form core particles with a particle size of 2.5 mm at 13℃; separately take 38 parts of the same batch of agent concentrate, 8 parts of milk powder, 6 parts of glucose, 1.2 parts of chitosan, 24 parts of modified bentonite, and sterile water to make up to 100 parts, and ultrasonically treat with 350W power for 12 min to make a coating liquid; place the core particles in a fluidized bed coating machine, spray the coating liquid evenly at 13℃ and 1.8 m / s wind speed, and let stand at 27℃ for 30 h to harden to obtain deodorizing agent particles; denitrifying agent particles and polyphosphate agent particles are prepared independently using the same method.

[0083] S4. Using a bottom sediment injection device, the compound bacterial agent is injected into the anaerobic layer of the bottom sediment in three stages. In the first stage, 350g of deodorizing bacterial agent granules are added per square meter of bottom sediment area. After injection, the dissolved oxygen in the water is controlled at 2.5mg / L. The water quality is monitored in real time until the chemical oxygen demand removal rate exceeds 90%, and then the mixture is left to stand for 10 days. In the second stage, 250g of denitrifying bacterial agent granules are added per square meter. The ammonia nitrogen concentration is monitored in real time until it is ≤5mg / L, and then the mixture is left to stand for 10 days. In the third stage, 200g of polyphosphate bacterial agent granules are added per square meter. The total phosphorus removal rate reaches 95%, and then the mixture is left to stand for 10 days to complete the repair.

[0084] The preparation steps of modified bentonite in step S3 are as follows:

[0085] A1. Add 12 parts of sodium-based bentonite to 45 parts of 0.8 mol / L oxalic acid solution, react at 70℃ and 380 rpm for 2.5 h, then filter, wash and dry to obtain acidified bentonite.

[0086] A2. Disperse 12 parts of acidified bentonite in 45 parts of anhydrous ethanol to form a suspension, add 16 parts of functional additives, and then add 6 parts of 24wt% ceryl acetylacetone solution. Sonicate at 50℃ for 50 min at 400W, then heat to 80℃ and stir at 280rpm for 2.4 h to obtain the reaction solution.

[0087] A3. Add 0.8 parts of glutaraldehyde to the reaction solution obtained in A2, adjust the pH to 5.2, stir at 220 rpm at 60℃ for 2 hours, filter, wash, and vacuum dry to obtain modified bentonite.

[0088] The preparation steps of the functional additives in step A2 are as follows:

[0089] (1) Dissolve 14 parts of 2-mercaptobenzothiazole, 25 parts of γ-chloropropyltrimethoxysilane and 6 parts of triethylamine in 55 parts of toluene, reflux at 90°C for 5 hours under nitrogen atmosphere, filter and wash, and then evaporate the filtrate by rotary evaporation to obtain the intermediate.

[0090] (2) 15 parts chitosan, 20 parts L-lactic acid and 0.9 parts p-toluenesulfonic acid were dispersed in 65 parts dimethyl sulfoxide. The mixture was stirred at 350 rpm for 8 h at 120 °C under nitrogen protection. After precipitation, filtration, washing and drying, the copolymer was obtained.

[0091] (3) Take 18 parts of the intermediate and dissolve them in a 100-part volume ratio of ethanol and water mixed solution. Let it stand at room temperature for 25 minutes to hydrolyze. Add 12 parts of the copolymer to it, adjust the pH to 5.2, stir at 400 rpm at 50℃ for 4 hours, and then precipitate, filter, wash and dry to obtain the functional additive.

[0092] Comparative Example 1

[0093] A method for in-situ remediation of black and odorous water bodies in rural areas differs from Example 4 in that strains are not screened, but commercially available room-temperature denitrifying bacteria, denitrifying bacteria, and polyphosphate-accumulating bacteria are used instead. That is, the sediment sampling, gradient dilution culture, and purification operations in step S1 are omitted, and commercially available strains are directly used for subsequent steps. Other conditions are the same as in Example 4.

[0094] Comparative Example 2

[0095] A method for in-situ remediation of black and odorous water bodies in rural areas differs from Example 4 in that the bacterial agent is in powder form instead of double-layered particles. Specifically, the core particle granulation and coating process in step S3 is omitted; instead, the concentrated bacterial agent solution is directly freeze-dried to produce powdered bacterial agent. Other conditions remain the same as in Example 4.

[0096] Comparative Example 3

[0097] A method for in-situ remediation of black and odorous water bodies in rural areas differs from Example 4 in that a mixed addition strategy is adopted instead of the three-stage precise addition. That is, in step S4, a mixture of deodorizing bacteria particles, denitrifying bacteria particles and polyphosphate bacteria particles is added at one time, omitting the staged injection and static curing process. Other conditions are the same as in Example 4.

[0098] Comparative Example 4

[0099] A method for in-situ remediation of black and odorous water bodies in rural areas differs from Example 4 in that the modified bentonite component in the coating layer is omitted, and unmodified sodium-based bentonite is used directly. That is, in step S3, the coating liquid is adjusted to 38 parts of bacterial agent concentrate, 8 parts of milk powder, 6 parts of glucose, 1.2 parts of chitosan and 24 parts of sodium-based bentonite, and sterile water is added to make up to 100 parts. Other conditions are the same as in Example 4.

[0100] Comparative Example 5

[0101] A method for in-situ remediation of black and odorous water bodies in rural areas differs from Example 4 in that the addition of functional additives is omitted in the preparation of modified bentonite, that is, the addition of functional additives is omitted in step A2, while other conditions are the same as in Example 4.

[0102] Comparative Example 6

[0103] A method for in-situ remediation of black and odorous water bodies in rural areas differs from Example 4 in that the copolymer obtained in step (2) is directly added to the reaction as a functional additive in the preparation of modified bentonite, i.e. steps (1) and (3) are omitted, while other conditions are the same as in Example 4.

[0104] Performance testing

[0105] Under environmental conditions of water temperature 8±2℃, initial COD=350mg / L, NH3-N=45mg / L, and TP=8mg / L, 30-day remediation tests were conducted on Examples 1-4 and Comparative Examples 1-6. The test results are shown in Table 1.

[0106] Table 1

[0107] Test item COD removal rate (%) Ammonia nitrogen removal rate (%) Total phosphorus removal rate (%) Bacterial agent survival rate (%) Example 1 91.15 92.72 89.17 83.37 Example 2 93.39 95.24 93.12 86.64 Example 3 96.07 97.63 96.84 89.15 Example 4 98.05 99.04 98.63 91.52 Comparative Example 1 57.21 63.53 60.26 58.33 Comparative Example 2 76.18 81.39 78.20 65.91 Comparative Example 3 81.12 86.44 75.43 73.62 Comparative Example 4 80.16 84.41 80.68 70.22 Comparative Example 5 82.46 85.17 78.92 71.83 Comparative Example 6 83.79 87.56 81.04 72.95

[0108] As shown in the table, the performance indicators of Examples 1 to 4 are significantly better than those of the comparative examples, with Example 4 showing the most outstanding performance. This result fully confirms the effectiveness of the process of the present invention, namely, by screening bacterial strains in the target water area, the metabolic activity of the bacterial community in the low-temperature environment is improved; the use of a double-layer particle structure with a modified bentonite coating layer optimizes the controlled release performance and compatibility of the bacterial agent with the bottom sediment, reducing the loss of the bacterial agent; and the staged precise dosing strategy strengthens the synergistic effect of the functional bacterial community, specifically improving the removal efficiency of different pollutants, and ultimately achieving efficient remediation of black and odorous water bodies.

[0109] The performance of the comparative examples was generally low. Comparative example 1, due to the lack of target water area strains, failed to adapt commercially available room-temperature strains to the low-temperature sediment environment, resulting in a significant decrease in the survival rate of the bacterial agent and a weakening of pollutant degradation capacity. Comparative example 2 used powdered bacterial agents, which lacked the controlled release and anchoring effect of double-layered particles, making the bacterial agent easily lost with the water flow and insufficiently contacting pollutants. Comparative example 3 adopted a mixed addition mode, without phased control according to the pollutant removal requirements, resulting in insufficient synergy of functional bacterial communities and uneven removal of pollutants such as total phosphorus. Comparative examples 4 to 6, due to imperfect bentonite modification processes (such as no modification, omission of functional additives, or simplification of the preparation process), weakened the controlled release capacity of the coating layer, pollutant adsorption performance, and bacterial community protection effect. Although they were better than the other comparative examples, they still could not achieve the remediation effect of the examples.

[0110] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A method for in-situ remediation of black and odorous water bodies in rural areas, characterized in that, Includes the following steps: S1. Samples were taken from the bottom sediment of the target water area and inoculated onto the culture medium by gradient dilution and spread method. After cultivation, single colonies of denitrifying bacteria, denitrifying bacteria and polyphosphate-accumulating bacteria were purified by streak plate method. S2. The strains obtained in S1 were prepared into a three-stage bacterial agent combination. The deodorizing bacterial agent consisted of denitrifying bacteria, lactic acid bacteria and yeast. The denitrifying bacterial agent consisted of denitrifying bacteria, Bacillus subtilis and actinomycetes. The polyphosphate-accumulating bacterial agent consisted of polyphosphate-accumulating bacteria, Bacillus coagulans and Pseudomonas. The antagonistic adaptability of each bacterial agent was enhanced by constant temperature shaking culture and alternating co-culture environment. S3. Centrifuge and concentrate the liquid cultures of the three types of bacterial agents obtained in S2. Mix the deodorizing bacterial agent concentrate with magnesium sulfate monohydrate and calcium oxide, and granulate to form core particles with a particle size of 1-3 mm. Separately, prepare a coating solution by taking the same batch of deodorizing bacterial agent concentrate, milk powder, glucose, chitosan, modified bentonite, and sterile water. Spray the solution evenly onto the surface of the core particles and harden to obtain bacterial agent particles. The denitrifying and polyphosphate-accumulating bacterial agent particles are prepared independently using the same method. S4. The compound bacterial agent is injected into the anaerobic layer of the bottom sediment in three stages through the bottom sediment injection device. In the first stage, deodorizing bacterial agent granules are added to control the dissolved oxygen in the water to 2-4 mg / L. After the chemical oxygen demand removal rate exceeds 85%, the second stage is entered to add denitrifying bacterial agent granules. After the ammonia nitrogen removal rate reaches more than 90%, the third stage is entered to add polyphosphate bacterial agent granules.

2. The in-situ remediation process for black and odorous water bodies in rural areas according to claim 1, characterized in that, In step S2, the deodorizing agent is composed of denitrifying bacteria, lactic acid bacteria and yeast in a weight ratio of (3-5):(2-3):(1-2), the denitrifying agent is composed of denitrifying bacteria, Bacillus subtilis and actinomycetes in a weight ratio of (4-6):(2-3):(1-2), and the polyphosphate-accumulating agent is composed of polyphosphate-accumulating bacteria, Bacillus coagulans and Pseudomonas in a weight ratio of (3-5):(2-3):(1-2).

3. The in-situ remediation process for black and odorous water bodies in rural areas according to claim 1, characterized in that, The preparation steps of the modified bentonite in step S3 are as follows: A1. Sodium-based bentonite is added to oxalic acid solution, and after reaction, it is filtered, washed, and dried to obtain acidified bentonite. A2. Disperse acidified bentonite in anhydrous ethanol to form a suspension, add functional additives, then add cerium acetylacetone solution dropwise, sonicate, heat to 75-80℃ and stir for 2-4 hours to obtain the reaction solution. A3. Add glutaraldehyde to the reaction solution obtained in A2, adjust the pH, stir the reaction at 50-60℃ for 1-3 hours, filter, wash, and vacuum dry to obtain modified bentonite.

4. The in-situ remediation process for black and odorous water bodies in rural areas according to claim 3, characterized in that, The preparation steps of the functional additives in step A2 are as follows: (1) Dissolve 2-mercaptobenzothiazole, γ-chloropropyltrimethoxysilane and triethylamine in toluene, reflux at 80-90℃ for 5-7 h under nitrogen atmosphere, filter and wash, and then evaporate the filtrate by rotary evaporation to obtain the intermediate. (2) Chitosan, L-lactic acid and p-toluenesulfonic acid are dispersed in dimethyl sulfoxide, and the reaction is carried out under nitrogen protection by heating and stirring. After precipitation, filtration, washing and drying, the copolymer is obtained. (3) Dissolve the intermediate in a mixed solution of ethanol and water with a volume ratio of (8-10):1, let it stand at room temperature for hydrolysis, add the copolymer, adjust the pH, stir at 40-50℃ for 3-5 hours, and then precipitate, filter, wash and dry to obtain the functional additive.

5. The in-situ remediation process for black and odorous water bodies in rural areas according to claim 1, characterized in that, In step S3, the core particles consist of 14-18 parts by weight of bacterial agent concentrate, 30-35 parts by weight of magnesium sulfate monohydrate and 20-24 parts by weight of calcium oxide, and the coating layer consists of 35-40 parts by weight of bacterial agent concentrate, 5-10 parts by weight of milk powder, 3-8 parts by weight of glucose, 0.5-1.5 parts by weight of chitosan, 20-25 parts by weight of modified bentonite and sterile water to make up to 100 parts.

6. The in-situ remediation process for black and odorous water bodies in rural areas according to claim 3, characterized in that, In step A1, the components are 8-12 parts by weight of sodium bentonite and 35-45 parts by weight of oxalic acid solution; in step A2, the components are 8-12 parts by weight of acidified bentonite, 35-45 parts by weight of anhydrous ethanol, 12-18 parts by weight of functional additives and 5-7 parts by weight of ceryl acetylacetone solution; and in step A3, the components are 0.6-0.9 parts by weight of glutaraldehyde.

7. The in-situ remediation process for black and odorous water bodies in rural areas according to claim 4, characterized in that, In step (1), the ingredients are 10-14 parts by weight of 2-mercaptobenzothiazole, 19-25 parts by weight of γ-chloropropyltrimethoxysilane, 4-7 parts by weight of triethylamine and 50-55 parts by weight of toluene.

8. The in-situ remediation process for black and odorous water bodies in rural areas according to claim 4, characterized in that, The (2) consists of 10-15 parts by weight of chitosan, 15-20 parts of L-lactic acid, 0.5-0.9 parts of p-toluenesulfonic acid and 60-65 parts of dimethyl sulfoxide.

9. The in-situ remediation process for black and odorous water bodies in rural areas according to claim 4, characterized in that, The step (3) consists of 12-18 parts by weight of intermediate, 90-100 parts by weight of mixed solution and 8-12 parts by weight of copolymer.