CN106754855A - Embedding type Nanoscale Iron/complex micro organism fungicide and preparation method thereof - Google Patents

Abstract

The invention relates to an embedded nano-iron/composite microbial bacterial agent and a preparation method thereof. In the preparation method, Agrobacterium, Enterobacter cloacae, bacillus, Gordonella, Pseudomonas putida and Pseudomonas stutzeri were firstly used to prepare nano-iron solution B, and to prepare embedding agent agar, PVA, _SiO2 Solution C, prepare the cross-linking agent aluminum sulfate saturated boric acid solution D; respectively take 15-18% solution B and 6-15% bacteria A in the 57-66% solution C according to the volume ratio, stir and mix evenly, Add it dropwise to 1‐22% solution D at room temperature in a protected environment, perform cross-linking treatment, wash, and store to obtain nano-iron/composite microbial bacterial agent. The present invention utilizes the synergistic effect between nano-iron and microorganisms to improve the degradation efficiency of triclosan; the prepared bacterial agent has high strength, low microbial toxicity and low raw material source price, and can be widely used in the treatment of water bodies polluted by triclosan .

Description

Embedded nano-iron/composite microbial agent and preparation method thereof

technical field

The invention relates to the field of triclosan wastewater treatment, in particular to an embedded nano-iron/composite microbial agent for degrading triclosan and a preparation method thereof.

Background technique

Triclosan has a good sterilization and disinfection effect, and has good safety, and even has the effect of promoting human skin metabolism, brightening and moisturizing. Since it was used in soap production in the 1970s, the application scope of triclosan has gradually expanded, and it has been widely used in the production process of personal care products and pharmaceuticals, such as detergents, deodorants, cosmetics, disinfection equipment, and Disinfection and sterilization of textiles before leaving the factory, etc. Triclosan is a polar hydrophobic organic matter, which is easy to deposit in solid phase substances such as soil and bottom mud. The lipophilicity of hydrophobic substances makes it easy to accumulate in organisms, which also increases the possibility of triclosan environmental residues and threatens human health through the accumulation of food chains in mammals. The easy adsorption and deposition, persistence and bioaccumulation of these pollutants bring long-term and unpredictable environmental risks to the surrounding ecological environment. The control and governance of this kind of pollution has aroused widespread concern.

Biological treatment is a commonly used wastewater treatment method at present. This method decomposes and absorbs the pollutants in wastewater through the metabolism of microorganisms, so as to achieve the purpose of pollution control. Compared with other methods, biological treatment has low cost, high efficiency, easy operation, and most importantly, no secondary pollution. Therefore, it has been widely used in wastewater treatment. With the development of the economy, the composition of wastewater is becoming more and more complex, especially when the wastewater contains toxic and refractory organic pollutants, due to the small number and types of microorganisms with special degradation ability for this type of organic substances in the environment, and it At a disadvantage in interspecies competition, the traditional biological treatment technology faces great challenges.

If microorganisms or certain substrates with specific functions are added to the traditional biological treatment system to enhance its ability to degrade specific pollutants, thereby improving the treatment effect of the entire sewage treatment system, we call this technology bioaugmentation technology. In recent years, due to its huge specific surface area and high activity, nanomaterials have improved the reaction rate, and have been used in contaminated soil and groundwater remediation and sewage treatment, and nano-scale zero-valent iron (nZVI ) has been relatively extensively studied. nZVI is an effective reducing agent for dehalogenation, which attracted people's attention as early as the 1980s. Nano-zero-valent iron can catalyze the reduction of various organic halides, such as: halogenated alkanes, halogenated olefins, halogenated aromatics and other refractory organic pollutants, and convert them into non-toxic and harmless compounds, while improving their biodegradability , can create favorable conditions for further biodegradation. Although nano-zero-valent iron has many advantages, it still encounters some problems in the process of its application, such as the poor stability of nano-zero-valent iron. Nano zero-valent iron is easily oxidized to form iron oxides or hydroxides deposited on the surface of nano-iron, so that nano-zero-valent iron is passivated.

Contents of the invention

The purpose of the present invention is to address the deficiencies in the prior art, to provide a kind of triclosan with high degradation efficiency, high inoculant strength, low microbial toxicity, low raw material source price embedded nano-iron/composite microbial inoculum and its preparation method.

The present invention uses chemical means to embed nano-iron and microorganisms to form a composite bacterial agent, which can form a synergistic effect on the treatment of triclosan pollutants, not only can use the high specific surface area and surface activity of nano-iron particles, It can also ensure the stability and activity of microorganisms; the prepared embedded bacterial agent is suitable for in-situ restoration and has no secondary pollution.

The purpose of the present invention is achieved through the following technical solutions:

The preparation method of embedded nano-iron/composite microbial bacterial agent comprises the following steps:

(1) Preparation of bacteria:

Agrobacterium sp., Enterobactercloacae., Bacillus sp., Gordonia sp., Pseudomonasputida. The 2 rings of Pseudomonas stutzeri. were transferred to the nutrient solution, the bacteria were cultured at 35-37°C for 1-3 days, and inoculated into the container containing the proliferation medium at a volume ratio of 5-18% In the medium, cultivated under the condition of 35-37 ℃ for 1-3 days, and centrifuged to obtain the logarithmic growth phase cells of the above-mentioned bacteria; after washing 1-2 times with phosphate buffer saline; according to volume percentage, 5 ~8% Agrobacterium, 4-6% Bacillus, 7-15% Enterobacter cloacae, 10-19% Gordonella, 13-27% Pseudomonas putida and 35-45% Pseudomonas stutzeri Bacteria were mixed to obtain thallus A for triclosan degradation;

(2) Preparation of nano-iron solution:

Using the liquid phase reduction method, in the liquid phase system protected by nitrogen, the strong reducing agent KBH ₄ reduces FeSO ₄ 7H ₂ O to obtain Fe ⁰ , and prepares a nano-iron solution with a concentration of 0.1-0.6 g/L with Fe ⁰ , which is recorded as Solution B;

(3) Embedding agent agar, PVA, SiO ₂ Preparation of solution:

Heat agar and PVA at a temperature of about 90-100°C to completely dissolve in water to obtain a solution with a mass percentage of 5-9% of agar and 7.5-15% of PVA, and then add SiO ₂ to control SiO ₂ The mass concentration in the mixture is 1-3mg/L, and it is alternately cooled to 50°C after mixing, which is recorded as solution C;

(4) Preparation of crosslinking agent aluminum sulfate saturated boric acid solution:

Dissolve the aluminum sulfate powder in a saturated boric acid solution to obtain a saturated boric acid solution of aluminum sulfate with a molar concentration of 0.1 to 1 mol/L, which is referred to as solution D;

(5) Preparation of nano-iron/composite microbial bacterial agent:

Under the condition of constant temperature water bath at 50-70°C, take 15-18% solution B and 6-15% bacterial cell A into 57-66% solution C according to the volume ratio, stir and mix evenly, under the environment of nitrogen protection Add dropwise to 1‐22% solution D at room temperature, cross-link, wash, and store to obtain nano-iron/composite microbial agent.

To further realize the object of the present invention, preferably, the main components of the nutrient solution are beef extract 6.0g/L, NaCl 5.0g/L, peptone 10.0g/L, soybean powder 2.0g/L, pH 6.5, and the rest is water.

Preferably, the main components of the proliferation medium are 20.0 g/L casein, 3.0 g/L potassium hydrogen phosphate, 3.0 g/L glucose, 4.0 g/L soybean powder, 5.0 g/L sodium chloride, and the rest is water .

Preferably, by volume percentage, the composition of the phosphate buffer is sodium chloride 9.0g/L, potassium chloride 0.3g/L, dipotassium hydrogen phosphate 1.2g/L and potassium dihydrogen phosphate 0.3g/L , and the rest is water.

Preferably, the preservation method of the embedded nano-iron/composite microbial agent refers to soaking in sterile physiological saline and storing in a refrigerator at 4°C.

Preferably, the preservation refers to soaking in sterile physiological saline and storing in a refrigerator at 4°C.

Preferably, the Agrobacterium sp., Enterobactercloacae., Bacillus sp., Gordonia sp., Pseudomonas putida. , Pseudomonas stutzeri. (Pseudomonas stutzeri.) 2 rings were transferred to nutrient solution containing 30-40mL.

Preferably, the cross-linking treatment refers to cross-linking at 4-6°C for 10-36 hours.

Preferably, the centrifugation described in step 1) refers to centrifugation at a speed of 4000-5000rpm for 15-30min.

Preferably, the cleaning is with 0.8-1.2% NaCl solution.

An embedded nano-iron/composite microbial bacterial agent for degrading triclosan is prepared by the above preparation method.

The present invention has the following advantages:

1) The embedded nano-iron/composite microbial bacterial agent of the present invention utilizes the strong reducibility of nano-iron to triclosan, the adsorption to microorganisms, and the effect of nano-iron on cytochrome c of mitochondria of microorganisms to change cytochrome The oxidation-reduction potential of c and the ability to enhance electron transfer, so the compound bacterial agent can produce a synergistic effect to jointly promote the degradation of triclosan. Under the range value conditions of the embodiments of the present invention, the composite bacterial agent prepared by the present invention is compared with a single microbial experiment and a single nano-iron experiment under the same experimental conditions, and it can be verified that the composite bacterial agent can produce a synergistic effect and jointly promote triclosan. The degraded nano-iron can interact with the cytochrome c of the mitochondria of microorganisms, change the oxidation-reduction potential of cytochrome c and strengthen the electron transfer ability.

2) The raw material of the embedding agent agar selected in the present invention has wide sources, is cheap and non-toxic, and has good biocompatibility. The prepared bacterial agent has high strength and low microbial toxicity. At the same time, it solves the unstable problem of microorganisms adsorbed on the inorganic porous _SiO2 material. The synergistic effect of nano-iron and microorganisms enhances the degradation efficiency of triclosan, which is suitable for large-scale industrial production.

3) This method is simple and convenient to use, and the prepared bacterial agent can be directly put into the polluted water body after activation, so as to realize the in-situ repair of the polluted water body, effectively avoid the loss of microorganisms, and there is no secondary pollution.

detailed description

In order to better understand the present invention, the present invention will be further described below in conjunction with the examples, but the protection scope of the present invention is not limited to the range expressed in the examples.

In the embodiment step 1) and step 6), the main components of the nutrient solution are beef extract 6.0g/L, NaCl 5.0g/L, peptone 10.0g/L, soybean powder 2.0g/L, pH 6.5, and the rest is water .

The main components of the proliferation medium are 20.0 g/L casein, 3.0 g/L potassium hydrogen phosphate, 3.0 g/L glucose, 4.0 g/L soybean powder, 5.0 g/L sodium chloride, and the rest is water.

Example 1

(1) Preparation of triclosan-degrading bacterial liquid

Agrobacterium sp., Enterobactercloacae., Bacillus sp., Gordonia sp., Pseudomonasputida. Pseudomonas (Pseudomonas stutzeri.) 2 rings were transferred to 30mL nutrient solution, the bacteria were cultured at 35°C for 2 days, and inoculated into the proliferation medium container at a volume ratio of 10%, at 35 Cultivate for 2 days under the condition of ℃, centrifuge at a speed of 5000rpm for 15min, and obtain the logarithmic growth phase cells of the above bacteria respectively. The logarithmic growth phase cells of the above-mentioned thalli are taken out, and with phosphate buffer (its main component sodium chloride 9.0g/L, potassium chloride 0.3g/L, dipotassium hydrogen phosphate 1.2g/L and potassium dihydrogen phosphate 0.3g/L, the rest is water) and washed twice. By volume percentage, get 5% Agrobacterium, 5% Bacillus, 10% Enterobacter cloacae, 20% Gordonella, 20% Pseudomonas putida and 40% Pseudomonas stutzeri and mix to obtain Bacteria for triclosan degradation. The bacteria were suspended in saline and refrigerated at 4°C for later use. Denoted as cell A;

(2) Preparation of nano-iron solution

Using the liquid phase reduction method, in the liquid phase system protected by nitrogen, the strong reducing agent KBH ₄ reduces FeSO ₄ ·7H ₂ O to obtain Fe ⁰ , and prepares a nano-iron solution with a concentration of 0.4g/L with Fe ⁰ . Denoted as solution B.

(3) Preparation of embedding agent agar, PVA, _SiO2 solution

Heat agar and PVA at a temperature of 90°C and completely dissolve them in clear water to obtain a solution with a mass percentage of agar of 5% and a mass percentage of PVA of 7.5%, and then add _SiO2 , and the mass concentration of _SiO2 in the mixture is 1mg/ L, to be mixed alternately cooled to 55 ° C, recorded as solution C;

(4) Preparation of crosslinking agent aluminum sulfate saturated boric acid solution

The aluminum sulfate powder is dissolved in a saturated boric acid solution to obtain a saturated boric acid solution of aluminum sulfate with a molar concentration of 0.5 mol/L. Record it as solution D.

(5) Preparation of embedded bacterial agent

Under the condition of constant temperature water bath at 60°C, take 15% 0.1mg/L solution B by volume ratio, and add 6% bacterium A to 57% solution C (containing 5% agar, 7.5% PVA, 1mg/L LSiO ₂ ), stir and mix well. Add dropwise to 22% solution D (room temperature) under nitrogen protection environment, cross-link at 4°C for 10h, then wash and store with 0.9wt% NaCl solution to obtain nano-iron/composite microbial bacterial agent.

(6) Degradation effect of triclosan pollution

Take 3 mg/L of nano-iron composite microbial bacterial agent and put it into nutrient liquid base for 6 hours of cultivation, make it activated and directly put it into 10 L of triclosan simulated polluted wastewater with a concentration of 5 mg/L, aerate for 5 days, and the aeration rate is 2 L/L h. In terms of mass concentration, the nutrient solution is beef extract 6.0g/L, NaCl 5.0g/L, peptone 10.0g/L, soybean powder 2.0g/L, pH 6.5, and the rest is water.

Triclosan was determined by Waters high-performance liquid chromatography, and the determination conditions were chromatographic column: Waters C ₁₈ column (150×4.6mm ID, 5 μm); 35°C column temperature, with acetonitrile/water (75:25, v/v) as For mobile phase, the total flow rate is 1.0 mL/min, the injection volume is 10 μL, and the detection wavelength is 230 nm. The peak eluting time of the target substance is about 7.2 minutes, and the total detection time of the sample is 12 minutes. The removal rate of triclosan was obtained by testing the initial concentration C ₀ of triclosan and the concentration C _t after the reaction in water samples.

Using the method of this example to treat 5L of triclosan in water with 5mg/L of triclosan, add 2g of nano-iron composite microbial bacterial agent; directly put the bacterial agent into the nutrient solution and cultivate it for 6 hours, and put it into use directly after activation. After 5 days of aeration treatment, the removal rate of triclosan reached 91%, which was significantly higher than the 28% of the control group of a single bacterial species (Pseudomonas stutzeri), indicating that the embedded nano-iron/composite microbial agent has a certain effect on triclosan. Good degradation effect, and obviously better than a single strain; nano-iron has strong reducibility to triclosan, adsorption to microorganisms, and nano-iron can interact with cytochrome c in the mitochondria of microorganisms to change the oxidation of cytochrome c Reduction potential and enhanced electron transfer capability; the compound bacterial agent can produce a synergistic effect to jointly promote the degradation of triclosan. The strong reducibility of nano-iron to triclosan, the adsorption of microorganisms, and the interaction of nano-iron with cytochrome c in the mitochondria of microorganisms, changing the oxidation-reduction potential of cytochrome c and enhancing the ability of electron transfer, the compound bacterial agent can produce The synergistic effect jointly promotes the degradation of triclosan.

Example 2

(1) Preparation of triclosan-degrading bacterial liquid

Agrobacterium sp., Enterobactercloacae., Bacillus sp., Gordonia sp., Pseudomonasputida. Pseudomonas (Pseudomonas stutzeri.) 2 rings were transferred to 30mL nutrient solution, the bacteria were cultured at 35°C for 2 days, and inoculated into the proliferation medium container at a volume ratio of 10%, at 35 Cultivate for 2 days under the condition of ℃, centrifuge at a speed of 5000rpm for 15min, and obtain the logarithmic growth phase cells of the above bacteria respectively.

The logarithmic growth phase cells of the above-mentioned thalli are taken out, and with phosphate buffer (its main component sodium chloride 9.0g/L, potassium chloride 0.3g/L, dipotassium hydrogen phosphate 1.2g/L and potassium dihydrogen phosphate 0.3g/L, the rest is water) and washed twice. By volume percentage, get 5% Agrobacterium, 5% Bacillus, 10% Enterobacter cloacae, 20% Gordonella, 20% Pseudomonas putida and 40% Pseudomonas stutzeri and mix to obtain Bacteria for triclosan degradation. The bacteria were suspended in saline and refrigerated at 4°C for later use. Denoted as cell A;

(2) Preparation of nano-iron solution

Using the liquid phase reduction method, in the liquid phase system protected by nitrogen, the strong reducing agent KBH ₄ reduces FeSO ₄ ·7H ₂ O to obtain Fe ⁰ , and prepares a nano-iron solution with a concentration of 0.4g/L with Fe ⁰ . Denoted as solution B.

(3) Preparation of embedding agent agar, PVA, _SiO2 solution

Heat agar and PVA at a temperature of 90°C and completely dissolve them in clear water to obtain a solution with a mass percentage of 7% agar and 11.5% by mass of PVA, then add SiO ₂ , and the mass concentration of SiO ₂ in the mixture is 2 mg/ L, to be mixed alternately cooled to 55 ° C, recorded as solution C;

(4) Preparation of crosslinking agent aluminum sulfate saturated boric acid solution

The aluminum sulfate powder is dissolved in a saturated boric acid solution to obtain a saturated boric acid solution of aluminum sulfate with a molar concentration of 0.5 mol/L. Record it as solution D.

(5) Preparation of embedded bacterial agent

Under the condition of constant temperature water bath at 60 ℃, take 16% 0.4mg/L solution B respectively by volume ratio, 10% thalline A joins 61% solution C (containing percentage by mass 7% agar, 11.5%PVA, 2mg/L L SiO ₂ ), stir and mix well. Add dropwise to 13% solution D (room temperature) under nitrogen protection environment, cross-link at 4°C for 23h, then wash and store with 0.9wt% NaCl solution to obtain nano-iron/composite microbial agent.

(6) Degradation effect of triclosan pollution

Take 3 mg/L of nano-iron composite microbial bacterial agent and put it into nutrient liquid base for 6 hours of cultivation, make it activated and directly put it into 10 L of triclosan simulated polluted wastewater with a concentration of 5 mg/L, aerate for 5 days, and the aeration rate is 2 L/L h. In terms of mass concentration, the composition of the nutrient solution is beef extract 6.0g/L, NaCl 5.0g/L, peptone 10.0g/L, soybean powder 2.0g/L, pH 6.5, and the rest is water.

Using the method of this example, add 2 g of nano-iron composite microbial compound bacterial agent to 5 L of waste water containing 5 mg/L triclosan; after activation, directly put the bacterial agent into the nutrient solution for 6 hours of cultivation, and put it into use directly. After 5 days of aeration treatment, the removal rate of triclosan reached 98%, which was significantly higher than the 32% of the control group of a single bacterial species (Pseudomonas stutzeri), indicating that the embedded nano-iron/composite microbial agent has a certain effect on triclosan. Good degradation effect, and obviously better than a single strain. Because of the strong reducibility of nano-iron to triclosan, the adsorption of microorganisms, and the interaction of nano-iron with cytochrome c in the mitochondria of microorganisms, changing the oxidation-reduction potential of cytochrome c and enhancing the ability of electron transfer. Therefore, the compound bacterial agent can produce a synergistic effect to jointly promote the degradation of triclosan.

Triclosan was determined by Waters high-performance liquid chromatography, and the determination conditions were chromatographic column: Waters C ₁₈ column (150×4.6mm ID, 5 μm); 35°C column temperature, with acetonitrile/water (75:25, v/v) as For mobile phase, the total flow rate is 1.0 mL/min, the injection volume is 10 μL, and the detection wavelength is 230 nm. The peak eluting time of the target substance is about 7.2 minutes, and the total detection time of the sample is 12 minutes.

Example 3

(1) Preparation of triclosan-degrading bacterial liquid

Agrobacterium sp., Enterobactercloacae., Bacillus sp., Gordonia sp., Pseudomonasputida. Pseudomonas (Pseudomonas stutzeri.) 2 rings were transferred to 30mL nutrient solution, the bacteria were cultured at 35°C for 2 days, and inoculated into the proliferation medium container at a volume ratio of 10%, at 35 Cultivate for 2 days under the condition of ℃, centrifuge at a speed of 5000rpm for 15min, and obtain the logarithmic growth phase cells of the above bacteria respectively.

The logarithmic growth phase cells of the above-mentioned thalli are taken out, and with phosphate buffer (its main component sodium chloride 9.0g/L, potassium chloride 0.3g/L, dipotassium hydrogen phosphate 1.2g/L and potassium dihydrogen phosphate 0.3g/L, the rest is water) and washed twice. By volume percentage, get 5% Agrobacterium, 5% Bacillus, 10% Enterobacter cloacae, 20% Gordonella, 20% Pseudomonas putida and 40% Pseudomonas stutzeri and mix to obtain Bacteria for triclosan degradation. The bacteria were suspended in saline and refrigerated at 4°C for later use. Denoted as cell A;

(2) Preparation of nano-iron solution

Using the liquid phase reduction method, in the liquid phase system protected by nitrogen, the strong reducing agent KBH ₄ reduces FeSO ₄ ·7H ₂ O to obtain Fe ⁰ , and prepares a nano-iron solution with a concentration of 0.4g/L with Fe ⁰ . Denoted as solution B.

(3) Preparation of embedding agent agar, PVA, _SiO2 solution

Heat agar and PVA at a temperature of 90°C and completely dissolve them in clear water to obtain a solution with a mass percent of agar of ₉ % and a mass percent of PVA of ₁₅ %. L, to be mixed alternately cooled to 55 ° C, recorded as solution C;

(4) Preparation of crosslinking agent aluminum sulfate saturated boric acid solution

The aluminum sulfate powder is dissolved in a saturated boric acid solution to obtain a saturated boric acid solution of aluminum sulfate with a molar concentration of 0.5 mol/L. Record it as solution D.

(5) Preparation of embedded bacterial agent

Under the condition of constant temperature water bath at 60°C, take 18% 0.6mg/L solution B by volume ratio, 15% thalline A is added to 66% solution C (containing mass percent 9% agar, 15% PVA, 3mg/L L SiO ₂ ), stir and mix well. Add dropwise to 1% solution D (room temperature) under nitrogen protection environment, cross-link at 4°C for 36h, then wash and store with 0.9wt% NaCl solution to obtain nano-iron/composite microbial bacterial agent.

(6) Degradation effect of triclosan pollution

Take 3 mg/L of nano-iron composite microbial bacterial agent and put it into nutrient liquid base for 6 hours of cultivation, make it activated and directly put it into 10 L of triclosan simulated polluted wastewater with a concentration of 5 mg/L, aerate for 5 days, and the aeration rate is 2 L/L h. In terms of mass concentration, the composition of the nutrient solution is beef extract 6.0g/L, NaCl 5.0g/L, peptone 10.0g/L, soybean powder 2.0g/L, pH 6.5, and the rest is water.

Using the method of this example, add 2 g of nano-iron composite microbial compound bacterial agent to 5 L of waste water containing 5 mg/L triclosan; after activation, directly put the bacterial agent into the nutrient solution for 6 hours of cultivation, and put it into use directly. After 5 days of aeration treatment, the removal rate of triclosan reached 96%, which was significantly higher than the 25% of the control group of a single bacterial species (Pseudomonas stutzeri), indicating that the embedded nano-iron/composite microbial agent has a certain effect on triclosan. Good degradation effect, and obviously better than a single strain. Because of the strong reducibility of nano-iron to triclosan, the adsorption of microorganisms, and the interaction of nano-iron with cytochrome c in the mitochondria of microorganisms, changing the oxidation-reduction potential of cytochrome c and enhancing the ability of electron transfer. Therefore, the compound bacterial agent can produce a synergistic effect to jointly promote the degradation of triclosan.

Triclosan was determined by Waters high-performance liquid chromatography, and the determination conditions were chromatographic column: Waters C ₁₈ column (150×4.6mm ID, 5 μm); 35°C column temperature, with acetonitrile/water (75:25, v/v) as For mobile phase, the total flow rate is 1.0 mL/min, the injection volume is 10 μL, and the detection wavelength is 230 nm. The peak eluting time of the target substance is about 7.2 minutes, and the total detection time of the sample is 12 minutes.

In the present invention, the synergistic effect of nano-iron and microorganisms, the experiment proves that under the same experimental conditions, the conforming bacterial agent prepared by the present invention is better than single microorganism Agrobacterium sp. and Enterobacter cloacae respectively. , Bacillus sp., Gordonia sp., Pseudomonas putida., Pseudomonas stutzeri. The strong reducibility of nano-iron to triclosan, the adsorption of microorganisms, and the interaction of nano-iron with cytochrome c in the mitochondria of microorganisms, changing the oxidation-reduction potential of cytochrome c and enhancing the ability of electron transfer, so the composite bacterial agent can A synergistic effect is produced to jointly promote the degradation of triclosan. It overcomes the problem that the bacteria are easily inhibited by intermediate metabolites in the process of degrading triclosan. At the same time, nano-iron and microorganisms are embedded to make bacterial agents, which can not only use the high specific surface area and surface activity of nano-iron particles, but also ensure The stability and activity of microorganisms make it form a synergistic effect on the treatment of triclosan pollutants, and the removal rate is significantly increased. The prepared embedded bacteria agent is suitable for resting in situ and has no secondary pollution.

According to the report of relevant bacteria degrading triclosan: the degradation rate of fungal laccases (laccases)/redox mediator system degrading triclosan is 90% [Murugesan K, Chang Y Y, Kim Y M, et al.Enhanced transformation of triclosanbylaccase in the presence of redox mediators[J].Water Research,2010,44(1):298‐308.], the degradation rate of white rot fungi to triclosan is also 90%[InoueY, Hata T, Kawai S, et al. Elimination and detoxification of triclosan bymanganese peroxidase from white rot fungus[J].Journal of Hazardous Materials,2010,180(1‐3):764‐767.]. The degradation efficiency of these bacteria to triclosan is lower than that of the bacterial agent of the present invention, so the bacterial agent has a good application prospect in treating triclosan wastewater. The embedded nano-iron single microbial agent prepared by the invention not only has advantages in degrading triclosan. Moreover, the embedding agent can utilize the high specific surface area and surface activity of the nano-iron particles, and can also ensure the stability and activity of microorganisms. The prepared embedded bacteria agent is suitable for in-situ repair and has no secondary pollution. The prepared embedding agent can be put into use only after simple activation, and has the prospect of large-scale industrial production.

The implementation of the present invention is not limited by the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods, including Within the protection scope of the present invention.

Claims (10)

1. the preparation method of embedded nano-iron/composite microbial bacterial agent is characterized in that comprising the steps: (1) Preparation of bacteria: Agrobacterium sp., Enterobactercloacae., Bacillus sp., Gordonia sp., Pseudomonas putida., The 2 rings of Pseudomonas stutzeri. were respectively transferred to the nutrient solution, and the bacteria were cultured at 35-37°C for 1-3 days, and inoculated into the growth medium-containing medium at a volume ratio of 5-18%. In the container, cultured at 35-37°C for 1-3 days, centrifuged to obtain the logarithmic growth phase cells of the above bacteria; washed with phosphate buffer for 1-2 times; calculated by volume percentage, respectively 5-8% Agrobacterium, 4-6% Bacillus, 7-15% Enterobacter cloacae, 10-19% Gordonella, 13-27% Pseudomonas putida and 35-45% Pseudomonas stutzeri Bacteria were mixed to obtain thallus A for triclosan degradation; (2) Preparation of nano-iron solution: Using the liquid phase reduction method, in the liquid phase system protected by nitrogen, the strong reducing agent KBH ₄ reduces FeSO ₄ 7H ₂ O to obtain Fe°, and prepares a nano-iron solution with a concentration of 0.1-0.6g/L with Fe°, which is recorded as Solution B; (3) Embedding agent agar, PVA, SiO ₂ Preparation of solution: Heat agar and PVA at a temperature of about 90-100°C to completely dissolve in water to obtain a solution with a mass percentage of 5-9% of agar and 7.5-15% of PVA, and then add SiO ₂ to control SiO ₂ The mass concentration in the mixture is 1-3mg/L, and it is alternately cooled to 50°C after mixing, which is recorded as solution C; (4) Preparation of crosslinking agent aluminum sulfate saturated boric acid solution: Dissolve the aluminum sulfate powder in a saturated boric acid solution to obtain a saturated boric acid solution of aluminum sulfate with a molar concentration of 0.1 to 1 mol/L, which is referred to as solution D; (5) Preparation of nano-iron/composite microbial bacterial agent: Under the condition of constant temperature water bath at 50-70°C, take 15-18% solution B and 6-15% bacterial cell A into 57-66% solution C according to the volume ratio, stir and mix evenly, under the environment of nitrogen protection Add dropwise to 1‐22% solution D at room temperature, cross-link, wash, and store to obtain nano-iron/composite microbial agent. 2. The preparation method according to claim 1, wherein the main components of the nutrient solution are beef extract 6.0g/L, NaCl 5.0g/L, peptone 10.0g/L, soybean powder 2.0g/L, pH 6.5, the rest is water. 3. The preparation method according to claim 1, wherein the main components of the proliferation medium are 20.0 g/L of casein, 3.0 g/L of potassium hydrogen phosphate, 3.0 g/L of glucose, and 4.0 g/L of soybean powder. L, sodium chloride 5.0g/L, the rest is water. 4. its preparation method according to claim 1, is characterized in that, by volume percentage, the composition of described phosphate buffer solution is sodium chloride 9.0g/L, potassium chloride 0.3g/L, hydrogen phosphate two Potassium 1.2g/L and potassium dihydrogen phosphate 0.3g/L, the rest is water. 5. The preparation method according to claim 1, wherein the preservation refers to soaking in sterile physiological saline and storing in a refrigerator at 4°C. 6. The preparation method according to claim 1, wherein said Agrobacterium sp., Enterobacter cloacae., Bacillus sp., Gordonia sp. .), Pseudomonas putida., and Pseudomonas stutzeri. 2 rings were transferred to 30‐40 mL of nutrient solution. 7. The preparation method according to claim 1, characterized in that the cross-linking treatment refers to cross-linking at 4-6°C for 10-36 hours. 8. The preparation method according to claim 1, wherein the centrifugation in step 1) refers to centrifugation at a speed of 4000-5000rpm for 15-30min. 9. The preparation method according to claim 1, characterized in that, the cleaning is to wash with 0.8-1.2% NaCl solution. 10. An embedded nano-iron/composite microbial agent for degrading triclosan, characterized in that it is prepared by the preparation method described in any one of claims 1-9.