CN106754855B - Embedded nano-iron/compound microbial agent and preparation method thereof - Google Patents

Embedded nano-iron/compound microbial agent and preparation method thereof Download PDF

Info

Publication number
CN106754855B
CN106754855B CN201611044542.4A CN201611044542A CN106754855B CN 106754855 B CN106754855 B CN 106754855B CN 201611044542 A CN201611044542 A CN 201611044542A CN 106754855 B CN106754855 B CN 106754855B
Authority
CN
China
Prior art keywords
solution
iron
nano
preparation
triclosan
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201611044542.4A
Other languages
Chinese (zh)
Other versions
CN106754855A (en
Inventor
陈元彩
张振
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China University of Technology SCUT
Original Assignee
South China University of Technology SCUT
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China University of Technology SCUT filed Critical South China University of Technology SCUT
Publication of CN106754855A publication Critical patent/CN106754855A/en
Application granted granted Critical
Publication of CN106754855B publication Critical patent/CN106754855B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/70—Treatment of water, waste water, or sewage by reduction
    • C02F1/705—Reduction by metals
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/34—Organic compounds containing oxygen
    • C02F2101/345—Phenols
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/36—Organic compounds containing halogen

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Environmental & Geological Engineering (AREA)
  • Water Supply & Treatment (AREA)
  • Hydrology & Water Resources (AREA)
  • Inorganic Chemistry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to an embedded nano-iron/compound microbial agent and a preparation method thereof. The preparation method comprises the steps of firstly preparing a nano iron solution B from agrobacterium tumefaciens, enterobacter cloacae, bacillus, gordonia, pseudomonas putida and pseudomonas stutzeri, and preparing embedding agents of agar, PVA and SiO2Preparing a crosslinking agent aluminum sulfate saturated boric acid solution D; and (2) respectively taking 15-18% of the solution B and 6-15% of the thallus A according to the volume ratio, adding the solution B and the thallus A into the 57-66% of the solution C, stirring and mixing uniformly, dropwise adding the solution A into the 1-22% of the solution D at room temperature under the nitrogen protection environment, and performing crosslinking treatment, cleaning and preservation to obtain the nano iron/composite microbial agent. The invention utilizes the synergistic effect between the nano-iron and the microorganism to improve the degradation efficiency of the triclosan; the prepared microbial inoculum has high strength, small microbial toxicity and low raw material source price, and can be widely used for treating water bodies polluted by triclosan.

Description

Embedded nano-iron/compound 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/compound microbial agent for degrading triclosan and a preparation method thereof.
Background
Triclosan has good sterilization and disinfection effects, good safety, and even has the effects of promoting metabolism of human skin, and brightening and moistening skin. Since the 20 th 70 th century, triclosan has been used in soap production, it has been widely used in personal care products and pharmaceutical products, such as detergents, deodorants, cosmetics, disinfection devices, and textile disinfection before delivery. Triclosan belongs to polar hydrophobic organic matters and is easy to deposit on solid-phase substances such as soil, bottom mud and the like. The lipophilicity of hydrophobic substances makes them susceptible to accumulation in organisms, also increases the likelihood of triclosan environmental residuals and threatens human health through mammalian food chain accumulation. The easy adsorption, deposition, persistence, and bio-enrichment of such contaminants pose long-term, unpredictable environmental risks to the surrounding ecological environment. The control and management of such pollution has attracted a great deal of attention.
Biological treatment is a commonly used wastewater treatment method at present, and pollutants in wastewater are decomposed and absorbed through the metabolism of microorganisms, so that the purpose of pollution treatment is achieved. Biological treatment is widely used in wastewater treatment because it is low in cost, high in efficiency, easy to operate, and most importantly, free of secondary pollution, compared with other methods. With the development of economy, the components of wastewater become increasingly complex, and particularly when the wastewater contains toxic and refractory organic pollutants, the traditional biological treatment technology faces great challenges because the types and the quantity of microorganisms with special degradation capability for the organic matters in the environment are small, and meanwhile, the microorganisms are at a disadvantage in interspecific competition.
If microorganism or some matrix with specific function is added into the traditional biological treatment system to enhance the degradation capability of the traditional biological treatment system to specific pollutants, thereby improving the treatment effect of the whole sewage treatment system, the technology is called as a biological strengthening technology. In recent years, the reaction rate of the nano material is improved due to the huge specific surface area and high activity of the nano material, and the nano-material is applied to polluted soil and groundwater remediation and sewage treatment, and nano-scale zero-value (nZVI) research is relatively more. nZVI is an effective dehalogenation reducing agent, which has attracted attention as early as the 80's in the 20 th century. The nanometer zero-valent iron can catalyze and reduce various organic halides, such as halogenated alkane, halogenated olefin, halogenated aromatic hydrocarbon and other refractory organic pollutants, convert the organic pollutants into non-toxic and harmless compounds, improve the biodegradability of the compounds, and create favorable conditions for further biodegradation. Although the nano zero-valent iron has many advantages, some problems are encountered in the application process, such as poor stability of the nano zero-valent iron. The nano zero-valent iron is easily oxidized to form iron oxide or hydroxide to deposit on the surface of the nano iron, so that the nano zero-valent iron is passivated.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an embedded nano-iron/compound microbial agent which has high triclosan degradation efficiency, high microbial agent strength, small microbial toxicity and low raw material source price and a preparation method thereof.
According to the invention, the nano-iron and the microorganisms are embedded by using a chemical means to prepare the composite microbial inoculum, so that a synergistic effect can be formed on the treatment of triclosan pollutants, the high specific surface area and the surface activity of nano-iron particles can be utilized, and the stability and the activity of the microorganisms can be ensured; the prepared embedded microbial inoculum is suitable for in-situ remediation and has no secondary pollution.
The purpose of the invention is realized by the following technical scheme:
the preparation method of the embedded nano-iron/compound microbial agent comprises the following steps:
(1) preparation of the thallus:
respectively picking and transferring 2 rings of Agrobacterium (Agrobacterium sp.), enterobacter cloacae (Enterobacter cloacae), Bacillus (Bacillus sp.), Gordonia sp.), Pseudomonas putida (Pseudomonas putida) and Pseudomonas stutzeri (Pseudomonas stutzeri) into a nutrient solution, culturing the bacteria at 35-37 ℃ for 1-3 days, inoculating the bacteria into a container containing a proliferation culture medium at a volume ratio of 5-18%, culturing at 35-37 ℃ for 1-3 days, and centrifuging to obtain cells of the bacteria in a logarithmic growth phase; washing with phosphate buffer solution for 1-2 times; respectively mixing 5-8% of agrobacterium, 4-6% of bacillus, 7-15% of enterobacter cloacae, 10-19% of gordonia bacteria, 13-27% of pseudomonas putida and 35-45% of pseudomonas stutzeri by volume percentage to obtain a thallus A for triclosan degradation;
(2) preparing a nano iron solution:
adopting a liquid phase reduction method, in a liquid phase system protected by nitrogen, using a strong reducing agent KBH4Reduction of FeSO4·7H2O to Fe0From Fe0Preparing a nano-iron solution with the concentration of 0.1-0.6 g/L, and marking as a solution B;
(3) embedding medium agar, PVA, SiO2Preparation of the solution:
heating agar and PVA at about 90-100 ℃ to completely dissolve in clear water to obtain a solution with the mass percent of the agar of 5-9% and the mass percent of the PVA of 7.5-15%, and then adding SiO2Control of SiO2The mass concentration of the mixed solution in the mixture is 1-3 mg/L, and the mixed solution is alternately cooled to 50 ℃ and marked as solution C;
(4) preparation of crosslinking agent aluminum sulfate saturated boric acid solution:
dissolving aluminum sulfate powder in a saturated boric acid solution to obtain a saturated boric acid solution of aluminum sulfate with the molar concentration of 0.1-1 mol/L, and marking as a solution D;
(5) preparing a nano iron/compound microbial agent:
under the condition of a constant-temperature water bath at 50-70 ℃, respectively taking 15-18% of solution B and 6-15% of thallus A according to the volume ratio, adding the solution B and the thallus A into 57-66% of solution C, uniformly stirring and mixing, dropwise adding the solution A into 1-22% of solution D at room temperature under the nitrogen protection environment, performing crosslinking treatment, cleaning and storing to obtain the nano-iron/composite microbial agent.
To further achieve the purpose of the invention, preferably, the main components of the nutrient solution are beef extract 6.0 g/L/L, peptone 10.0 g/L, soybean meal 2.0 g/L, pH 6.5 and the balance of water.
Preferably, the main components of the proliferation medium are casein 20.0 g/L, potassium hydrogen phosphate 3.0 g/L, glucose 3.0 g/L, soybean meal 4.0 g/L, sodium chloride 5.0 g/L and the balance of water.
Preferably, the phosphate buffer solution comprises 9.0 g/L g of sodium chloride, 0.3 g/L g of potassium chloride, 1.2 g/L g of dipotassium phosphate and 0.3 g/L g of monopotassium phosphate, and the balance of water in percentage by volume.
Preferably, the method for storing the embedded nano-iron/compound microbial agent is to soak the embedded nano-iron/compound microbial agent in sterile physiological saline and store the embedded nano-iron/compound microbial agent in a refrigerator at 4 ℃.
Preferably, the preservation refers to soaking in sterile physiological saline and storing in a refrigerator at 4 ℃.
Preferably, the Agrobacterium sp, Enterobacter cloacae sp, Bacillus sp, Gordonia sp, Pseudomonas putida and Pseudomonas stutzeri 2 loops are transferred to a nutrient solution containing 30-40m L, respectively.
Preferably, the crosslinking treatment is crosslinking for 10-36 h at 4-6 ℃.
Preferably, the centrifugation treatment in step 1) is centrifugation at 4000-.
Preferably, the washing is with 0.8-1.2% NaCl solution.
An embedded nano-iron/compound microbial agent for degrading triclosan is prepared by the preparation method.
The invention has the following advantages:
1) according to the embedded nano-iron/compound microbial agent, the strong reducibility of nano-iron to triclosan and the adsorbability of nano-iron to microorganisms are utilized, and the nano-iron can act with cytochrome c of mitochondria of the microorganisms to change the oxidation-reduction potential of the cytochrome c and enhance the electron transfer capacity, so that the compound microbial agent can generate a synergistic effect to jointly promote the degradation of triclosan. Under the condition of the range value of the embodiment of the invention, the composite microbial inoculum prepared by the invention is compared with a single microorganism experiment and a single nano-iron experiment under the same experiment condition, and the composite microbial inoculum can be verified to generate a synergistic effect to jointly promote the effect of the degraded nano-iron of the triclosan on the cytochrome c of mitochondria of the microorganism, change the oxidation-reduction potential of the cytochrome c and enhance the electron transfer capability.
2) The embedding agent agar used in the invention has wide raw material source, low price, no toxicity and good biocompatibility. The prepared microbial inoculum has high strength and low microbial toxicity. Simultaneously solves the problem of adsorption on inorganic porous SiO2The instability of the material microorganism. The nano iron and the microorganism form a synergistic effect to enhance the degradation efficiency of the triclosan, and the method is suitable for large-scale industrial production.
3) The method is simple and convenient to use, and the prepared microbial inoculum can be directly put into the polluted water body after being activated, so that the in-situ remediation of the polluted water body is realized, the loss of microorganisms is effectively avoided, and no secondary pollution exists.
Detailed Description
The invention will be further illustrated by the following examples for a better understanding of the invention, but the scope of the invention as claimed is not limited to the examples.
In the embodiment, in the step 1) and the step 6), the main components of the nutrient solution are beef extract 6.0 g/L/L, peptone 10.0 g/L, soybean meal 2.0 g/L, pH 6.5 and the balance of water.
The main components of the proliferation culture medium are casein 20.0 g/L, potassium hydrogen phosphate 3.0 g/L, glucose 3.0 g/L, soybean meal 4.0 g/L, sodium chloride 5.0 g/L and the balance of water.
Example 1
(1) Preparation of triclosan degradation bacterium liquid
Respectively picking Agrobacterium (Agrobacterium sp.), enterobacter cloacae (enterobacter cloacae), Bacillus (Bacillus sp.), Gordonia sp.), Pseudomonas putida (Pseudomonas putida), Pseudomonas stutzeri (Pseudomonas stutzeri) 2 rings, respectively transferring the 2 rings into 30m of nutrient solution, culturing the bacteria at 35 ℃ for 2 days, inoculating the bacteria into a container of a propagation culture medium in a volume ratio of 10%, culturing the bacteria at 35 ℃ for 2 days, centrifuging the bacteria at 5000rpm for 15min, respectively obtaining logarithmic cells of the bacteria, taking out the cells of the bacteria in a growth phase, using a phosphate buffer solution (the main component of which is sodium chloride 9.0 g/L, potassium chloride 0.3 g/L, dipotassium hydrogen phosphate 1.2 g/L g and potassium dihydrogen phosphate 0.3 g/539 for standby bacteria, respectively washing the bacteria at 20% of physiological saline, and obtaining the bacterial strain after refrigerating at 35 ℃ for 20%, and mixing the bacteria in a growth phase, and obtaining the bacterial strain degradation at 20% by using 20% of physiological saline, 20% and 20% of the bacteria A;
(2) preparation of nano-iron solution
Adopting a liquid phase reduction method, in a liquid phase system protected by nitrogen, using a strong reducing agent KBH4Reduction of FeSO4·7H2O to Fe0From Fe0A nano-iron solution with a concentration of 0.4 g/L was prepared and recorded as solution B.
(3) Embedding medium agar, PVA, SiO2Preparation of the solution
Heating agar and PVA at 90 deg.C to dissolve completely in clear water to obtain solution containing agar 5 wt% and PVA 7.5 wt%, and adding SiO2,SiO2The mass concentration of the mixture is 1 mg/L, and the mixture is alternately cooled to 55 ℃ to be marked as solution C;
(4) preparation of cross-linking agent aluminum sulfate saturated boric acid solution
Aluminum sulfate powder was dissolved in a saturated boric acid solution to give a saturated boric acid solution of aluminum sulfate having a molar concentration of 0.5 mol/L, denoted as solution D.
(5) Preparation of embedded microbial inoculum
Under the condition of constant temperature water bath at 60 ℃, 15 percent of solution B of 0.1 mg/L and 6 percent of thallus A are respectively added into 57 percent of solution C (containing agar of 5 percent by mass, PVA of 7.5 percent by mass and SiO of 1 mg/L percent by volume2) And stirring and mixing uniformly. Dropwise adding the mixture into a 22% solution D (room temperature) under the nitrogen protection environment, crosslinking for 10 hours at 4 ℃, and then washing and storing the mixture by using a 0.9 wt% NaCl solution to obtain the nano-iron/composite microbial agent.
(6) Degradation effect of triclosan pollution
Adding 3 mg/L of nano-iron compound microbial agent into a nutrient solution medium for culturing for 6h, directly adding 10L of triclosan simulated polluted wastewater with the concentration of 5 mg/L after activating, carrying out aeration treatment for 5d, wherein the aeration amount is 2L/h, and the nutrient solution comprises 6.0 g/L/L of beef extract, 10.0 g/L of peptone, 2.0 g/L of soybean meal, the pH value is 6.5 and the balance of water according to mass concentration.
Triclosan was measured by Waters high performance liquid chromatography with the following column: waters C18Column (150 × 4.6.6 mm I.D., 5 μm), column temperature of 35 deg.C, acetonitrile/water (75:25, v/v) as mobile phase, total flow rate of 1.0m L/min, sample injection amount of 10 μ L, detection wavelength of 230nm, target substance peak-off time of 7.2min, total sample detection time of 12min, and determination of initial concentration C of triclosan in water sample by testing0And post-reaction concentration CtAnd obtaining the triclosan removal rate.
The method comprises the steps of adding 2g of nano-iron compound microbial agent into triclosan 5L wastewater of 5 mg/L in water, directly adding the microbial agent into nutrient solution for culturing for 6 hours, and directly adding the microbial agent for use after activation, wherein the removal rate of the triclosan after 5d aeration treatment is 91%, which is obviously higher than 28% of a control group of a single strain (Pseudomonas stutzeri), which indicates that the embedded nano-iron/compound microbial agent has a good degradation effect on the triclosan and is obviously superior to the single strain, the strong reducibility of the nano-iron on the triclosan, the adsorbability of the nano-iron on microorganisms, the effect of the nano-iron on cytochrome c of mitochondria of the microorganisms, the change of the redox potential of the cytochrome c and the enhancement of the electron transfer capacity, the compound microbial agent can generate a synergistic effect to jointly promote the degradation of the triclosan, the strong reducibility of the nano-iron on the triclosan, the adsorbability of the microorganisms, and the nano-iron can act on the cytochrome c of the mitochondria of the microorganisms, the change of the redox potential of the cytochrome c and the.
Example 2
(1) Preparation of triclosan degradation bacterium liquid
Agrobacterium sp, Enterobacter cloacae, Bacillus sp, Gordonia sp, Pseudomonas putida, Pseudomonas stuartii 2 rings were picked up and transferred to 30m L nutrient solution, respectively, and the bacteria were cultured at 35 ℃ for 2 days, inoculated at 10% by volume into a growth medium container, cultured at 35 ℃ for 2 days, and centrifuged at 5000rpm for 15min to obtain logarithmic growth phase cells of the bacteria, respectively.
Taking out cells in the logarithmic growth phase of the thalli, washing the cells for 2 times by using a phosphate buffer solution (the main components of the phosphate buffer solution are 9.0 g/L, 0.3 g/L, 1.2 g/L of dipotassium hydrogen phosphate and 0.3 g/L of monopotassium phosphate, and the balance of water), respectively taking 5% of agrobacterium tumefaciens, 5% of bacilli, 10% of enterobacter cloacae, 20% of gordonia, 20% of pseudomonas putida and 40% of pseudomonas stutzeri according to volume percentage, mixing to obtain thalli for triclosan degradation, suspending the thalli in physiological saline, and refrigerating the thalli at 4 ℃ for later use, namely, marking as thalli A;
(2) preparation of nano-iron solution
Adopting a liquid phase reduction method, in a liquid phase system protected by nitrogen, using a strong reducing agent KBH4Reduction of FeSO4·7H2O to Fe0From Fe0A nano-iron solution with a concentration of 0.4 g/L was prepared and recorded as solution B.
(3) Embedding medium agar, PVA, SiO2Preparation of the solution
Heating agar and PVA at 90 deg.C to dissolve completely in clear water to obtain solution containing agar 7 wt% and PVA 11.5 wt%, and adding SiO2,SiO2The mass concentration of the mixture is 2 mg/L, and the mixture is alternately cooled to 55 ℃ to be marked as solution C;
(4) preparation of cross-linking agent aluminum sulfate saturated boric acid solution
Aluminum sulfate powder was dissolved in a saturated boric acid solution to give a saturated boric acid solution of aluminum sulfate having a molar concentration of 0.5 mol/L, denoted as solution D.
(5) Preparation of embedded microbial inoculum
Under the condition of constant temperature water bath at 60 ℃, 16 percent of solution B of 0.4 mg/L and 10 percent of thallus A are respectively added into the solution B and the thallus A according to the volume ratio61% solution C (containing 7% agar, 11.5% PVA, 2 mg/L SiO by mass%2) And stirring and mixing uniformly. Dropwise adding the mixture into a 13% solution D (room temperature) under the nitrogen protection environment, crosslinking for 23h at 4 ℃, and then washing and storing the mixture by using a 0.9 wt% NaCl solution to obtain the nano-iron/composite microbial agent.
(6) Degradation effect of triclosan pollution
Adding 3 mg/L of nano-iron compound microbial agent into nutrient solution medium for culturing for 6h, directly adding 10L of triclosan simulated polluted wastewater with the concentration of 5 mg/L after activating, carrying out aeration treatment for 5d, wherein the aeration amount is 2L/h, and the nutrient solution comprises 6.0 g/L/L of beef extract, 10.0 g/L of peptone, 2.0 g/L of soybean meal, the pH value is 6.5 and the balance of water by mass concentration.
By adopting the method of the embodiment, 2g of nano-iron composite microbial inoculum is added into wastewater containing 5 mg/L triclosan 5L, after activation, the microbial inoculum is directly put into nutrient solution for culturing for 6h and is directly put into use, the removal rate of the triclosan after 5d of aeration treatment reaches 98 percent and is obviously higher than 32 percent of a control group of a single strain (Pseudomonas stutzeri), which indicates that the embedded nano-iron/composite microbial inoculum has good degradation effect on the triclosan and is obviously superior to the single strain.
Triclosan was measured by Waters high performance liquid chromatography with the following column: waters C18A column (150 × 4.6.6 mm I.D., 5 μm), a column temperature of 35 ℃, acetonitrile/water (75:25, v/v) as a mobile phase, a total flow rate of 1.0m L/min, a sample injection amount of 10 μ L, a detection wavelength of 230nm, a target substance peak-off time of about 7.2min, and a total sample detection time of 12 min.
Example 3
(1) Preparation of triclosan degradation bacterium liquid
Agrobacterium sp, Enterobacter cloacae, Bacillus sp, Gordonia sp, Pseudomonas putida, Pseudomonas stuartii 2 rings were picked up and transferred to 30m L nutrient solution, respectively, and the bacteria were cultured at 35 ℃ for 2 days, inoculated at 10% by volume into a growth medium container, cultured at 35 ℃ for 2 days, and centrifuged at 5000rpm for 15min to obtain logarithmic growth phase cells of the bacteria, respectively.
Taking out cells in the logarithmic growth phase of the thalli, washing the cells for 2 times by using a phosphate buffer solution (the main components of the phosphate buffer solution are 9.0 g/L, 0.3 g/L, 1.2 g/L of dipotassium hydrogen phosphate and 0.3 g/L of monopotassium phosphate, and the balance of water), respectively taking 5% of agrobacterium tumefaciens, 5% of bacilli, 10% of enterobacter cloacae, 20% of gordonia, 20% of pseudomonas putida and 40% of pseudomonas stutzeri according to volume percentage, mixing to obtain thalli for triclosan degradation, suspending the thalli in physiological saline, and refrigerating the thalli at 4 ℃ for later use, namely, marking as thalli A;
(2) preparation of nano-iron solution
Adopting a liquid phase reduction method, in a liquid phase system protected by nitrogen, using a strong reducing agent KBH4Reduction of FeSO4·7H2O to Fe0From Fe0A nano-iron solution with a concentration of 0.4 g/L was prepared and recorded as solution B.
(3) Embedding medium agar, PVA, SiO2Preparation of the solution
Heating agar and PVA at 90 deg.C to dissolve in clear water completely to obtain solution containing agar 9 wt% and PVA 15 wt%, and adding SiO2,SiO2The mass concentration of the mixture is 3 mg/L, and the mixture is alternately cooled to 55 ℃ to be marked as solution C;
(4) preparation of cross-linking agent aluminum sulfate saturated boric acid solution
Aluminum sulfate powder was dissolved in a saturated boric acid solution to give a saturated boric acid solution of aluminum sulfate having a molar concentration of 0.5 mol/L, denoted as solution D.
(5) Preparation of embedded microbial inoculum
Under the condition of constant temperature water bath at 60 ℃, respectively taking 18 percent of solution B of 0.6 mg/L and 15 percent of solution B according to volume ratioAdding the thallus A into 66% solution C (containing 9% agar, 15% PVA, 3 mg/L SiO by mass percent)2) And stirring and mixing uniformly. Dropwise adding the solution into a 1% solution D (room temperature) under the nitrogen protection environment, crosslinking for 36h at 4 ℃, and then cleaning and storing by using a 0.9 wt% NaCl solution to obtain the nano-iron/composite microbial agent.
(6) Degradation effect of triclosan pollution
Adding 3 mg/L of nano-iron compound microbial agent into nutrient solution medium for culturing for 6h, directly adding 10L of triclosan simulated polluted wastewater with the concentration of 5 mg/L after activating, carrying out aeration treatment for 5d, wherein the aeration amount is 2L/h, and the nutrient solution comprises 6.0 g/L/L of beef extract, 10.0 g/L of peptone, 2.0 g/L of soybean meal, the pH value is 6.5 and the balance of water by mass concentration.
By adopting the method of the embodiment, 2g of nano-iron composite microbial inoculum is added into wastewater containing 5 mg/L triclosan 5L, after activation, the microbial inoculum is directly put into nutrient solution for culturing for 6h and is directly put into use, the removal rate of the triclosan after 5d of aeration treatment reaches 96 percent and is obviously higher than 25 percent of a control group of a single strain (Pseudomonas stutzeri), which indicates that the embedded nano-iron/composite microbial inoculum has good degradation effect on the triclosan and is obviously better than the single strain.
Triclosan was measured by Waters high performance liquid chromatography with the following column: waters C18A column (150 × 4.6.6 mm I.D., 5 μm), a column temperature of 35 ℃, acetonitrile/water (75:25, v/v) as a mobile phase, a total flow rate of 1.0m L/min, a sample injection amount of 10 μ L, a detection wavelength of 230nm, a target substance peak-off time of about 7.2min, and a total sample detection time of 12 min.
In the invention, the nano-iron and the microorganism have synergistic effect, and experiments prove that the prepared consistent microbial inoculum has better effects than single microorganism agrobacteria (Agrobacterium sp.), Enterobacter cloacae (Enterobacteriaceae), Bacillus sp., Gordonia sp., Pseudomonas putida (Pseudomonas putrescentia) and Pseudomonas stutzeri (Pseudomonas stutzeri) respectively under the same experimental conditions. The strong reducibility of the nano-iron to the triclosan and the adsorbability to the microorganism, and the nano-iron can act with the cytochrome c of mitochondria of the microorganism to change the oxidation-reduction potential of the cytochrome c and enhance the electron transfer capability, so that the compound microbial inoculum can generate a synergistic effect to jointly promote the degradation of the triclosan. The problem that the bacteria are easily inhibited by intermediate metabolites in the process of degrading triclosan is solved, and the nano-iron and the microorganisms are embedded to prepare the microbial inoculum, so that the high specific surface area and surface activity of nano-iron particles can be utilized, the stability and activity of the microorganisms can be ensured, a synergistic effect is formed on the treatment of triclosan pollutants, and the removal rate is obviously increased. The prepared embedded microbial inoculum is suitable for in-situ rest and has no secondary pollution.
According to reports of related bacteria degrading triclosan: the degradation rate of fungal laccase (laccases)/redox mediator system for triclosan is 90% [ Murugesan K, Chang Y, Kim Y M, et al, engineering dtransformation of triclosan in the presence of redox mediators [ J ]. Water Research,2010,44(1): 298- > 308 ], and the degradation rate of white rot fungi for triclosan is also 90% [ InouY, hatA T, Kawai S, et al, Elimation and determination of triclosan dehydrogenase from white rot root fusion [ J ]. Journal of triclosan Materials,2010,180 (1-3): 764- > 7 ]. The degradation efficiency of the bacteria to the triclosan is lower than that of the microbial inoculum of the invention, so the microbial inoculum has good application prospect in the aspect of treating triclosan wastewater. The embedded nano-iron single microbial agent prepared by the invention has advantages in the degradation of triclosan. And 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 microbial inoculum is suitable for in-situ remediation and has no secondary pollution. The prepared embedding agent can be put into use only by simple activation, and has the prospect of large-scale industrial production.
The embodiments of the present invention are not limited to the above-described embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and they are included in the scope of the present invention.

Claims (10)

1. The preparation method of the embedded nano-iron/compound microbial agent is characterized by comprising the following steps:
(1) preparation of the thallus:
respectively pick out Agrobacterium (Agrobacterium sp.) Enterobacter cloacae: (A), (B), (C)Enterobactercloacae.) Bacillus bacteria (1)Bacillus sp.) Gordoniae (A. sp.), (B. sp.), (C. spGordonia sp.) Pseudomonas putida (b)Pseudomonas putida.) Pseudomonas stutzeri (A), (B) and (C)Pseudomonas stutzeri.) Transferring 2 rings into nutrient solution, culturing bacteria at 35-37 deg.C for 1-3 days, inoculating 5-18% volume of bacteria into a container containing proliferation culture medium, culturing at 35-37 deg.C for 1-3 days, and centrifuging to obtain cells of logarithmic growth phase of the bacteria; washing with phosphate buffer solution for 1-2 times; respectively mixing 5-8% of agrobacterium, 4-6% of bacillus, 7-15% of enterobacter cloacae, 10-19% of gordonia bacteria, 13-27% of pseudomonas putida and 35-45% of pseudomonas stutzeri by volume percentage to obtain a thallus A for triclosan degradation;
(2) preparing a nano iron solution:
adopting a liquid phase reduction method, in a liquid phase system protected by nitrogen, using a strong reducing agent KBH4Reduction of FeSO4·7H2O to Fe0From Fe0Preparing a nano-iron solution with the concentration of 0.1-0.6 g/L, and marking as a solution B;
(3) embedding medium agar, PVA, SiO2Preparation of the solution:
heating agar and PVA at 90-100 ℃ to completely dissolve in clear water to obtain a solution with the mass percent of the agar of 5-9% and the mass percent of the PVA of 7.5-15%, and then adding SiO2Control of SiO2The mass concentration of the mixed solution in the mixture is 1-3 mg/L, and the mixed solution is alternately cooled to 50 ℃ and marked as solution C;
(4) preparation of crosslinking agent aluminum sulfate saturated boric acid solution:
dissolving aluminum sulfate powder in a saturated boric acid solution to obtain a saturated boric acid solution of aluminum sulfate with the molar concentration of 0.1-1 mol/L, and marking as a solution D;
(5) preparing a nano iron/compound microbial agent:
under the condition of a constant-temperature water bath at 50-70 ℃, respectively taking 15-18% of solution B and 6-15% of thallus A according to the volume ratio, adding the solution B and the thallus A into 57-66% of solution C, uniformly stirring and mixing, dropwise adding the solution A into 1-22% of solution D at room temperature under the nitrogen protection environment, performing crosslinking treatment, cleaning and storing to obtain the nano-iron/composite microbial agent.
2. The preparation method of claim 1, wherein the nutrient solution comprises beef extract 6.0 g/L/L, peptone 10.0 g/L, soybean meal 2.0 g/L, pH 6.5, and water as the rest.
3. The method according to claim 1, wherein the proliferation medium comprises casein 20.0 g/L, potassium hydrogen phosphate 3.0 g/L, glucose 3.0 g/L, soybean powder 4.0 g/L, sodium chloride 5.0 g/L, and water as the main component.
4. The method according to claim 1, wherein the phosphate buffer comprises, in terms of volume percentage, 9.0 g/L g of sodium chloride, 0.3 g/L g of potassium chloride, 1.2 g/L g of dipotassium phosphate and 0.3 g/L g of monopotassium phosphate, and the balance being water.
5. The method according to claim 1, wherein the preservation is performed by immersing in a sterile physiological saline and storing in a refrigerator at 4 ℃.
6. The method according to claim 1, wherein the Agrobacterium is selected from the group consisting of (A), (B), (C) and (C)Agrobacterium sp.) Enterobacter cloacae: (A), (B), (C)Enterobactercloacae.) Bacillus bacteria (1)Bacillus sp.) Gordoniae (A. sp.), (B. sp.), (C. spGordonia sp.) Fake sheet with bad smellBacteria (C)Pseudomonas putida.) Pseudomonas stutzeri (A), (B) and (C)Pseudomonas stutzeri.) The 2 loops were transferred to 30-40m L nutrient solutions, respectively.
7. The preparation method according to claim 1, wherein the crosslinking treatment is crosslinking at 4-6 ℃ for 10-36 h.
8. The method as claimed in claim 1, wherein the centrifugation treatment in step (1) is performed at 4000-.
9. The method according to claim 1, wherein the washing is performed with 0.8 to 1.2% NaCl solution.
10. An embedded nano-iron/compound microbial agent for degrading triclosan, which is prepared by the preparation method of any one of claims 1-9.
CN201611044542.4A 2016-06-29 2016-11-24 Embedded nano-iron/compound microbial agent and preparation method thereof Active CN106754855B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610508531 2016-06-29
CN2016105085310 2016-06-29

Publications (2)

Publication Number Publication Date
CN106754855A CN106754855A (en) 2017-05-31
CN106754855B true CN106754855B (en) 2020-07-28

Family

ID=58975195

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611044542.4A Active CN106754855B (en) 2016-06-29 2016-11-24 Embedded nano-iron/compound microbial agent and preparation method thereof

Country Status (1)

Country Link
CN (1) CN106754855B (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109277405B (en) * 2018-11-28 2021-02-02 青岛理工大学 Method for remediating arsenic-contaminated soil by using nano zero-valent iron and bacteria
CN109570227B (en) * 2018-11-28 2020-12-01 青岛理工大学 Method for restoring acidified arsenic-polluted soil by using charcoal-loaded nano zero-valent iron and bacteria
CN112077138A (en) * 2019-06-12 2020-12-15 天津大学 Application of nano-iron in treating phthalate ester in sediment or soil
CN111204884A (en) * 2020-02-27 2020-05-29 上海澄域环保工程有限公司 Method for degrading chlorinated aliphatic hydrocarbon in underground water

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101343629A (en) * 2007-07-10 2009-01-14 南京理工大学 Improved process for polyvinyl alcohol aluminum salt immobilization microorganism
CN101993868A (en) * 2010-09-30 2011-03-30 南开大学 Embedded nano iron/microbe microspheres and preparation method thereof
CN103627657A (en) * 2013-11-11 2014-03-12 华南理工大学 Composite microflora for degrading lignin wastewater and preparation method thereof
CN103642712A (en) * 2013-11-08 2014-03-19 华南理工大学 Composite flora for degrading papermaking waste water and preparation method thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101343629A (en) * 2007-07-10 2009-01-14 南京理工大学 Improved process for polyvinyl alcohol aluminum salt immobilization microorganism
CN101993868A (en) * 2010-09-30 2011-03-30 南开大学 Embedded nano iron/microbe microspheres and preparation method thereof
CN103642712A (en) * 2013-11-08 2014-03-19 华南理工大学 Composite flora for degrading papermaking waste water and preparation method thereof
CN103627657A (en) * 2013-11-11 2014-03-12 华南理工大学 Composite microflora for degrading lignin wastewater and preparation method thereof

Also Published As

Publication number Publication date
CN106754855A (en) 2017-05-31

Similar Documents

Publication Publication Date Title
Li et al. Algae-mediated antibiotic wastewater treatment: a critical review
CN106754855B (en) Embedded nano-iron/compound microbial agent and preparation method thereof
Kakooei et al. Mechanisms of microbiologically influenced corrosion: a review
Lin et al. Use of bacteria-immobilized cotton fibers to absorb and degrade crude oil
CN104099274B (en) The greedy copper bacterium of one strain aerobic degradation indole and application thereof
Mohite et al. Isolation, selection and biodegradation profile of phenol degrading bacteria from oil contaminated soil
CN105969760B (en) Embedded nano iron/single microorganism composite microbial agent and preparation method thereof
Sahebnazar et al. Enhancement of Pseudomonas aeruginosa growth and rhamnolipid production using iron-silica nanoparticles in low-cost medium
Zhao et al. Efficacy of forming biofilms by Pseudomonas migulae AN-1 toward in situ bioremediation of aniline-contaminated aquifer by groundwater circulation wells
Torrentó et al. Characterization of attachment and growth of Thiobacillus denitrificans on pyrite surfaces
CN112358880A (en) Microorganism-nano material composite microbial agent and preparation method and application thereof
CN107699513A (en) A kind of special degradation bacteria of black and odorous water and its application
Singh et al. Microbial biofilms: development, structure, and their social assemblage for beneficial applications
Khanpour-Alikelayeh et al. Enhanced biodegradation of light crude oil by immobilized Bacillus licheniformis in fabricated alginate beads through electrospray technique
Yu et al. Anti-biological contamination strategies for enhanced uranium extraction from seawater
Zhao et al. Iodine from bacterial iodide oxidization by Roseovarius spp. inhibits the growth of other bacteria
Liu et al. Study on a novel immobilized microbe pellets constructed with Alcaligenes sp. R3 and its ability to remove tetracycline
Shan et al. Enhanced treatment of synthetic wastewater by bioaugmentation with a constructed consortium
CN105925560B (en) Embedded nano-iron/two microbial agents and preparation method thereof
Sun et al. Enhanced removal of metronidazole from aqueous solutions via bioelectrochemical systems
CN106434408A (en) Citric acid bacillus Y3 with function of degrading brominated flame retardants and application thereof
CN106399200B (en) Alcaligenes and application thereof in high-salt high-polymer wastewater
CN106186365B (en) A kind of biological sponge iron and its application in sewage treatment
Zhang et al. Selective assembly of microbe on patterned porous interfaces as potential membrane reactors for enhanced biodegradation of phenol
CN102029285B (en) Method for degrading chlorobenzene organic pollutants by using double-liquid-phase system

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant