CN105969760B - Embedded nano iron/single microorganism composite microbial agent and preparation method thereof - Google Patents
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 title claims abstract description 98
- 229910052742 iron Inorganic materials 0.000 title claims abstract description 76
- 244000005700 microbiome Species 0.000 title claims abstract description 64
- 239000002131 composite material Substances 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 26
- 230000000813 microbial effect Effects 0.000 title claims abstract description 20
- XEFQLINVKFYRCS-UHFFFAOYSA-N Triclosan Chemical compound OC1=CC(Cl)=CC=C1OC1=CC=C(Cl)C=C1Cl XEFQLINVKFYRCS-UHFFFAOYSA-N 0.000 claims abstract description 63
- 229960003500 triclosan Drugs 0.000 claims abstract description 63
- 239000002068 microbial inoculum Substances 0.000 claims abstract description 43
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 41
- 241000589614 Pseudomonas stutzeri Species 0.000 claims abstract description 29
- 230000015556 catabolic process Effects 0.000 claims abstract description 25
- 238000006731 degradation reaction Methods 0.000 claims abstract description 25
- 238000011282 treatment Methods 0.000 claims abstract description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 22
- 229920001817 Agar Polymers 0.000 claims abstract description 20
- 239000008272 agar Substances 0.000 claims abstract description 20
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229910052681 coesite Inorganic materials 0.000 claims abstract description 15
- 229910052906 cristobalite Inorganic materials 0.000 claims abstract description 15
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 15
- 229910052682 stishovite Inorganic materials 0.000 claims abstract description 15
- 229910052905 tridymite Inorganic materials 0.000 claims abstract description 15
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 11
- 238000004132 cross linking Methods 0.000 claims abstract description 10
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 8
- -1 aluminum sulfate saturated boric acid Chemical class 0.000 claims abstract description 8
- 238000004140 cleaning Methods 0.000 claims abstract description 6
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 6
- 238000003756 stirring Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 96
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 31
- 241000894006 Bacteria Species 0.000 claims description 22
- 235000015097 nutrients Nutrition 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 20
- 239000011780 sodium chloride Substances 0.000 claims description 15
- 238000012258 culturing Methods 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 12
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 10
- 235000019764 Soybean Meal Nutrition 0.000 claims description 10
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 claims description 10
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical class OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 10
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 10
- 239000007791 liquid phase Substances 0.000 claims description 10
- 230000035755 proliferation Effects 0.000 claims description 10
- 239000004455 soybean meal Substances 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 230000003698 anagen phase Effects 0.000 claims description 8
- 235000015278 beef Nutrition 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 239000008055 phosphate buffer solution Substances 0.000 claims description 7
- 239000003638 chemical reducing agent Substances 0.000 claims description 6
- 230000000593 degrading effect Effects 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- 239000005018 casein Substances 0.000 claims description 5
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 5
- 235000021240 caseins Nutrition 0.000 claims description 5
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 229910052603 melanterite Inorganic materials 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- 239000001103 potassium chloride Substances 0.000 claims description 5
- 235000011164 potassium chloride Nutrition 0.000 claims description 5
- 230000009467 reduction Effects 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- 239000002504 physiological saline solution Substances 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims 4
- 230000002195 synergetic effect Effects 0.000 abstract description 9
- 239000002994 raw material Substances 0.000 abstract description 3
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 238000003860 storage Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 10
- 238000001514 detection method Methods 0.000 description 10
- 239000003643 water by type Substances 0.000 description 10
- 238000005273 aeration Methods 0.000 description 9
- 239000002351 wastewater Substances 0.000 description 9
- 102100030497 Cytochrome c Human genes 0.000 description 8
- 108010075031 Cytochromes c Proteins 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 230000004913 activation Effects 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 238000011065 in-situ storage Methods 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 238000005067 remediation Methods 0.000 description 5
- 239000013076 target substance Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003344 environmental pollutant Substances 0.000 description 4
- 210000003470 mitochondria Anatomy 0.000 description 4
- 230000033116 oxidation-reduction process Effects 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 231100000719 pollutant Toxicity 0.000 description 4
- 230000027756 respiratory electron transport chain Effects 0.000 description 4
- 235000010469 Glycine max Nutrition 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002086 nanomaterial Substances 0.000 description 3
- 239000002957 persistent organic pollutant Substances 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000004065 wastewater treatment Methods 0.000 description 3
- 108010029541 Laccase Proteins 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000010865 sewage Substances 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 241000233866 Fungi Species 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 238000005282 brightening Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005695 dehalogenation reaction Methods 0.000 description 1
- 239000002781 deodorant agent Substances 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000003673 groundwater Substances 0.000 description 1
- 150000004820 halides Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 235000014413 iron hydroxide Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
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- 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
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- 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
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- 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
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Abstract
The invention discloses an embedded nano-iron/single microorganism composite microbial inoculum and a preparation method thereof. The preparation method comprises the steps of firstly preparing pseudomonas stutzeri thallus to obtain thallus A, then preparing nano-iron solution B, and then preparing embedding agents of agar, PVA and SiO2And preparing a crosslinking agent aluminum sulfate saturated boric acid solution D, respectively adding 15-18% of the solution B and 6-15% of the thallus A into the 57-66% of the solution C according to the volume ratio under the condition of a constant-temperature water bath at 50-70 ℃, stirring and mixing uniformly, dropwise adding the solution into the 1-22% of the solution D at room temperature under the nitrogen protection environment, and performing crosslinking treatment, cleaning and storage to obtain the nano iron/single microorganism composite microbial inoculum. 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
Technical Field
The invention relates to the field of triclosan wastewater treatment, in particular to an embedded nano-iron/single microorganism composite 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/single microorganism composite 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 surface activity of nano iron particles can be utilized, the stability and activity of the microorganisms can be ensured, and 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/single microorganism composite microbial inoculum comprises the following steps:
(1) preparation of the thallus:
selecting a Pseudomonas stutzeri (Pseudomonas stutzeri) 2 ring, transferring the 2 ring into a nutrient solution, culturing bacteria at 35-37 ℃ for 1-3 days, inoculating the bacteria to a proliferation culture medium in 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 to obtain pseudomonas stutzeri thallus for triclosan degradation, and marking as thallus A;
(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 50-60 ℃ 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/single microorganism composite 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/single-microorganism composite microbial inoculum.
For further achieving the purpose of the invention, preferably, the nutrient solution of the pseudomonas stutzeri is 6.0g/L of beef extract, 5.0g/L of NaCl5.0g/L, 10.0g/L of peptone, 2.0g/L of soybean meal, pH 6.5 and the balance of water.
Preferably, the proliferation medium of the pseudomonas stutzeri is 20.0g/L of casein, 3.0g/L of potassium hydrogen phosphate, 3.0g/L of glucose, 4.0g/L of soybean meal, 5.0g/L of sodium chloride and the balance of water.
Preferably, the phosphate buffer solution comprises 9.0g/L of sodium chloride, 0.3g/L of potassium chloride, 1.2g/L of dipotassium hydrogen phosphate and 0.3g/L of monopotassium phosphate in percentage by volume, and the balance of water.
Preferably, the method for preserving the embedded nano-iron/single microorganism composite microbial inoculum is to soak in sterile normal saline and preserve in a refrigerator at 4 ℃; the Pseudomonas stutzeri 2 loop was transferred to 30-40 mL of nutrient solution.
Preferably, the centrifugation treatment in step 1) is centrifugation at 4000-.
Preferably, the number of phosphate buffer washes of step 1) is 1-2;
preferably, the washing in step 5) is washing with 0.8-1.2% NaCl solution.
Preferably, the crosslinking treatment in the step 5) is crosslinking for 10-36 h at 4-6 ℃.
An embedded nano-iron/single microorganism composite microbial agent for degrading triclosan is prepared by the preparation method.
Compared with the prior art, the invention has the following advantages:
1) according to the embedded nano-iron/single microorganism composite 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 composite microbial agent can generate a synergistic effect to jointly promote the degradation of the triclosan. Compared with a single microorganism experiment and a single nano-iron experiment under the same experiment conditions, the compound microbial inoculum prepared by the invention can be verified to generate a synergistic effect to jointly promote the degradation of triclosan.
2) The embedding agent agar adopted by the invention has wide raw material source, low price, no toxicity and good biocompatibility, the prepared microbial inoculum has high strength and low toxicity of microorganisms, can be repeatedly utilized for 4-5 times, solves the problem of instability of the microorganisms, and enhances the degradation efficiency of the triclosan by the synergistic effect formed by the nano iron and the microorganisms, thereby being 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.
Example 1
(1) Preparation of triclosan degradation bacterium liquid
Picking 2 rings of Pseudomonas stutzeri, transferring the 2 rings into 30mL of nutrient solution, culturing bacteria at 35 ℃ for 2 days, inoculating the bacteria into a container of a proliferation culture medium of Pseudomonas stutzeri in a volume ratio of 10%, culturing at 35 ℃ for 2 days, centrifuging at 5000rpm for 15min, and obtaining cells of the bacteria in a logarithmic growth phase;
taking out cells in logarithmic growth phase of the above thallus, washing with phosphate buffer solution (its main components are 9.0g/L sodium chloride, 0.3g/L potassium chloride, 1.2g/L dipotassium hydrogen phosphate and 0.3g/L potassium dihydrogen phosphate, and the rest is water) for 2 times, suspending in physiological saline, and refrigerating at 4 deg.C for use, and marking as thallus A;
the nutrient solution of the pseudomonas stutzeri comprises 6.0g/L of beef extract, 5.0g/L of NaCl5, 10.0g/L of peptone, 2.0g/L of soybean meal, pH 6.5 and the balance of water.
The proliferation culture medium of Pseudomonas stutzeri comprises casein 20.0g/L, potassium hydrogen phosphate 3.0g/L, glucose 3.0g/L, soybean powder 4.0g/L, sodium chloride 5.0g/L, and water in balance.
(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 0.4g/L was prepared and is noted 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 SiO2SiO of the2The mass concentration of the mixture is 1mg/L, and the mixture is alternately cooled to 55 ℃ after being mixed, and is marked as solution C;
(4) preparation of cross-linking agent aluminum sulfate saturated boric acid solution
Dissolving aluminum sulfate powder in saturated boric acid solution to obtain saturated boric acid solution of aluminum sulfate with the molar concentration of 0.5mol/L, and marking as solution D.
(5) Preparation of embedded microbial inoculum
Under the condition of constant temperature water bath at 60 ℃, 15 percent of 0.1mg/L solution B and 6 percent of thallus A are respectively added into 57 percent of solution C (containing 5 percent of agar and 7.5 percent of PVA in percentage by mass and 1mg/LSiO in percentage by volume2) And then, uniformly stirring and mixing, dropwise adding the mixture into a 22% solution D (room temperature) under the nitrogen protection environment, crosslinking for 10 hours at 4 ℃, and then cleaning and storing the solution by using a 0.9 wt% NaCl solution to obtain the nano-iron/single microorganism composite microbial inoculum.
(6) Degradation effect of triclosan pollution
3mg of the prepared nano-iron/single microorganism composite microbial agent is put into nutrient solution for culturing for 6h, 10L of triclosan simulated polluted wastewater with the concentration of 5mg/L is directly put into the activated nano-iron/single microorganism composite microbial agent after activation, the aeration treatment is carried out for 5d, and the aeration amount is 2L/h. According to mass concentration, the nutrient solution comprises 6.0g/L of beef extract, 5.0g/L of NaCl5, 10.0g/L of peptone, 2.0g/L of soybean meal, pH 6.5 and the balance of water.
The control group used a single pseudomonas stutzeri under the same experimental conditions, but did not form inclusion bodies with the nano-iron.
Triclosan was measured by Waters high performance liquid chromatography with the following column: waters C18Columns (150 × 4.6mmi.d., 5 μm); column temperature of 35 ℃, acetonitrile/water (75:25, v/v) as a mobile phase, total flow rate of 1.0mL/min, sample amount of 10 muL, and detection wavelength of 230 nm. The peak time of the target substance is about 7.2min, and the total detection time of the sample is 12 min. And obtaining the triclosan removal rate by testing the initial concentration C0 and the concentration Ct after reaction of the triclosan in the water sample.
By adopting the method of the embodiment, 2g of nano-iron/single microorganism composite microbial inoculum is added into 5L of wastewater containing 5mg/L of triclosan, and the microbial inoculum is directly put into a nutrient solution (see the embodiment) for culturing for 6h and is directly put into use after being activated. After 5 days of aeration treatment, the removal rate of the triclosan reaches 83 percent, which is obviously higher than 20 percent of a control group of a single strain (the single Pseudomonas stutzeri under the same experimental conditions does not form an embedded body with the nano-iron), and the embedded nano-iron/single microorganism composite microbial agent has a good degradation effect on the triclosan. Because the nano iron has strong reducibility to the triclosan and adsorbability to microorganisms, and the nano iron can act with the cytochrome c of mitochondria of the microorganisms to change the oxidation-reduction potential of the cytochrome c and enhance the electron transfer capability, the compound microbial inoculum can generate a synergistic effect to jointly promote the degradation of the triclosan.
The embedded nano-iron/single microorganism composite microbial inoculum prepared by the invention has advantages in degrading triclosan, can utilize the high specific surface area and surface activity of nano-iron particles, can also ensure the stability and activity of microorganisms, is suitable for in-situ remediation and has no secondary pollution, and the prepared embedded microbial inoculum can be put into use only by simple activation, thereby having the prospect of large-scale industrial production.
Example 2
(1) Preparation of triclosan degradation bacterium liquid
Picking 2 rings of Pseudomonas stutzeri (from the same source as in example 1), transferring the rings into 30mL of nutrient solution, culturing bacteria at 35 ℃ for 2 days, inoculating the bacteria into a container of proliferation culture medium of Pseudomonas stutzeri at a volume ratio of 10%, culturing at 35 ℃ for 2 days, centrifuging at 5000rpm for 15min, and obtaining cells of the bacteria in logarithmic growth phase;
taking out cells in logarithmic growth phase of the above thallus, washing with phosphate buffer solution (its main components are 9.0g/L sodium chloride, 0.3g/L potassium chloride, 1.2g/L dipotassium hydrogen phosphate and 0.3g/L potassium dihydrogen phosphate, and the rest is water) for 2 times, suspending in physiological saline, and refrigerating at 4 deg.C for use, and marking as thallus A;
the nutrient solution of the pseudomonas stutzeri comprises 6.0g/L of beef extract, 5.0g/L of NaCl5, 10.0g/L of peptone, 2.0g/L of soybean meal, pH 6.5 and the balance of water.
The proliferation culture medium of Pseudomonas stutzeri comprises casein 20.0g/L, potassium hydrogen phosphate 3.0g/L, glucose 3.0g/L, soybean powder 4.0g/L, sodium chloride 5.0g/L, and water in balance.
(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 Fe0Preparation of a concentration of 0.4g/LNano-iron solution, noted 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 mixed solution in the mixture is 2mg/L, and the mixed solution is alternately cooled to 55 ℃ and is marked as solution C;
(4) preparation of cross-linking agent aluminum sulfate saturated boric acid solution
Dissolving aluminum sulfate powder in saturated boric acid solution to obtain saturated boric acid solution of aluminum sulfate with the 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 0.4mg/L solution B and 10 percent of thallus A are respectively added into 61 percent of solution C (containing 7 percent of agar, 11.5 percent of PVA and 2mg/L of SiO in percentage 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 23 hours at the temperature of 4 ℃, and then cleaning and storing the mixture by using a 0.9 wt% NaCl solution to obtain the nano-iron/single microorganism composite microbial inoculum.
(6) Degradation effect of triclosan pollution
3mg of the prepared nano-iron/single microorganism composite microbial agent is put into a nutrient solution medium for culturing for 6 hours, 10L of triclosan simulated polluted wastewater with the concentration of 5mg/L is directly put into the activated nano-iron/single microorganism composite microbial agent after activation, aeration treatment is carried out for 5 days, and the aeration amount is 2L/h. According to mass concentration, the nutrient solution comprises 6.0g/L of beef extract, 5.0g/L of NaCl5, 10.0g/L of peptone, 2.0g/L of soybean meal, 6.5 of pH and the balance of water.
Triclosan was measured by Waters high performance liquid chromatography with the following column: waters C18Columns (150 × 4.6mmi.d., 5 μm); column temperature of 35 ℃, acetonitrile/water (75:25, v/v) as a mobile phase, total flow rate of 1.0mL/min, sample amount of 10 muL, and detection wavelength of 230 nm. The peak time of the target substance is about 7.2min, and the total detection time of the sample is 12 min. By testing the initial concentration C of triclosan in a water sample0And the concentration Ct after the reaction to obtainTo the triclosan removal rate.
By adopting the method of the embodiment, 2g of nano-iron/single microorganism composite microbial inoculum is added into 5L of wastewater containing 5mg/L of triclosan; after activation, the microbial inoculum was directly added to the nutrient solution (see this example) and cultured for 6h, and then directly added for use. After 5 days of aeration treatment, the removal rate of the triclosan reaches 83 percent, which is obviously higher than 28 percent of a control group of a single strain (the single Pseudomonas stutzeri under the same experimental conditions does not form an embedded body with the nano-iron), and the embedded nano-iron/single microorganism composite microbial inoculum has a good degradation effect on the triclosan. Because the nano iron has strong reducibility to the triclosan and adsorbability to microorganisms, and the nano iron can act with the cytochrome c of mitochondria of the microorganisms to change the oxidation-reduction potential of the cytochrome c and enhance the electron transfer capability, the compound microbial inoculum can generate a synergistic effect to jointly promote the degradation of the triclosan.
Triclosan was measured by Waters high performance liquid chromatography with the following column: waters C18Columns (150 × 4.6mmi.d., 5 μm); column temperature of 35 ℃, acetonitrile/water (75:25, v/v) as a mobile phase, total flow rate of 1.0mL/min, sample amount of 10 muL, and detection wavelength of 230 nm. The peak time of the target substance is about 7.2min, and the total detection time of the sample is 12 min.
Example 3
(1) Preparation of triclosan degradation bacterium liquid
Picking 2-ring Pseudomonas stutzeri (from the same source as in example 1), transferring the 2-ring Pseudomonas stutzeri (from the same source as in example 1) into 30mL of nutrient solution, culturing bacteria at 35 ℃ for 2 days, inoculating the bacteria into a container of a proliferation medium in a volume ratio of 10%, culturing the bacteria at 35 ℃ for 2 days, and centrifuging the bacteria at 5000rpm for 15min to obtain cells of the logarithmic growth phase of the bacteria;
the cells of the above cells in the logarithmic growth phase were taken out, washed 2 times with a phosphate buffer (containing 9.0g/L sodium chloride, 0.3g/L potassium chloride, 1.2g/L dipotassium hydrogenphosphate, 0.3g/L potassium dihydrogenphosphate and the balance water), suspended in physiological saline, and refrigerated at 4 ℃ for future use. Marking as thallus A;
the nutrient solution of the pseudomonas stutzeri comprises 6.0g/L of beef extract, 5.0g/L of NaCl5, 10.0g/L of peptone, 2.0g/L of soybean meal, pH 6.5 and the balance of water.
The proliferation culture medium of Pseudomonas stutzeri comprises casein 20.0g/L, potassium hydrogen phosphate 3.0g/L, glucose 3.0g/L, soybean powder 4.0g/L, sodium chloride 5.0g/L, and water in balance.
(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 Fe0Preparing nano-iron solution with the concentration of 0.4 g/L. Denoted 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 3mg/L, and the mixture is alternately cooled to 55 ℃ after being mixed, and is marked as solution C;
(4) preparation of cross-linking agent aluminum sulfate saturated boric acid solution
Dissolving aluminum sulfate powder in saturated boric acid solution to obtain saturated boric acid solution of aluminum sulfate with the 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 0.6mg/L solution B and 6 percent of thallus A are respectively added into 66 percent of solution C (containing 9 percent of agar, 15 percent of PVA and 3mg/L of SiO in percentage by mass)2) And stirring and mixing uniformly. Dropwise adding the mixture into a 1% solution D (room temperature) under the nitrogen protection environment, crosslinking for 36h at 4 ℃, and then cleaning and storing the mixture by using a 0.9 wt% NaCl solution to obtain the nano-iron/single microorganism composite microbial inoculum.
(6) Degradation effect of triclosan pollution
3mg of the prepared nano-iron/single microorganism composite microbial agent is put into nutrient solution for culturing for 6h, 10L of triclosan simulated polluted wastewater with the concentration of 5mg/L is directly put into the activated nano-iron/single microorganism composite microbial agent after activation, the aeration treatment is carried out for 5d, and the aeration amount is 2L/h. According to mass concentration, the nutrient solution comprises 6.0g/L of beef extract, 5.0g/L of NaCl5, 10.0g/L of peptone, 2.0g/L of soybean meal, 6.5 of pH and the balance of water.
Triclosan was measured by Waters high performance liquid chromatography with the following column: waters C18Columns (150 × 4.6mmi.d., 5 μm); column temperature of 35 ℃, acetonitrile/water (75:25, v/v) as a mobile phase, total flow rate of 1.0mL/min, sample amount of 10 muL, and detection wavelength of 230 nm. The peak time of the target substance is about 7.2min, and the total detection time of the sample is 12 min. By testing the initial concentration C of triclosan in a water sample0And the concentration Ct after the reaction is obtained to obtain the removal rate of the triclosan
By adopting the method of the embodiment, 2g of nano-iron/single microorganism composite microbial inoculum is added into 5L of wastewater containing 5mg/L of triclosan; after activation, the microbial inoculum was directly added to the nutrient solution (see this example) and cultured for 6h, and then directly added for use. After 5 days of aeration treatment, the removal rate of the triclosan reaches 83 percent and is obviously higher than 21 percent of a control group of a single strain (a single Pseudomonas stutzeri under the same experimental conditions but does not form an embedded body with the nano-iron), which indicates that the embedded nano-iron/single microorganism composite microbial agent has a good degradation effect on the triclosan. Because the nano-iron has strong reducibility to triclosan, adsorbability to microorganisms and can act with cytochrome c of mitochondria of microorganisms, the oxidation-reduction potential of the cytochrome c is changed and the electron transfer capacity is enhanced. Therefore, the compound microbial inoculum can generate a synergistic effect to jointly promote the degradation of the triclosan.
Triclosan was measured by Waters high performance liquid chromatography with the following column: waters C18Columns (150 × 4.6mmi.d., 5 μm); column temperature of 35 ℃, acetonitrile/water (75:25, v/v) as a mobile phase, total flow rate of 1.0mL/min, sample amount of 10 muL, and detection wavelength of 230 nm. The peak time of the target substance is about 7.2min, and the total detection time of the sample is 12 min.
The invention overcomes the problem that a single strain is easily inhibited by intermediate metabolites in the process of degrading triclosan, and simultaneously, the nano-iron and the microorganism 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 microorganism 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 biochemical from bottom corner fusion [ J ]. Journal of Hazards Materials 2010, 180 (1-3): 764- > 764 ]. The degradation efficiency of the bacteria on the triclosan is obviously lower than that of the microbial inoculum of the invention, the embedded nano-iron single microbial inoculum prepared by the invention has advantages on the degradation of the triclosan, and the embedded agent can utilize the high specific surface area and surface activity of nano-iron particles, and can also ensure the stability and activity of microorganisms, and 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/single microorganism composite microbial inoculum is characterized by comprising the following steps:
(1) preparation of the thallus:
selecting a Pseudomonas stutzeri (Pseudomonas stutzeri) 2 ring, transferring the 2 ring into a nutrient solution, culturing bacteria at 35-37 ℃ for 1-3 days, inoculating the bacteria to a proliferation culture medium in 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 to obtain pseudomonas stutzeri thallus for triclosan degradation, and marking as thallus A;
(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 50-60 ℃ 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/single microorganism composite 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/single-microorganism composite microbial inoculum.
2. The method for preparing embedded nano-iron/single microorganism composite microbial inoculum according to claim 1, wherein the nutrient solution of pseudomonas stutzeri is beef extract 6.0g/L, NaCl5.0g/L, peptone 10.0g/L, soybean meal 2.0g/L, pH 6.5, and the balance water.
3. The method for preparing embedded nano-iron/single microorganism composite bacterial agent according to claim 1, wherein the proliferation culture medium of pseudomonas stutzeri comprises casein 20.0g/L, potassium hydrogen phosphate 3.0g/L, glucose 3.0g/L, soybean meal 4.0g/L, sodium chloride 5.0g/L, and the balance of water.
4. The preparation method of the embedded nano-iron/single microorganism composite microbial inoculum according to claim 1, wherein the phosphate buffer solution comprises 9.0g/L of sodium chloride, 0.3g/L of potassium chloride, 1.2g/L of dipotassium hydrogen phosphate and 0.3g/L of potassium dihydrogen phosphate, and the balance of water.
5. The method for preparing the embedded nano-iron/single microorganism composite microbial inoculum according to claim 1, wherein the method for storing the embedded nano-iron/single microorganism composite microbial inoculum is to soak in sterile physiological saline and store the immersed nano-iron/single microorganism composite microbial inoculum in a refrigerator at 4 ℃; the Pseudomonas stutzeri 2 loop was transferred to 30-40 mL of nutrient solution.
6. The method for preparing embedded nano-iron/single microorganism composite bacterial agent as claimed in claim 1, wherein the centrifugation treatment in step 1) is centrifugation at 4000-.
7. The method for preparing the embedded nano-iron/single microorganism composite bacterial agent according to claim 1, wherein the number of times of washing with the phosphate buffer solution in step 1) is 1-2.
8. The method for preparing the embedded nano-iron/single microorganism composite bacterial agent according to claim 1, wherein the washing in step 5) is washing with 0.8-1.2% NaCl solution.
9. The preparation method of the embedded nano-iron/single microorganism composite microbial inoculum according to claim 1, wherein the crosslinking treatment in the step 5) is crosslinking for 10-36 h at 4-6 ℃.
10. An embedded nano-iron/single microorganism composite microbial inoculum for degrading triclosan, which is characterized by being prepared by the preparation method of any one of claims 1 to 9.
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