CN105925560B - Embedded nano-iron/two microbial agents and preparation method thereof - Google Patents
Embedded nano-iron/two microbial agents and preparation method thereof Download PDFInfo
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- CN105925560B CN105925560B CN201610508394.0A CN201610508394A CN105925560B CN 105925560 B CN105925560 B CN 105925560B CN 201610508394 A CN201610508394 A CN 201610508394A CN 105925560 B CN105925560 B CN 105925560B
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 title claims abstract description 82
- 229910052742 iron Inorganic materials 0.000 title claims abstract description 59
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 28
- 230000000813 microbial effect Effects 0.000 title claims abstract description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 35
- 241000589614 Pseudomonas stutzeri Species 0.000 claims abstract description 28
- 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
- 241000589776 Pseudomonas putida Species 0.000 claims abstract description 21
- 229920001817 Agar Polymers 0.000 claims abstract description 19
- 239000008272 agar Substances 0.000 claims abstract description 19
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229910052681 coesite Inorganic materials 0.000 claims abstract description 17
- 229910052906 cristobalite Inorganic materials 0.000 claims abstract description 17
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 17
- 229910052682 stishovite Inorganic materials 0.000 claims abstract description 17
- 229910052905 tridymite Inorganic materials 0.000 claims abstract description 17
- 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
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 6
- 238000004140 cleaning Methods 0.000 claims abstract description 5
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 90
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- 235000015097 nutrients Nutrition 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 18
- 241000894006 Bacteria Species 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 13
- 239000011780 sodium chloride Substances 0.000 claims description 13
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 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
- 239000007791 liquid phase Substances 0.000 claims description 10
- 235000019764 Soybean Meal Nutrition 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 9
- 239000004455 soybean meal Substances 0.000 claims description 9
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 8
- 230000035755 proliferation Effects 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 235000015278 beef Nutrition 0.000 claims description 6
- 239000003638 chemical reducing agent Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 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
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- 239000001103 potassium chloride Substances 0.000 claims description 5
- 235000011164 potassium chloride Nutrition 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 230000009467 reduction Effects 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000012071 phase Substances 0.000 claims description 4
- 239000008363 phosphate buffer Substances 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
- 238000004321 preservation Methods 0.000 claims description 4
- 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 3
- 239000005018 casein Substances 0.000 claims description 3
- 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 3
- 235000021240 caseins Nutrition 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- XEFQLINVKFYRCS-UHFFFAOYSA-N Triclosan Chemical compound OC1=CC(Cl)=CC=C1OC1=CC=C(Cl)C=C1Cl XEFQLINVKFYRCS-UHFFFAOYSA-N 0.000 abstract description 63
- 229960003500 triclosan Drugs 0.000 abstract description 63
- 244000005700 microbiome Species 0.000 abstract description 33
- 239000002068 microbial inoculum Substances 0.000 abstract description 32
- 238000006731 degradation reaction Methods 0.000 abstract description 25
- 230000015556 catabolic process Effects 0.000 abstract description 24
- 230000002195 synergetic effect Effects 0.000 abstract description 11
- 238000003756 stirring Methods 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 241001052560 Thallis Species 0.000 abstract 1
- 238000003860 storage Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 15
- 239000002351 wastewater Substances 0.000 description 11
- 102100030497 Cytochrome c Human genes 0.000 description 10
- 108010075031 Cytochromes c Proteins 0.000 description 10
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N Iron oxide Chemical compound [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 238000005273 aeration Methods 0.000 description 9
- 230000000593 degrading effect Effects 0.000 description 8
- 230000003698 anagen phase Effects 0.000 description 7
- 239000002131 composite material Substances 0.000 description 7
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- 238000005067 remediation Methods 0.000 description 6
- 239000003643 water by type Substances 0.000 description 6
- 238000011065 in-situ storage Methods 0.000 description 5
- 210000003470 mitochondria Anatomy 0.000 description 5
- 230000033116 oxidation-reduction process Effects 0.000 description 5
- 230000027756 respiratory electron transport chain Effects 0.000 description 5
- 230000004913 activation Effects 0.000 description 4
- 239000003344 environmental pollutant Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
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- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000004128 high performance liquid chromatography 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
- 239000013076 target substance Substances 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
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004060 metabolic 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
- 238000001179 sorption measurement Methods 0.000 description 2
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 101100450149 Dictyostelium discoideum hatA gene Proteins 0.000 description 1
- 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
- 235000014413 iron hydroxide Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 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
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- 239000004753 textile Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
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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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- 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
- C12N11/04—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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- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
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- Engineering & Computer Science (AREA)
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- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
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- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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- Dispersion Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses embedded nano-iron/two microbial agents and a preparation method thereof. Firstly, preparing thalli A of two microbial agents by using a 2-ring of Pseudomonas putida (Pseudomonas putida) and Pseudomonas stutzeri (Pseudomonas stutzeri); preparing nano iron solution B, preparing embedding agent agar, PVA and SiO2Preparing a crosslinking agent aluminum sulfate saturated boric acid solution D; and 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, dripping the solution A into a 1-22% solution D at room temperature under the nitrogen protection environment, and performing crosslinking treatment, cleaning and storage to obtain the nano-iron/two microbial agents. The invention utilizes the synergistic effect between the nano-iron and the microorganism to improve the degradation efficiency of the triclosan, and the prepared microbial inoculum has high strength, small toxicity of the microorganism and low price of raw material sources, and can be widely used for treating the water body polluted by the triclosan.
Description
Technical Field
The invention relates to the field of triclosan wastewater treatment, in particular to an embedded nano-iron/two microbial agents 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/two microbial agents for degrading triclosan, which has obvious triclosan degradation effect and no secondary pollution, 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/two microbial agents comprises the following steps:
(1) preparation of the thallus:
picking 2 rings of Pseudomonas putida (Pseudomonas putida.) and Pseudomonas stutzeri (Pseudomonas stutzeri.), respectively transferring into a nutrient solution, culturing bacteria at 35-37 ℃ for 1-3 days, inoculating 5-18% of the bacteria in a container containing a proliferation culture medium in volume proportion, 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, mixing 37-45% of pseudomonas putida and 55-63% of pseudomonas stutzeri by volume percentage to obtain pseudomonas stutzeri thallus used 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 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) preparation of nano-iron/two microbial agents:
under the condition of a constant-temperature water bath at 50-70 ℃, 15-18% of solution B and 6-15% of thallus A are respectively added into 57-66% of solution C according to the volume ratio, stirred and mixed uniformly, and are dripped into 1-22% of solution D at room temperature under the nitrogen protection environment, and the nano-iron/two microbial agents are obtained after cross-linking treatment, cleaning and preservation.
To further achieve the purpose of the invention, preferably, the main components of the nutrient solution are 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.
Preferably, the main components of the proliferation medium are 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 preservation refers to soaking in sterile physiological saline and storing in a refrigerator at 4 ℃.
Preferably, the Pseudomonas putida (Pseudomonas putida.) and Pseudomonas stutzeri (Pseudomonas stutzeri.)2 rings are transferred to 30-40mL of nutrient solution, 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/two microbial agents is prepared by the preparation method.
Compared with the prior art, the invention has the following advantages:
1) according to the embedded nano-iron/two-microorganism composite microbial agent, the strong reducibility of nano-iron to triclosan and the adsorbability of nano-iron to microorganisms are utilized, 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, the composite microbial agent can generate a synergistic effect to jointly promote the degradation of the triclosan, and compared with a single microorganism experiment and a single nano-iron experiment under the same experiment condition, the prepared composite microbial agent can generate the synergistic effect to jointly promote the degradation of the triclosan, and the effect is obvious.
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, and simultaneously solves the problem of adsorption on inorganic porous SiO2The material has the problem of instability of microorganisms, and the nano iron and the microorganisms form a synergistic effect to enhance the degradation efficiency of the triclosan, so that 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, the main components of the nutrient solution in the step 1) and the step 6) are 6.0g/L of beef extract, 5.0g/L of NaCl5.0g/L, 10.0g/L of peptone, 2.0g/L of soybean meal, 6.5 of pH and the balance of water.
The main components of the proliferation culture medium are 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.
Example 1
(1) Preparation of triclosan degradation bacterium liquid
Picking 2 rings of Pseudomonas putida (Pseudomonas putida.) and Pseudomonas stutzeri (Pseudomonas stutzeri.), transferring into 30mL of nutrient solution, culturing bacteria at 35 deg.C for 2 days, inoculating into proliferation medium at 10% volume ratio, culturing at 35 deg.C for 2 days, centrifuging at 5000rpm for 15min, and obtaining cells of logarithmic growth phase of the bacteria.
The cells of the above cells in the logarithmic growth phase were taken out and washed 2 times with a phosphate buffer (containing 9.0g/L of sodium chloride, 0.3g/L of potassium chloride, 1.2g/L of dipotassium hydrogenphosphate and 0.3g/L of potassium dihydrogenphosphate as the main components, and the balance water). According to the volume percentage, 37 percent of pseudomonas putida and 63 percent of pseudomonas stutzeri are mixed to obtain pseudomonas stutzeri thallus for degrading triclosan. The cells were suspended in physiological saline and refrigerated at 4 ℃ for future use. Marking as thallus 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 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 completely in clear water to obtain solution containing agar 5 wt% and PVA 7.5 wt%, and adding SiO2Control of SiO2The 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 0.5 mol/L. Denoted as solution D.
(5) Preparation of embedded microbial inoculum
Under the condition of a 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 according to the volume ratio, and are stirred and mixed evenly. Dropwise adding the mixture into a room-temperature 22% solution D (the sum of the amounts of A, B, C, D is 100%) under the protection of nitrogen, crosslinking for 10 hours at 4 ℃, and then washing and storing the solution by using a 0.9 wt% NaCl solution to obtain the nano-iron/microorganism (two) microbial inoculum.
(6) Degradation effect of triclosan pollution
3mg/L of the prepared nano-iron two-microorganism composite microbial inoculum 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 two-microorganism composite microbial inoculum after the 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.
Triclosan was measured by Waters high performance liquid chromatography with the following column: waters C18A column (150 × 4.6mm i.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 post-reaction concentration CtAnd obtaining the triclosan removal rate.
2g of embedded nano-iron/two microbial agents are added into 5L of triclosan wastewater with 5mg/L of water treated by the method in the embodiment; the microbial inoculum is directly put into nutrient solution for culturing for 6h, and is directly put into use after being activated, the removal rate of the triclosan after 5d of aeration treatment reaches 87 percent, which is obviously higher than that of a control group of a single strain (Pseudomonas stutzeri), and the removal rate is 24 percent, which shows that the embedded nano iron/two microbial inoculum has good degradation effect on the triclosan and is obviously better than that of the single strain. 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, the oxidation-reduction potential of the cytochrome c is changed, the electron transfer capacity is enhanced, and the composite microbial inoculum can generate a synergistic effect to jointly promote the degradation of the triclosan.
The embedded nano-iron/two microbial agents prepared by the invention have 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, are suitable for in-situ remediation, have no secondary pollution, can be put into use after being simply activated, and have the prospect of large-scale industrial production.
Example 2
(1) Preparation of triclosan degradation bacterium liquid
Pseudomonas putida (Pseudomonas putida.) and Pseudomonas stutzeri (Pseudomonas stutzeri.)2 rings were picked up, transferred to 30mL of 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 cells of the cells in the logarithmic growth phase.
The cells of the above cells in the logarithmic growth phase were taken out and washed 2 times with a phosphate buffer (containing 9.0g/L of sodium chloride, 0.3g/L of potassium chloride, 1.2g/L of dipotassium hydrogenphosphate and 0.3g/L of potassium dihydrogenphosphate as the main components, and the balance water). According to the volume percentage, 37 percent of pseudomonas putida and 63 percent of pseudomonas stutzeri are mixed to obtain pseudomonas stutzeri thallus for degrading triclosan. The cells were suspended in physiological saline and refrigerated at 4 ℃ for future use. Marking as thallus 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 0.4g/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 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 23h at 4 ℃, and then cleaning and storing the mixture by using a 0.9 wt% NaCl solution to obtain the nano iron/microorganism (two) microbial inoculum.
(6) Degradation effect of triclosan pollution
3mg/L of the microbial inoculum is taken and 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 microbial inoculum, and 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.
2g of embedded nano-iron/two microbial agents are added into 5L of triclosan wastewater with 5mg/L of water treated by the method in the embodiment; the microbial inoculum is directly put into nutrient solution for 6h for culture and directly put into use after activation, the removal rate of the triclosan after 5d of aeration treatment reaches 96 percent and is obviously higher than that of a control group of a single strain (Pseudomonas stutzeri), which shows that the embedded nano-iron/microorganism (two) microbial inoculum has good degradation effect on the triclosan and is obviously superior to the single strain. 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 C18A column (150 × 4.6mm i.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 rings of Pseudomonas putida (Pseudomonas putida.) and Pseudomonas stutzeri (Pseudomonas stutzeri.), transferring into 30mL of nutrient solution, culturing bacteria at 35 deg.C for 2 days, inoculating into proliferation medium container at 10%, culturing at 35 deg.C for 2 days, centrifuging at 5000rpm for 15min, and obtaining cells of the cells in logarithmic growth phase.
The cells of the above cells in the logarithmic growth phase were taken out and washed 2 times with a phosphate buffer (containing 9.0g/L of sodium chloride, 0.3g/L of potassium chloride, 1.2g/L of dipotassium hydrogenphosphate and 0.3g/L of potassium dihydrogenphosphate as the main components, and the balance water). According to the volume percentage, 37 percent of pseudomonas putida and 63 percent of pseudomonas stutzeri are mixed to obtain pseudomonas stutzeri thallus for degrading triclosan. The cells were suspended in physiological saline and refrigerated at 4 ℃ for future use. Marking as thallus 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 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 ℃,18 percent of 0.6mg/L solution B and 15 percent of thallus A are respectively added into 66 percent of thallus A according to volume ratio% of solution C (containing 9% agar, 15% PVA, 3mg/L SiO by mass percent)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/microorganism (two) microbial inoculum.
(6) Degradation effect of triclosan pollution
3mg/L of the microbial inoculum is taken and 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 microbial inoculum, and 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.
The method is adopted to treat 5mg/L of triclosan wastewater in the water, and 2g of two microorganism compound microbial agents of nano-iron are added into the 5L of triclosan wastewater; after activation, the microbial inoculum is directly put into nutrient solution for culturing for 6h and directly put into use. The removal rate of the triclosan after 5 days of aeration treatment reaches 95 percent and is obviously higher than that of a control group of a single strain (pseudomonas stutzeri) by 24 percent, which shows that the embedded nano-iron/microorganism (two) microbial inoculum has good degradation effect on the triclosan and is obviously better than that of the single strain. 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 C18A column (150 × 4.6mm i.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.
In the invention, the nano iron and the microorganism have synergistic effect, the nano iron has strong reducibility to triclosan and adsorbability to the microorganism, and the nano iron can act with cytochrome c of mitochondria of the microorganism to change the oxidation-reduction potential of the cytochrome c and enhance the electron transfer capability. Therefore, 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 triclosan degradation process is solved, and simultaneously, 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, the 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 remediation 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 degrading triclosan; the embedding agent can utilize the high specific surface area and surface activity of the nano iron particles, can also ensure the stability and activity of microorganisms, and the prepared embedding 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/two microbial agents is characterized by comprising the following steps:
(1) preparation of the thallus:
selecting Pseudomonas putida (B)Pseudomonas putida.) And Pseudomonas stutzeri (Pseudomonas stutzeri.)2, respectively transferring the cells into nutrient solution, culturing the bacteria at 35-37 ℃ for 1-3 days, inoculating the bacteria into a container containing 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 logarithmic phase; washing with phosphate buffer solution, and mixing 37-45% of pseudomonas putida and 55-63% of pseudomonas stutzeri by volume percentage to obtain thallus A of two microbial agents;
(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) preparation of nano-iron/two microbial agents:
under the condition of a constant-temperature water bath at 50-70 ℃, 15-18% of solution B and 6-15% of thallus A are respectively added into 57-66% of solution C according to the volume ratio, the mixture is stirred and mixed uniformly, the mixture is dripped into 1-22% of solution D at room temperature under the nitrogen protection environment, and then crosslinking treatment, cleaning and preservation are carried out to obtain the nano-iron/two microbial agents.
2. The preparation method of claim 1, wherein the nutrient solution comprises beef extract 6.0g/L, NaCl5.0g/L, peptone 10.0g/L, soybean meal 2.0g/L, pH 6.5, and water in balance.
3. The method according to claim 1, wherein the proliferation medium 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 water in balance.
4. The method according to claim 1, wherein the phosphate buffer comprises, in terms of volume percentage, 9.0g/L of sodium chloride, 0.3g/L of potassium chloride, 1.2g/L of dipotassium hydrogenphosphate and 0.3g/L of potassium dihydrogenphosphate, and the balance of 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 said Pseudomonas putida (P), (B) and (C)Pseudomonas putida.) And Pseudomonas stutzeri (Pseudomonas stutzeri.) Transfer 2 rings to 30-40mL of nutrient solution, 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/two microbial agents, characterized in that it is prepared by the preparation method of any one of claims 1-9.
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| CN101993868A (en) * | 2010-09-30 | 2011-03-30 | 南开大学 | Embedded nano iron/microbe microspheres and preparation method thereof |
| CN103013885A (en) * | 2012-12-26 | 2013-04-03 | 重庆绿色智能技术研究院 | Benzene composite degrading microbe and immobilized benzene composite microbial inoculum, and preparation method thereof |
| CN103862037A (en) * | 2014-02-27 | 2014-06-18 | 浙江大学 | Preparation method and preprocessing method of biomaterial-embedded zero-valent-iron-ferroferric-oxide double-nanometer system |
| CN105132402A (en) * | 2015-07-18 | 2015-12-09 | 常州大学 | Preparation method of microbial macromolecular balls for nitrogen removal from wastewater |
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| CN101993868A (en) * | 2010-09-30 | 2011-03-30 | 南开大学 | Embedded nano iron/microbe microspheres and preparation method thereof |
| CN103013885A (en) * | 2012-12-26 | 2013-04-03 | 重庆绿色智能技术研究院 | Benzene composite degrading microbe and immobilized benzene composite microbial inoculum, and preparation method thereof |
| CN103862037A (en) * | 2014-02-27 | 2014-06-18 | 浙江大学 | Preparation method and preprocessing method of biomaterial-embedded zero-valent-iron-ferroferric-oxide double-nanometer system |
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