CN105779356B - A kind of microorganism self-assembled nanometer material and its preparation method and application - Google Patents
A kind of microorganism self-assembled nanometer material and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of microorganism self-assembled nanometer materials and its preparation method and application.Specifically, the present invention can efficiently remove phosphorus in high-salt wastewater using one plant under aerobic condition, while raw material synthesizes bacterium Shewanella (Shewanella sp.) CF8 6 of calcium phosphorus nano particle under the conditions of low saturation in graywater, calcium phosphorus nano material has been synthesized while waste water dephosphorization, and calcium phosphorus nano particle is especially synthesized under the conditions of phosphorus low saturation.This method and its preparation-obtained nano material play an important roll in removal environmental pollution and biomedicine etc. fields.
Description
Technical field
The invention belongs to field of nano material preparation, and in particular to a kind of microorganism self-assembled nanometer material and its preparation side
Method and application.
Background technology
Nano material refers at least one-dimensional in nanoscale range or substantially single by their conducts in three-dimensional material
The material that member is constituted.Nano material has some unique characteristics such as skin effect, small-size effect, quantum effect, to quilt
It is widely used in the industries such as the energy, catalysis, biosensor, biomedicine.The side of traditional physical chemistry nano materials
Method mainly has a two phase process, reverse microemulsion method, photochemical syntheses method, electrolysis method and heating, ultrasonic method etc., but this
A little traditional synthetic methods have various defects for being difficult to overcome, and such as cost of material height, energy consumption is high, severe reaction conditions or difficulty
In large-scale production, while chemical synthesis process generally requires presoma, however these presomas are expensive and may have
Toxicity, and chemical synthesis process is required for carrying out in the solution of high saturation, needing to add a large amount of chemical reagent causes into
This is higher, these defects all significantly limit the application of chemical method.In contrast, biosynthesis nano material has clearly
It is clean, reaction condition is mild, the advantages such as at low cost, easy to operate, and the nano material of biosynthesis has good dispersibility, steady
The advantages that qualitative, biocompatibility and controllability, has become domestic and international research hotspot.What is had now been found that there is synthesis to receive
Rice material microbe species it is very limited, mainly include prokaryotes and eucaryote, as bacterium, saccharomycete, it is certain virus from
Son, fungi and plant etc. have the ability of extracellular, intracellular synthesis or nanoassemble, while also having and utilizing plant extracts individually
And the report of the nano materials such as natural polysaccharide marine polysaccharide.But the nanometer material synthesized using biological method reported
Material focus primarily upon noble metal include and nano metal sulfide material in terms of, such as golden (Au), silver-colored (Ag), platinum (Pt) and cadmium sulfide
(CdS), cadmium selenide (CdSe) etc..However about the bacterial strain report of biosynthesis calcium phosphorus nano particle especially nanometer hydroxyapatite
Road is less.
Nanometer hydroxyapatite is had been to be concerned by more and more people due to it in fields such as environment, biological medicines.
(1) Heavy-Metal-Contaminated Environments reparation：Heavy metal element can occur with sulphur atom, the nitrogen-atoms on amino acid side chain
Effect has very high toxicity.The existing restorative procedure of Heavy-Metal-Contaminated Environments mainly has peripheral doses, chemical remediation and biology to repair
Multiple method.Wherein chemical remediation needs to add chemical agent to pollution environment such as soil, water body etc. is additional and make heavy metal ion
Occur absorption, redox reaction, precipitate the effects that, the method although it is easy to operate it is with obvious effects be also easy to produce secondary pollution
And it is costly.It is a kind of effective method to Heavy-Metal-Contaminated Environments repair using phosphorated material.Document in recent years
The phosphate mineral of the biosynthesis of report such as zinc-phosphorus and uranyl phosphate etc. can adsorb a series of metal, and have
There are the potentiality that can be operated within the scope of wider pH.
(2) biological medicine utilizes：Nano material has great answer in life sciences such as biomedical pharmacy, human healths
With as conveyed drug to lesions position, using nano material as biomedical checkout and diagnosis using nano particle as carrier
With material etc..Synthos such as hydroxyapatite is the host inorganic mineralogical composition of animal and skeleton and tooth, is had good
Good activity and biocompatibility, such as hydroxylapatite ceramic is a kind of very promising artificial bone and artificial oral-cavity material.
There is one plant of Serratieae Serratia sp. of document report that can synthesize difference under different condition of culture
The hydroxyapatite nanoparticle of grain size and characteristic, the Serratieae Serratia sp. can only be in the solution (P of high saturation：
Calcium phosphorus nano particle 5mM) and in biological buffer is generated, and the generation of such calcium phosphorus nano particle is not stricti jurise
On biosynthesis, but a kind of biodegradation process, the Serratieae Serratia sp. are a kind of atypical by generating
Acid phosphatase decomposes the sodium glycero-phosphate in matrix to release a large amount of inorganic phosphate radical ions, the phosphate radical of high concentration
Ion forms hydroxyapatite nanoparticle with calcium ion in cell surface or extracellular polymeric, it is then applied to water
The removal of radionuclide in solution.However the condition of its formation nano particle is still more harsh.How by simple method
It is effective to obtain calcium phosphorus nano material, it is still the technical barrier faced in the art.
Nano material is assembled into the macro-scale material with hierarchical organization as micro-nano structure unit can make its generation
Superior whole collaboration property is the effective way for improving nano material actual application ability.In recent years, oneself has developed more
Kind packaging strategy, such as electrochemical deposition method, surface-functionalized method and micro-embossing technology etc..But there are equipment requirement height, reaction items
Part is harsh, the shortcomings of easily causing secondary pollution and is of high cost.Therefore, a kind of efficient, low cost of development, environmental-friendly group accommodate
The technology of meter Dan Yuan prepares the material with certain structure and function for solving the problems, such as nano material in practical applications
It is of great significance.
Compared with physical-chemical process, by microbial technique be applied to nano material self assembly have it is environmental-friendly, it is at low cost,
Mild condition and simple operation and other advantages.There is document report to utilize microorganism secretion bacteria cellulose induced oxidation graphene nano
The compound ball material of piece assembling biology, Sr is realized using carbonate mineralized bacterium2+Immobilization etc..But it is had no so far about utilization
The report of microorganism self-assembled nanometer calcium phosphate material, therefore realize that the self assembly of nano-calcium-phosphor material is still by microbial technique
This field needs the technical barrier solved.
Invention content
Inventor is found surprisingly that in the research of biological phosphate-eliminating bacterial strain, efficient dephosphorization bacterium Shewanella strain
(Shewanella sp.) CF8-6 can be self-assembly of nano particle in incubation around thalline, the Shewanella
(Shewanella sp.) CF8-6 is preserved in China typical culture collection center, preservation address on March 29th, 2016
For：Luojiashan, Wuchang, Wuhan City, Hubei Province, preserving number are：CCTCC M 2016154.The bacterial strain can pollute in removing waste water
Calcium phosphorus is formed using raw material in waste water to precipitate, so it can be applied to the reparation of Heavy-Metal-Contaminated Environments, have while object
Easy to operate and lower-cost advantage.
Specifically, the present invention relates to following technical schemes：
First, one of the objects of the present invention is to provide Shewanella strain (Shewanella sp.) CF8-6 to be received in preparation
Application in rice material.
Shewanella strain (Shewanella sp.) CF8-6 of the present invention can prepare calcium phosphorus by routine culture mode
Nano particle (nanometer hydroxyapatite) forms calcium phosphorus nano particle especially with raw material in waste water.
Secondly, the present invention provides a kind of preparation method of microorganism self-assembled nanometer material, including：
(1) by Shewanella strain (Shewanella sp.) CF8-6 activation cultures, activation bacterium solution is made；
(2) activation bacterium solution is inoculated into phosphorous cultivating system and is cultivated, obtained nano particle and be distributed in somatic cells
Surface and surrounding microorganism nanoassemble system；
(3) separation obtains nano material.
The phosphorous cultivating system of the present invention can be phosphorus-containing wastewater, and bacterial strain of the present invention is in high P content waste water and low
Calcium phosphorus nano-calcium-phosphor particle can be formed in phosphorus-containing wastewater and can be self-assembled into the effigurate nano material of tool.
In the preparation method of the microorganism self-assembled nanometer material, it is preferred that step (1) is by bacterial strain Shewanella
Sp.CF8-6, at 25 DEG C, is cultivated 24 hours in seawater LB liquid medium under conditions of 200rpm, and activation bacterium solution is made；Seawater
The group of LB liquid medium becomes：1% peptone, 0.3% yeast powder, Chen Haishui are prepared.
Preferably, step (2) is that will tap into phosphorus-containing wastewater in 10% ratio after bacterium solution centrifuge washing that step (1) obtains
In, at 25 DEG C, is cultivated 48 hours under conditions of 200rpm, centrifuge and be distributed in somatic cells surface and surrounding microorganism to obtain the final product
Nano particle and its self-assembly system.
Preferably, centrifugal rotational speed 4000rpm, time are 10 minutes, (when centrifugation bacterium solution volume difference, when required centrifugation
Between it is different, but rotating speed is 4000rpm and following), centrifugal rotational speed is controlled, to prevent the nano particle of phage surface and surrounding
Fall off.
Phosphorus-containing wastewater can be actual domestic wastewater in above-mentioned preparation method, such as seawater toilet-flushing waste water；Wherein adopted in the present invention
With simulated wastewater, simulation high-salt wastewater formula (one) (low-phosphorous waste water)：C6H12O6·H2O 1.5g/L, CH3COONa 0.75g/L,
MgSO4·7H2O 1.18g/L,NH4Cl 0.9g/L,KH2PO4·2H2O 0.066g/L are (in terms of P:10mg/L),NaCl 30g/
L, with originally water dissolution.
Simulate high-salt wastewater formula (two) (high phosphorus waste water):C6H12O6·H2O 1.5g/L, CH3COONa 0.75g/L,
MgSO4·7H2O 1.18g/L,NH4Cl 0.9g/L, phosphoglycerol disodium salt (C3H6NaO7P, the 50mg/L in terms of P)：335.5mg/
L, NaCl 30g/L, CaCl2(in terms of Ca：80mg/L) 222mg/L uses deionized water dissolving.
It is this field routine techniques that above-mentioned preparation method, which detaches the method for obtaining nano material, such as by calcining removal certainly
Organic matter in assembly system is to obtain nano material.
Furthermore the present invention also aims to provide a kind of microorganism self-assembled nanometer material obtained by above-mentioned preparation method
Material.The nano material that its self assembly of different microorganisms obtains often differs greatly, including the grain size of nano particle, nano particle
Form assembling arrangement mode, the nano material appearance etc. of nano material.In the nano material of the present invention, nano particle diameter is
100-200nm is tightly covered on cell surface, and bacterium is surrounded.
In addition, the application of the microorganism self-assembled nanometer material is also an object of the present invention, application includes：Water
Heavy-Metal-Contaminated Environments reparation prepares carrier or material etc. that biological medicine utilizes.
The invention has the advantages that：
(1) bacterial strain of the present invention has environmental condition very high adaptability, can not saliferous, less salt, it is with high salt with
And wide pH, temperature, growth in nutrition, and nano materials while can efficiently remove Phosphorus From Wastewater；
(2) bacterial strain of the present invention can realize culture and dephosphorization under single aerobic condition；
(3) bacterial strain of the present invention can be using raw material in waste water in low saturation condition while removing Phosphorus From Wastewater
Lower synthesis calcium phosphorus nano particle, and the ability with nanoassemble, need not additionally add chemical reagent, environmental-friendly cost
It is low；
(4) present invention is not necessarily to high temperature and pressure when synthesizing calcium phosphorus nano particle, is not required to adjust pH, does not also need presoma,
Has many advantages, such as easy to operate, non-secondary pollution；
(5) nano material particle diameter distribution of the present invention is uniform, is covered in phage surface, and bacterial strain condition is cultivated by control
Required particle size and appearance and size can be prepared.
Description of the drawings
Fig. 1 is the nano material picture (AFM) that bacterial strain CF8-6 is synthesized under the conditions of waste water formula (one)；
Fig. 2 is the nano material picture that bacterial strain CF8-6 is synthesized under the conditions of waste water formula (two), Fig. 2 a:Atomic force microscopy
Mirror picture (AFM), Fig. 2 b, Fig. 2 c:Nano particle transmission electron microscope picture (TEM), Fig. 2 d, Fig. 2 e, Fig. 2 f:Nano-particles self assemble
Transmission electron microscope
Specific implementation mode
1 Shewanella of embodiment (Shewanella sp.) CF8-6 is detached and identification
The screening separation process of Shewanella strain (Shewanella sp.) CF8-6 is as follows：
(1) be separated to multi-strain bacteria strain from South China Sea bed mud, will be partially separated from the inoculation of South China Sea bed mud to
It in seawater LB liquid medium, at 25 DEG C, is cultivated 24 hours under conditions of 200rpm, obtains activation bacterium solution；
(2) the activation bacterium solution that above-mentioned steps (1) obtain is inoculated into 10% ratio in simulation high-salt wastewater, 25
DEG C, it is cultivated under conditions of 200rpm, several plants of bacterium for being above 90% in 12h and for 24 hours removal rate is selected to carry out secondary screening；
(3) step (2) obtained bacterial strain is screened again according to step (1) (2), increases sampling time point, selection removes
Phosphorus speed and one plant of optimal bacterium of dephosphorizing rate, are named as CF8-6.
Above-mentioned seawater LB medium components：Peptone 10g/L, yeast extract 3g/L, Chen Haishui preparation, wherein seawater salt
Degree is 3.5%.
It is as follows to simulate sanitary wastewater ingredient with high salt：
C6H12O6·H2O 1.5g/L, CH3COONa 0.75g/L, MgSO4·7H2O 1.18g/L, NH4Cl 0.9g/L,
KH2PO4·2H2O 0.066g/L(P:10mg/L), NaCl 30g/L.
All culture mediums equal 121 DEG C of high-temperature sterilization 20min before the use.It is seeded in clean bench and carries out.Strain
(bacterium solution containing 600uL and 300uL glycerine), -80 DEG C of long-term preservations in ultra low temperature freezer are stored in 1.5mL centrifuge tubes.
The Physiology and biochemistry of the bacterial strain characterizes：Bacterial strain CF8-6 can be 5-35 DEG C in temperature, pH5.8-9.8, salinity 0-12%, tight
It being grown under the aerobic condition of culture of lattice, and there is preferable phosphorus ligands effect, morphological features are negative for Gram's staining,
Observed under electron microscope is bacillus, there is pod membrane and flagellum.24 hours colony characteristics of bacterial strain solid culture are circle, milky.
The molecular biology identification of the bacterial strain：Using the DNA of kit extraction bacterial strain CF8-6, expanded by PCR
16S rDNA sequences obtain the 16S rDNA sequences of bacterial strain CF8-6, such as sequence table SEQ ID NO:Shown in 1.Use blast program
Nucleotide homology comparison is carried out to listed 16S rNDA sequences in the 16S rDNA sequences and GenBank of bacterial strain CF8-6,
It learns that bacterial strain CF8-6 belongs to genus Shewanella (Shewanella), therefore the bacterium is named as Shewanella sp.CF8-6.
Shewanella (Shewanella sp.) CF8-6 is preserved in Chinese Typical Representative culture guarantor on March 29th, 2016
Tibetan center, preservation address are：Luojiashan, Wuchang, Wuhan City, Hubei Province, preserving number are：CCTCC M 2016154.
The preparation of 2 microorganism self-assembled nanometer material of embodiment
The method that nano material is prepared using Shewanella strain (Shewanella sp.) CF8-6 is as follows：
(1) by bacterial strain Shewanella sp.CF8-6 in seawater LB liquid medium at 25 DEG C, under conditions of 200rpm
Activation bacterium solution is made in culture 24 hours；Activate bacterium solution parameter of noncentricity：10min is centrifuged under the conditions of 10000rpm；
(2) it will be tapped into simulated wastewater in the ratio of 10% (v/v) after bacterium solution centrifuge washing that step (1) obtains, 25
DEG C, it is cultivated 48 hours under conditions of 200rpm, centrifugation obtains thalline, and nano particle is distributed in cell surface and its surrounding.From
Heart rotating speed is 4000rpm, and the time is 10 minutes；
(3) thalline containing nano particle that step (2) obtains is washed with deionized water twice, rotating speed 4000rpm, when
Between be 15 minutes.Then it takes away and does transmission electron microscope (TEM).
(4) atomic force microscope is done after handling the thalline containing nano particle that step (2) obtains, observes granulated
Shape and size.
Simulate high-salt wastewater formula (one) (low-phosphorous waste water)：C6H12O6·H2O 1.5g/L, CH3COONa 0.75g/L,
MgSO4·7H2O 1.18g/L,NH4Cl 0.9g/L,KH2PO4·2H2O 0.066g/L are (in terms of P 10mg/L:),NaCl 30g/
L, with originally water dissolution.
Simulate high-salt wastewater formula (two) (high phosphorus waste water):C6H12O6·H2O 1.5g/L, CH3COONa 0.75g/L,
MgSO4·7H2O 1.18g/L,NH4Cl 0.9g/L, phosphoglycerol disodium salt (C3H6NaO7P, in terms of P：50mg/L),NaCl
30g/L, CaCl2(in terms of Ca：80mg/L), deionized water dissolving is used.
Its nano material picture prepared is shown in that Fig. 1 and Fig. 2, wherein Fig. 1 are bacterial strain CF8-6 in waste water formula (one) item respectively
The nano material picture (AFM) synthesized under part, Fig. 2 are the nano material that bacterial strain CF8-6 is synthesized under the conditions of waste water formula (two)
Picture, Fig. 2 a:Atomic force microscope images (AFM), Fig. 2 b, Fig. 2 c:Nano particle transmission electron microscope picture (TEM), Fig. 2 d, figure
2e, Fig. 2 f:Nano-particles self assemble transmission electron microscope.
In the nano material of the present invention, calcium phosphorus nano particle diameter is 100-200nm.
It should be noted that above example is only used to illustrate the technical scheme of the present invention rather than is limited.Although ginseng
It is described the invention in detail according to given example, but those skilled in the art can be as needed to this hair
Bright technical solution is modified or replaced equivalently, without departing from the spirit of the technical scheme of the invention and range.
Claims (6)
1. applications of Shewanella strain (Shewanella sp.) CF8-6 in preparing nano material, the Shewanella strain
(Shewanella sp.) CF8-6 was preserved in China typical culture collection center on March 29th, 2016, and preservation address is：
Luojiashan, Wuchang, Wuhan City, Hubei Province, preserving number are：CCTCC M 2016154；The nano material is calcium phosphorus nano material.
2. a kind of preparation method of microorganism self-assembled nanometer material, including：
(1) by Shewanella strain (Shewanella sp.) CF8-6 activation cultures, activation bacterium solution is made；
(2) activation bacterium solution is inoculated into phosphorous cultivating system and is cultivated, obtained nano particle and be distributed in somatic cells surface
And surrounding microorganism nanoassemble system；Phosphorous cultivating system is phosphorus-containing wastewater；
(3) separation obtains nano material；
Shewanella strain (Shewanella sp.) CF8-6 is preserved in Chinese Typical Representative culture guarantor on March 29th, 2016
Tibetan center, preservation address are：Luojiashan, Wuchang, Wuhan City, Hubei Province, preserving number are：CCTCC M 2016154.
3. preparation method according to claim 2, which is characterized in that step (1) is by bacterial strain Shewanella
Sp.CF8-6, at 25 DEG C, is cultivated 24 hours in seawater LB liquid medium under conditions of 200rpm, and activation bacterium solution is made；Seawater
The group of LB liquid medium becomes：1% peptone, 0.3% yeast powder, Chen Haishui are prepared.
4. preparation method according to claim 2, which is characterized in that step (2) is that the bacterium solution for obtaining step (1) centrifuges
It taps into phosphorus-containing wastewater in 10% ratio after washing, at 25 DEG C, is cultivated 48 hours under conditions of 200rpm, centrifuged up to nanometer
Distribution of particles is in somatic cells surface and surrounding microorganism nanoassemble system.
5. preparation method according to claim 4, which is characterized in that centrifugal rotational speed 3000-4000rpm.
6. preparation method according to claim 2, which is characterized in that step (3) is removed by calcining in self-assembly system
Organic matter to obtain nano material.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
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| CN201610236255.7A CN105779356B (en) | 2016-04-14 | 2016-04-14 | A kind of microorganism self-assembled nanometer material and its preparation method and application |
| PCT/CN2017/075497 WO2017177773A1 (en) | 2016-04-14 | 2017-03-02 | Highly efficient aerobic phosphorus-removing bacteria capable of synthesizing nanoparticles by microbial self-assembly using waste water |
| US16/093,639 US20190071335A1 (en) | 2016-04-14 | 2017-03-02 | Highly efficient aerobic phosphorus-removing bacteria capable of synthesizing nanoparticles by microbial self-assembly using waste water |
| US17/179,449 US20210317023A1 (en) | 2016-04-14 | 2021-02-19 | Highly efficient aerobic phosphorus-removing bacteria capable of synthesizing nanoparticles by microbial self-assembly using waste water |
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| US20190071335A1 (en) * | 2016-04-14 | 2019-03-07 | Shandong University | Highly efficient aerobic phosphorus-removing bacteria capable of synthesizing nanoparticles by microbial self-assembly using waste water |
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