WO2017177773A1 - 一类能够利用废水进行微生物自组装合成纳米颗粒的高效好氧除磷菌 - Google Patents

一类能够利用废水进行微生物自组装合成纳米颗粒的高效好氧除磷菌 Download PDF

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WO2017177773A1
WO2017177773A1 PCT/CN2017/075497 CN2017075497W WO2017177773A1 WO 2017177773 A1 WO2017177773 A1 WO 2017177773A1 CN 2017075497 W CN2017075497 W CN 2017075497W WO 2017177773 A1 WO2017177773 A1 WO 2017177773A1
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phosphorus
strain
wastewater
self
microbial
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PCT/CN2017/075497
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English (en)
French (fr)
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周维芝
蒋丽
黄兆松
王艳茹
赵海霞
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山东大学
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Priority claimed from CN201610232231.4A external-priority patent/CN105858596B/zh
Priority claimed from CN201610232204.7A external-priority patent/CN105821082B/zh
Priority claimed from CN201610232227.8A external-priority patent/CN105802885B/zh
Priority claimed from CN201610236259.5A external-priority patent/CN105754902B/zh
Priority claimed from CN201610236255.7A external-priority patent/CN105779356B/zh
Priority claimed from CN201610240140.5A external-priority patent/CN105754904B/zh
Priority claimed from CN201610239347.0A external-priority patent/CN105858599B/zh
Application filed by 山东大学 filed Critical 山东大学
Priority to US16/093,639 priority Critical patent/US20190071335A1/en
Publication of WO2017177773A1 publication Critical patent/WO2017177773A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/341—Consortia of bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/08—Seawater, e.g. for desalination
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2305/00—Use of specific compounds during water treatment
    • C02F2305/08—Nanoparticles or nanotubes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/30—Aerobic and anaerobic processes
    • C02F3/308—Biological phosphorus removal
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the invention relates to the technical field of wastewater phosphorus treatment and nano material preparation, in particular to a high-efficiency aerobic phosphorus removal bacteria capable of synthesizing nanoparticles by using microbial self-assembly by using wastewater.
  • Phosphorus is an important element in the formation of living organisms, and it is also an essential nutrient for biological growth. However, if excessive phosphorus is released into water bodies, it will cause damage to water eutrophication and red tide, affecting water quality and ecological balance. It has brought great harm to China's tourism, industry, agriculture and aquaculture industry. Therefore, effectively reducing the phosphorus concentration in wastewater is an urgent problem to solve water pollution.
  • domestic and international sewage phosphorus removal technologies mainly include adsorption, chemical precipitation and biological methods.
  • the adsorption method is a process of dephosphorization by utilizing the affinity of some porous or large specific surface area solid materials for phosphate ions in water.
  • the phosphorus removal by adsorption method has many problems in the anti-interference, dissolution loss and regeneration of the adsorbent. Due to the low adsorption capacity of traditional adsorbents, the adsorption method has not been widely used as a separate phosphorus removal method, and is often used as an auxiliary means in combination with other phosphorus removal methods.
  • the chemical precipitation method is a process in which a metal cation is combined with a phosphate to form a precipitate, but this method generates a large amount of chemical sludge to cause secondary pollution, and at the same time, the high cost of the treatment results in a high treatment cost and a high residual metal ion concentration. And the chemical precipitation method is not suitable for the treatment of low-phosphorus wastewater.
  • the biological phosphorus removal method Compared with the adsorption method and the chemical precipitation method, the biological phosphorus removal method has the advantages of high efficiency, low cost and environmental friendliness.
  • the enhanced biological phosphorus removal system which relies on the action of polyphosphate bacteria, is currently the most widely used biological phosphorus removal method.
  • the principle is to use phosphorus-accumulating bacteria to release phosphorus under anaerobic conditions, excessive phosphorus intake under good conditions, and finally achieve the purpose of phosphorus removal by means of sludge discharge.
  • this method only stores phosphorus in the cells, and further anaerobic digestion and chemical precipitation are needed to achieve stable recovery of phosphorus.
  • the high salt environment will inhibit the microorganisms, even if the wastewater is diluted to a salinity of 1%, it is difficult to treat, compared with nitrifying bacteria and denitrifying bacteria.
  • the phosphorus-removing bacteria are more sensitive to salinity. It has been reported in the literature that when the salinity increases from 0% to 0.4%, the salinity has no effect on nitrogen removal, and the phosphorus removal rate decreases from 85% to 25%.
  • the traditional biological phosphorus removal method is greatly limited in the treatment of high-salt wastewater, and the biological sludge has the defects of long acclimation period and difficulty in starting, so the screening uses phosphorus removal with salt tolerance in high salt environment. Strains, exploring its application in the further removal of phosphorus have important theoretical and practical significance.
  • Nanomaterials refer to materials in which at least one dimension of a three-dimensional material is in the nanometer scale or composed of them as basic units. Nanomaterials have many unique properties such as surface effects, small size effects, and quantum effects, and are widely used in energy, catalysis, biosensors, and biomedical industries.
  • biosynthetic nanomaterials have the advantages of cleanness, mild reaction conditions, low cost, and simple operation, and the biosynthesized nanomaterials have good dispersibility, stability, biocompatibility and adjustability. It has become a research hotspot at home and abroad.
  • the types of microorganisms with synthetic nanomaterials that have been discovered so far are limited, including prokaryotes and eukaryotes, such as bacteria, yeasts, certain viral ions, fungi, and plants with extracellular, intracellular, or nano-self-assembly.
  • prokaryotes and eukaryotes such as bacteria, yeasts, certain viral ions, fungi, and plants with extracellular, intracellular, or nano-self-assembly.
  • Capabilities, as well as reports on the use of synthetic nanomaterials such as plant extracts and natural polysaccharides such as marine polysaccharides.
  • the reported nanomaterials synthesized by biological methods mainly focus on precious metals including metal sulfide minerals such as gold (Au), silver (Ag), platinum (Pt) and cadmium sulfide (CdS), and cadmium selenide ( CdSe) and so on.
  • metal sulfide minerals such as gold (Au), silver (Ag), platinum (Pt) and cadmium sulfide (CdS), and cadmium selenide ( CdSe) and so on.
  • phosphorus is a limited and important resource, and excessive emission of phosphorus into water bodies can cause damage to water eutrophication and red tides, and waste of phosphorus resources.
  • proven phosphorus resources on land will be mined in the coming decades. Therefore, the recycling of phosphorus, especially the recovery of phosphorus from wastewater, has received increasing attention.
  • nano-hydroxyapatite has attracted more and more attention as a product in the fields of environment and biomedicine.
  • the chemical synthesis of phosphorus-containing nanomaterials was carried out in supersaturated phosphate ionic liquids and precursors. Phosphate chemical precipitation at room temperature, neutral pH and below 4000 ⁇ M has not been reported.
  • the object of the present invention is to provide a high-efficiency aerobic phosphorus-removing bacteria capable of synthesizing nanoparticles by microbial self-assembly using wastewater.
  • Another object of the present invention is to provide an application of the above strain in the preparation of a self-assembling biomaterial.
  • the present invention adopts the following technical solutions:
  • a highly efficient aerobic phosphorus-removing bacteria capable of synthesizing nanoparticles by microbial self-assembly using wastewater.
  • the high-efficiency aerobic phosphorus-removing bacteria capable of synthesizing nanoparticles by microbial self-assembly using waste water of the invention include: Shewanella sp. CF8-6, Psychrobacter aquimaris X3-1403 and lemon color Erythrobacter citreus X3-1411.
  • Shewanella sp. CF8-6 was deposited with the China Center for Type Culture Collection on March 29, 2016. The deposit address is: Lushan, Wuchang, Wuhan, Hubei province, and the deposit number is: CCTCC M 2016154.
  • the strain can be grown at a temperature of 5-35 ° C, pH 5.8-9.8, salinity 0-12%, strict aerobic culture conditions, and has a good phosphorus removal effect, the morphological characteristics of the cells are Gram stain It was negative, observed under electron microscope as bacillus, with capsule and flagella. The colony of the strain was solid for 24 hours and characterized by round and milky white.
  • the Psychrobacter aquimaris X3-1403 was deposited with the China Center for Type Culture Collection on March 29, 2016. Address: Lushan, Wuchang, Wuhan, deposit number: CCTCC M 2016155.
  • the strain of the present invention Psychrobacter aquimaris X3-1403 can be cultured at 15-30 ° C, the pH of the medium is 7-8, and the salinity is 0-12% (optimally 1% to 5%). Under the growth, the morphological characteristics of the cells were negative for Gram staining, and were observed under electron microscope as cocci or Brevibacterium, which were isolated, doubled or aggregated, with capsules and no flagella. The colony of the strain solid LB medium cultured for 24 hours was characterized by round, smooth and creamy color.
  • Erythrobacter citreus X3-1411 was deposited with the China Center for Type Culture Collection on March 29, 2016. Address: Lushan, Wuchang, Wuhan, China, deposit number: CCTCC M 2016156.
  • the strain can be grown under the conditions of 15-30 ° C, pH 7-8, salinity 0-12% (optimal 1%-5%), and the morphological characteristics of the strain are: Gram stain is negative, Observed under the electron microscope as a bacterium, alone, in double or short chain, with capsules and no flagella. The colonies of the strain solid LB cultured for 24 hours were characterized by round, smooth and yellow.
  • a microbial agent wherein the active ingredient of the microbial agent is the above-mentioned Shewanella sp. At least one of CF8-6, Psychrobacter aquimaris X3-1403, and Erythrobacter citreus X3-1411.
  • the microbial agent may further comprise a carrier; the carrier may be a solid carrier or a liquid carrier.
  • the solid carrier or liquid carrier is a conventional carrier material, wherein the solid carrier may be selected from the group consisting of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, corn flour, soy flour, polyvinyl alcohol and/or poly
  • the diol; the liquid carrier can be a vegetable oil, mineral oil or water.
  • the active ingredient may be present as a cultured living cell, a fermentation broth of a living cell, a filtrate of a cell culture, or a mixture of a cell and a filtrate.
  • the dosage form of the microbial agent may be a plurality of dosage forms such as a liquid preparation, a suspension, a powder, a granule, a wettable powder or a water-dispersible granule.
  • a method comprising the above-mentioned Shewanella sp. CF8-6, Psychrobacter aquimaris X3-1403 and Erythrobacter citreus X3-1411.
  • the biofilm is specifically composed of artificial filler or natural material as a carrier, and Erythrobacter citreus X3-1411 is attached to flocculate a film formed on the surface thereof.
  • the strain or the microbial agent of the invention can be used for phosphorus removal in salty wastewater or salt-free wastewater, in particular, the strain or the microbial agent of the invention is extremely effective for high salinity wastewater treatment, such as seawater flushing wastewater, the invention
  • the salinity in the high salinity wastewater can be up to 15%.
  • the strain or the microbial agent of the present invention is used for the treatment of phosphorus in wastewater of 0%-10% salinity.
  • a method for removing phosphorus from a salty wastewater is provided, the steps are as follows:
  • the above strain is inoculated into the LB medium, and after activation and culture, the activated bacterial liquid is prepared, and the activated bacterial liquid is added to the wastewater to be treated according to a volume fraction of 8-12%;
  • the bacterial agent of the above strain is added to the wastewater to be treated at a dosage of 5-20 mg/L.
  • the above-mentioned high-efficiency aerobic phosphorus-removing bacteria capable of synthesizing nanoparticles by using microbial self-assembly of wastewater or Pseudoalteromonas sp. DSBS with the accession number CCTCC M2013652 in preparing nano materials. Applications, especially in the preparation of self-assembled nanomaterials under low phosphorus conditions.
  • the above-mentioned high-efficiency aerobic phosphorus-removing bacteria can utilize the different concentrations of phosphorus in the wastewater (including high-phosphorus conditions and low-phosphorus conditions) to carry out microbial self-assembly and synthesis of nano-materials; in particular, micro-particle self-assembly and synthesis of nanoparticles can be carried out under low phosphorus conditions.
  • the low phosphorus condition means that the concentration of phosphorus is low saturation or unsaturated.
  • a bio-nanomaterial synthesized and self-assembled by the above-mentioned strain the bio-nanomaterial being the above-mentioned high-efficiency aerobic phosphorus-removing bacteria capable of synthesizing nanoparticles by using microbial self-assembly of wastewater or having the preservation number CCTCC M2013652 Pseudoalteromonas sp.
  • DSBS was synthesized in phosphorus-containing wastewater and prepared by self-assembly.
  • the concentration of the phosphorus element is from 0.3 mM to 1.3 mM.
  • a method for producing the above self-assembled bio-nanomaterial comprising the step of activating the strain, and the step of cultivating and self-assembling the activated strain in the phosphorus-containing wastewater.
  • the step of activating the strain comprises: inoculating the above strain in LB medium, 180-220 rpm, 20-30 ° C, and activating culture for 18-30 h; preferably, the activation culture condition is 200 rpm, 25 ° C, Activated for 24 h.
  • the formulation of the LB medium is: 1% peptone, 0.3% yeast powder, and seawater.
  • the step of culturing and self-assembly comprises: inoculating the activated strain in phosphorus-containing wastewater, 180-220 rpm, 15-30 ° C, culture for 42-54 h; preferably, 200 rpm, 25 ° C, culture 48h.
  • the inoculated amount of the activated strain was 8-12% (v/v).
  • the step of culturing and self-assembling further comprises: centrifuging the culture solution after the culture, and removing the supernatant to obtain a cell containing the nano material, that is, a bio-nano material.
  • the centrifugation speed was 5000 rpm and the centrifugation time was 10 min.
  • the phosphorus-containing wastewater may be seawater flushing wastewater or domestic wastewater or a non-low saturated system containing cadmium and phosphorus; in the non-low saturated system containing cadmium and phosphorus, the concentration of cadmium and phosphorus is ⁇ M level to nM level.
  • the use of the bio-nanomaterial the application of which mainly includes applications in the field of the environment and in the field of biomedicine.
  • the heavy metal element can interact with the sulfur atom and the nitrogen atom on the side chain of the amino acid, and has high toxicity.
  • environmental heavy metal pollution and repair problems have received widespread attention.
  • Environmental heavy metal pollution There are repair methods mainly including physical repair, chemical repair and bioremediation.
  • chemical repair needs to add additional chemical agents to the polluted environment such as soil and water to make heavy metal ions adsorb, redox reaction and precipitate. This method is easy to produce secondary pollution and high cost despite simple operation. It is an effective method to repair environmental heavy metal pollution by using phosphorus-containing materials.
  • the bio-nano material of the invention comprises the nano-hydroxyapatite produced by the micro-organism, so it can be applied to the repair of environmental heavy metal pollution, has the advantages of simple operation and low cost; and the bio-nano material also includes active bacteria Body, the bacteria have a further adsorption effect on heavy metals.
  • Biomedical field The application of the nano-biomaterial for removing organic matter (retaining nano-hydroxyapatite after removing organic matter) in the preparation of a drug carrier, an antitumor drug, a hard tissue repairing material, an artificial bone, an artificial tooth.
  • Nanomaterials have important applications in biomedical pharmacy, human health and other life sciences, such as the use of nanoparticles as a carrier to transport drugs to the lesions, and the use of nanomaterials as biomedical diagnostic materials.
  • Calcium phosphate salts such as hydroxyapatite are the main inorganic mineral components of animals and human bones and teeth. They have good activity and biocompatibility. For example, hydroxyapatite ceramics are promising artificial bones and artificial oral cavity. material.
  • Biosynthesized nano-calcium phosphate materials not only have the characteristics of nano-materials, but also have better biocompatibility and adaptability. For example, nano-hydroxyapatite has broad application prospects in biomedical fields.
  • a method for preparing a nano-hydroxyapatite wherein the biological nano-material is purified and separated to obtain nano-hydroxyapatite, and the specific method for purifying and separating may be performed by burning the biological nano-material. The organic matter is removed to obtain nano-hydroxyapatite.
  • the nano-hydroxyapatite material obtained by the above method has a uniform particle size distribution, and the required particle size and shape size can be prepared by controlling the conditions of the preparation method, and the nano-hydroxyl group obtained by the self-assembly method of the strain of the present invention is obtained.
  • Apatite has good film-forming properties and has a good application in the preparation of thin film materials.
  • the strain of the present invention has high adaptability to environmental conditions, can grow in the range of no salt, high salt and wide pH, temperature and nutrition, and can efficiently remove phosphorus in wastewater and make it in wastewater. Phosphorus falls below 0.5mg/L to meet emission standards.
  • the strain of the present invention has a good phosphorus removal and purification effect on high salinity wastewater such as seawater flushing wastewater, and solves the shortage of fresh water resources and establishes effective seawater flushing. Wastewater utilization system has good utilization value.
  • strain of the present invention only needs to be inoculated into the phosphorus-containing wastewater after being activated in the process of removing phosphorus, and the step is simple, the operation is easy, and the cost is low;
  • the strain of the present invention can realize phosphorus removal under a single aerobic condition, simplifies the phosphorus removal process, improves the operability of the phosphorus removal process, and provides a new way for biological phosphorus removal;
  • the strain of the present invention is a metal phosphate in a non-low saturated wastewater system (the calcium phosphorus deposit in the system of the present invention)
  • the precipitated form of the lake achieves the removal of phosphorus.
  • the strain of the invention has the functions of self-flocculation and self-assembly, and the strain can synthesize calcium-phosphorus nanoparticles at low concentration conditions by using raw materials in the wastewater while degrading the pollutants in the wastewater, and has nano-self-assembly.
  • the ability does not require additional chemical reagents, and the environmentally friendly cost is low, realizing the recycling of phosphorus resources.
  • the preparation method of the bio-nano material of the invention has the advantages of mild conditions, simple operation, clean and pollution-free, low cost, high efficiency and large-scale popularization and application.
  • the bio-nano material prepared by the invention has nanometer particles distributed on the surface of the bacterial cell and surrounding, and the removal effect is remarkable when the bio-nano material is used to remove fluorine in water, adsorb phenol, remove heavy metals such as lead and cadmium, and clean radioactive waste. Improvement. After the cells of the bio-nano material are removed, a porous nano-material can be formed, which can be used as a drug carrier or the like.
  • Figure 1 Phylogenetic tree of strain CF8-6
  • Figure 2 Psychrobacter aquimaris X3-1403 Gram staining results
  • Figure 3 AFM pattern of Psychrobacter aquimaris X3-1403 cells
  • Figure 4 Figure of the results of Gram staining of Erythrobacter citreus X3-1411;
  • Figure 5 AFM map of the morphology of Erythrobacter citreus X3-1411;
  • Figure 6a Growth curve of CF8-6 at different salinities
  • FIG. 7 Psychrobacter aquimaris X3-1403 removes TP, COD, NH 4 + -N and TN in simulated seawater flushing wastewater;
  • Figure 8 Removal of TP, COD, NH 4 + -N and TN in simulated seawater flushing wastewater by Erythrobacter citreus X3-1411;
  • FIG 9 Picture of nanomaterials synthesized by strain CF8-6 under wastewater formulation (1) (AFM);
  • Figure 10 Picture of nanomaterials synthesized by strain CF8-6 under wastewater formulation (II), Figure 10a: Atomic Force Microscope Image (AFM), Figure 10b, Figure 10c: Nanoparticle Transmission Electron Microscope Image (TEM), Figure 10d, Figure 10e, FIG. 10f: nanoparticle self-assembled transmission electron microscope;
  • II wastewater formulation
  • Figure 10a Atomic Force Microscope Image
  • Figure 10b Atomic Force Microscope Image
  • Figure 10c Nanoparticle Transmission Electron Microscope Image
  • Figure 10d Figure 10e
  • FIG. 10f nanoparticle self-assembled transmission electron microscope
  • Figure 11 Electron micrograph of self-flocculation of bacteria Psychrobacter aquimaris X3-1403 in simulated seawater flushing wastewater;
  • Figure 12 Electron micrograph of self-assembled and synthetic nanomaterials of Psychrobacter aquimaris X3-1403 in simulated seawater flushing wastewater;
  • Figure 13 Self-flocculation electron micrograph of Erythrobacter citreus X3-1411 in simulated seawater flushing wastewater
  • Figure 14 Transmission electron micrograph of synthetic nanomaterials in the simulated seawater flushing wastewater of Erythrobacter citreus X3-1411.
  • Figure 15 Atomic force microscopy images (A), scanning electron microscopy images (B, C, D) and energy spectrum analysis (E) of Pseudoalteromonas sp. DSBS in the formation of nanoparticles in low salt wastewater.
  • Figure 16 Phosphorus removal from Pseudoalteromonas sp. DSBS in high salt wastewater.
  • Figure 17 Atomic force microscopy images (A), scanning electron microscopy images (B) and energy spectrum analysis (C) of strains forming nanoparticles in high salt wastewater.
  • test materials used in the examples of the present invention are all conventional test materials in the art, and are commercially available.
  • Example 1 Isolation and Identification of High-efficiency Phosphorus-removing Bacteria capable of self-assembly of microorganisms under low phosphorus conditions
  • the formula of the seawater LB liquid medium is: 1% peptone, 0.3% yeast powder, and seawater.
  • composition of simulated high-salt domestic wastewater is as follows:
  • All media were sterilized at 121 ° C for 20 min before use. Inoculation is carried out in a clean bench. The strains were stored in a 1.5 mL centrifuge tube (containing 600 uL of bacterial solution and 300 uL of glycerol) and stored in an ultra-low temperature freezer at -80 °C for a long time.
  • strain CF8-6 strain X3-1403
  • Strain X3-1411 strain CF8-6, strain X3-1403
  • the strain CF8-6 can be grown at a temperature of 5-35 ° C, pH 5.8-9.8, salinity 0-12%, strict aerobic culture conditions, and has a good phosphorus removal effect.
  • the morphological characteristics of the cells were negative for Gram staining, and the cells were observed under an electron microscope, with capsules and flagella.
  • the strain was cultured in solid state for 24 hours, and the colony was characterized by a round shape and milky white.
  • the DNA of the strain CF8-6 was extracted using a kit, and the 16S rDNA sequence was amplified by PCR to obtain the 16S rDNA sequence of the strain CF8-6 as shown in SEQ ID NO: 1 of the Sequence Listing.
  • the nucleotide sequence homology between the 16S rDNA sequence of strain CF8-6 and the 16S rNDA sequence registered in GenBank was compared by BLAST program, and it was found that strain CF8-6 belongs to Shewanella, so the strain was Named Shewanella sp. CF8-6, the phylogenetic tree of this strain is shown in Figure 1.
  • Shewanella sp. CF8-6 was deposited with the China Center for Type Culture Collection on March 29, 2016. The deposit address is: Lushan, Wuchang, Wuhan, Hubei province, and the deposit number is: CCTCC M 2016154 .
  • strain X3-1403 can be grown at 15-30 ° C, pH 7-8, salinity 0-12% (optimal 1% ⁇ 5%) culture conditions, the morphology of the bacteria is Gram staining was negative, as shown in Figure 2, observed under electron microscope as cocci or Brevibacterium, alone, in pairs or aggregated, with capsules, no flagella.
  • the colony of the solid LB cultured for 24 hours was characterized by round, smooth and creamy color, as shown in Fig. 3.
  • the sequence of the 16s rDNA of strain X3-1403 is shown in SEQ ID NO. After comparing the 16S rDNA nucleotide sequence tested with the similarity sequence entered into the GenBank database of the NCBI website, the results showed that Psychrobacter aquimaris X3-1403 and Psychrobacter were on the smallest branch. The similarity between its 16S rDNA sequence and Psychrobacter aquimaris was 99.64%. Combined with colony morphology and 16S rDNA sequence analysis, it was identified as Psychrobacter aquimaris.
  • strain Psychrobacter aquimaris X3-1403 was further confirmed to be Psychrobacter aquimaris, and Psychrobacter aquimaris X3-1403 was deposited in the China Center for Type Culture Collection. Address: Wushan, Wuchang, Wuhan, deposit number: CCTCC M2016155.
  • strain X3-1411 The main biological characteristics of strain X3-1411 were: Gram staining was negative (results shown in Figure 4), observed under electron microscope as bacilli, alone, in double or short chain, with capsule, no flagella (results) As shown in Figure 5). The colonies of the strain solid LB cultured for 24 hours were characterized by round, smooth and yellow.
  • the strain can be grown under culture conditions of 15 to 30 ° C, pH 7 to 8, and salinity of 0 to 12% (optimally 1% to 5%).
  • the sequence of the 16s rDNA of strain X3-1411 is shown in SEQ ID NO.
  • the sequence was aligned with BLAS (http://blast.ncbi.nlm.nih.gov/Blast.cgi) in the GenBank database, and the similarity of the sequence to the Erythrobacter citreus strain was 99.26%.
  • the strain X3-1411 was identified as a lemon. Erythrobacter citreus has been deposited with the China Center for Type Culture Collection on March 29, 2016. Address: Lushan, Wuchang, Wuhan, deposit number: CCTCC M 2016156.
  • Example 2 Study on phosphorus removal effect of the strain of the present invention
  • strain Shewanella sp. CF8-6 in water treatment is as follows:
  • strain Shewanella sp. CF8-6 was cultured in a seawater LB liquid medium at 25 ° C, 200 rpm for 24 hours to prepare an activated bacterial solution.
  • the activated bacterial solution obtained in the step (1) is inoculated into the wastewater of different salinity in the simulated wastewater (the salinity ranges from 0% to 20%) at a ratio of 10%, and cultured at 25 ° C, 200 rpm.
  • the concentration of phosphorus in the supernatant was measured at different times and the biomass was measured at a wavelength of 600 nm.
  • the removal efficiency of the strain and the growth curve of the strain under different salinity ranges were obtained.
  • the water treatment effect data are shown in Fig. 6a and Fig. 6b. Shown.
  • the strain Shewanella sp. CF8-6 of the present invention has a very high phosphorus removal efficiency in the salty wastewater, especially in wastewater having a salinity of 10% or less, and phosphorus in 10 hours.
  • the removal rate can reach above 99%; even for 12%, 15% salinity wastewater, the strain Shewanella sp. CF8-6 of the present invention also has an excellent phosphorus removal rate.
  • the simulated wastewater formulation and the screening strains have the same concentration of the other components except the salinity value of the wastewater formulation, and the phosphorus concentration (in terms of P) is 10 mg/L.
  • Psychrobacter aquimaris X3-1403 was cultured in LB for 24 hours, then inoculated into simulated seawater flushing wastewater with 10% inoculation amount, and the strain was tested for TP, COD, NH 4 + by time point . -N and TN removal effects. It can be seen from Fig. 7 that the strain has a high removal effect on TP and COD, the removal rates are 70.5% and 75.5%, respectively, and the removal rates at 48h are 0.57 mg/(L ⁇ h) and 18.7 mg/( L ⁇ h). The removal effect of NH 4 + -N and TN was poor, only 17.8% and 19.4%.
  • the strain of Erythrobacter citreus X3-1411 isolated and screened in Example 1 was cultured on LB medium for 24 hours, and then inoculated into simulated seawater flushing wastewater with 10% inoculation amount, and cultured at 25 ° C and 200 rpm. The strains were sampled at time to determine the removal effect of the strain on TP, COD, NH 4 + -N and TN. The result is shown in Figure 8.
  • the formulation of the simulated seawater flushing wastewater in this embodiment is the same as that in the third embodiment.
  • Table 2 The composition of the simulated domestic wastewater is (deionized water):
  • Example 5 Application of the strain of the invention in preparing self-assembled nanomaterials
  • strain Shewanella sp. CF8-6 was cultured in seawater LB liquid medium at 25 ° C, 200 rpm for 24 hours to obtain an activated bacterial solution; the activated bacterial liquid centrifugation parameter: centrifuged at 10,000 rpm for 10 min;
  • the bacterial liquid obtained in the step (1) is centrifuged and washed, and then introduced into the simulated wastewater at a ratio of 10% (v/v), and cultured at 25 ° C, 200 rpm for 48 hours, and the cells are obtained by centrifugation.
  • the particles are distributed on and around the cell surface.
  • the centrifugal speed is 4000 rpm and the time is 10 minutes;
  • the nanoparticle-containing cells obtained in the step (2) were washed twice with deionized water at a number of revolutions of 4000 rpm for 15 minutes. Then take it as a transmission electron microscope (TEM).
  • TEM transmission electron microscope
  • the nanoparticle-containing cells obtained in the step (2) were treated and subjected to an atomic force microscope to observe the shape and size of the particles.
  • Simulated high-salt wastewater formulation (1) (low-phosphorus wastewater): C 6 H 12 O 6 ⁇ H 2 O 1.5g/L, CH 3 COONa 0.75g/L, MgSO 4 ⁇ 7H 2 O 1.18g/L, NH 4 Cl 0.9 g/L, KH 2 PO 4 ⁇ 2H 2 O 0.066 g/L (based on P 10 mg/L:), NaCl 30 g/L, dissolved in tap water.
  • Simulated high-salt wastewater formulation (2) (high-phosphorus wastewater): C 6 H 12 O 6 ⁇ H 2 O 1.5g/L, CH 3 COONa 0.75g/L, MgSO 4 ⁇ 7H 2 O 1.18g/L, NH 4 Cl 0.9g / L, disodium glyceryl phosphate (C 3 H 6 NaO 7 P, P: 50mg / L), NaCl 30g / L, CaCl 2 (calculated as Ca: 80mg / L), dissolved in deionized water .
  • Figure 9 shows the strain CF8-6 in the wastewater formulation (1).
  • the picture of the nanomaterials synthesized under the conditions (AFM)
  • Figure 10 is a picture of the nanomaterials synthesized by the strain CF8-6 under the conditions of the wastewater formulation (II)
  • Figure 10a Atomic Force Microscope Image
  • Figure 10b Figure 10c: Nanoparticles Transmission electron microscopy (TEM)
  • Figure 10d Figure 10e
  • Figure 10f Nanoparticle self-assembled transmission electron microscopy.
  • the calcium phosphate nanoparticle has a particle diameter of 100 to 200 nm.
  • Psychrobacter aquimaris X3-1403 was activated in LB medium for 24 h, and the culture conditions were 200 rpm and 25 °C. Then 1 in a sterile operating table with 25 mL of activated culture solution using a previously sterilized centrifuge tube, centrifuged at 10,000 rpm for 10 min; 2 to remove the supernatant, and then resuspend the bacterial solution with 10 mL of sterile deionized water, centrifuged at 10,000 rpm for 10 min; 3 Repeat step 2 once.
  • the resuspended bacterial liquid was inoculated separately into simulated seawater flushing wastewater (formulation composition is the same as Table 1) and simulated domestic wastewater (formulation composition is the same as Table 2) (inoculation amount 10%), and cultured at 200 rpm and 25 ° C for 48 hours.
  • the culture solution was centrifuged at 5000 rpm for 10 min, and the supernatant was discarded.
  • the cells at the bottom of the centrifuge tube were washed twice with deionized water to obtain a cell containing nano materials. A part of the cells were resuspended, fixed and stained on a copper grid, and finally dried for transmission electron microscope observation.
  • the above LB medium formula is: 1% peptone, 0.3% yeast powder, and seawater.
  • FIG. 11 An electron micrograph of the self-flocculation of the bacteria in simulated seawater flushing wastewater is shown in Fig. 11.
  • the electron micrographs of the cells synthesized in the simulated seawater flushing wastewater and self-assembled are shown in Fig. 12.
  • Fig. 11 and Fig. 12 show that the nanoparticle material is honeycomb-shaped, the particle size is nanometer and uniform, and the structure is compact and easy. A layered nanomaterial is produced.
  • the strain Erythrobacter citreus X3-1411 was activated in LB for 24 h, and the culture conditions were 200 rpm and 25 °C. Then 1 in a sterile operating table with 25 mL of activated culture solution using a previously sterilized centrifuge tube, centrifuged at 10,000 rpm for 10 min; 2 to remove the supernatant, and then resuspend the bacterial solution with 10 mL of sterile deionized water, centrifuged at 10,000 rpm for 10 min; 3 Repeat step 2 once.
  • the resuspended bacterial liquid was inoculated separately into simulated seawater flushing wastewater (formulation composition is the same as Table 1) and simulated domestic wastewater (formulation composition is the same as Table 2) (inoculation amount 10%), and cultured at 200 rpm and 25 ° C for 48 hours.
  • the culture solution was centrifuged at 5000 rpm for 10 min, and the supernatant was discarded.
  • the cells at the bottom of the centrifuge tube were washed twice with deionized water to obtain a cell containing nano materials. A part of the cells were resuspended, fixed and stained on a copper grid, and finally dried for transmission electron microscope observation.
  • the above LB formula is: 1% peptone, 0.3% yeast powder, and seawater.
  • FIG. 13 An electron micrograph of the self-flocculation of the bacteria in simulated seawater flushing wastewater is shown in Fig. 13.
  • the synthetic nanomaterials of the bacteria in the simulated seawater flushing wastewater and the self-assembled transmission electron micrographs are shown in Fig. 14, and Fig. 13 and Fig. 14 show that the material has good dispersibility and uniform particle size distribution.
  • Pseudoalteromonas sp. DSBS was inoculated into liquid LB medium at 0.9% (v/v), and cultured at 25 ° C, 200 rpm on a constant temperature shaker for 20 hours to obtain an enriched cell.
  • liquid LB medium components are as follows:
  • Peptone 10g / L yeast powder 3g / L, prepared in seawater, seawater salinity of 3.5%.
  • the low salt wastewater components are as follows:
  • Glucose monohydrate 5.06g/L, NaAC 1.5g/L, NaCl 3.5g/L, NH 4 Cl 2.6g/L, MgSO 4 ⁇ 7H 2 O 2.4g/L, total cadmium (Cd(NO 3 ) 2 ⁇ 4H 2 O) and total phosphorus (K 2 HPO 4 ) concentrations were 8 mg/L and 9 mg/L, respectively, initial pH 7.2, salinity 0.35%.
  • the concentration of cadmium and phosphorus is the concentration in the general industrial cadmium-containing wastewater. Under this pH and ion concentration, it is not enough to form inorganic chemical cadmium precipitate, which belongs to the ⁇ M-grade unsaturated low-salt environment of cadmium and phosphorus.
  • atomic force microscopy and scanning electron microscopy were used to observe the cells after removal of phosphorus and cadmium.
  • the results of the atomic force microscope observation are shown in A of Fig. 15.
  • the surface of the cells is uniformly aggregated with nanoparticles, and a small number of particles are scattered and scattered around, and the diameter of the particles is 25-100 nm.
  • Some of the cells in the figure have flagella, some are not, and may be shed during culture or sample pretreatment.
  • the bacteria-synthesized nanoparticles have three different forms: smaller particles with a diameter of 25-60 nm uniformly dispersed on the surface of the cells, spherical aggregates of smaller particles having a diameter of 100 nm adhered to the surface of the cells, and aggregates detached from the bacteria.
  • the piles are adhered to the polysaccharide fibers between the cells.
  • the energy spectrum analysis of the points in the figure shows that the particles mainly contain C, O, Cd, P, S elements.
  • the nanoparticles are cadmium-phosphorus-sulfur nanoparticles containing polysaccharides. Wherein Na is a precipitated soluble ion, and Al is a sample stage element, which is not an element in the nanoparticle.
  • the bacterium can simultaneously remove phosphorus cadmium in a low-salt wastewater with unsaturated phosphorus and cadmium concentration, and form cadmium-phosphorus-sulfur nanoparticles with three forms of inclusion polysaccharides in the extracellular space, with a diameter of 25-60 nm.
  • Pseudoalteromonas sp. DSBS was inoculated into liquid LB medium at 0.9% (v/v), and cultured at 25 ° C, 200 rpm on a constant temperature shaker for 20 hours to obtain an enriched cell.
  • the washed bacterial suspension was inoculated into three groups of high-salt wastewater according to 0.2 ⁇ (v/v), and cultured in a constant temperature shaker at 25°C and 200 rpm for 48 hours to obtain cadmium-phosphorus-sulfur nanoparticles. Bacterial suspension.
  • the bacterial suspension containing the cadmium-phosphorus-sulfur nanoparticles was washed with deionized water at a centrifugal speed of 3000 rpm, and the nano-material was prepared by using low-speed centrifugation to prevent particle surface elution of the cells.
  • the liquid LB medium component is as follows:
  • Peptone 10g / L yeast powder 3g / L, prepared in seawater, seawater salinity of 3.5%.
  • the high salt wastewater components are as follows:
  • the concentration of total phosphorus in the supernatant was determined by molybdate spectrophotometry in three groups of wastewater, and the results are shown in FIG. In a high-salt environment, the bacteria had a removal rate of 46.24% and 72.48% for 9 mg/L and 5 mg/L phosphorus, respectively.
  • the cells were also observed using atomic force microscopy and scanning electron microscopy.
  • the results of the atomic force microscope are shown in Fig. 17A, and the surface of the cells is uniformly dispersed with nanoparticle materials, and the diameters are all below 10 nm.
  • the concentration of phosphorus and cadmium in synthetic seawater is nM grade, and the lower cadmium concentration results in smaller particle size of the nanoparticles.
  • the cells in this field of view have no flagella and may not be observed due to breakage.
  • the bacterium can also form cadmium-phosphorus-sulfur nanoparticles with inclusion polysaccharides in the extracellular salt in the high-salt wastewater with unsaturated phosphorus and cadmium concentration, and the diameter is 10 nm.

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Abstract

提供了一类能够利用废水进行微生物自组装合成纳米颗粒的好氧除磷菌，包括：希瓦氏菌(Shewanella sp.)CF8-6、海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403和柠檬色赤杆菌(Erythrobacter citreus)X3-1411，含有所述希瓦氏菌(Shewanella sp.)CF8-6、海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403和柠檬色赤杆菌(Erythrobacter citreus)X3-1411中的至少一种的菌剂、生物膜或生物反应器，以及利用上述菌株除去含盐废水中的磷的方法。还提供了一种由上述菌株或保藏编号为CCTCC M2013652的假交替单胞菌(Pseudoalteromonas sp.)DSBS在含磷废水中合成并经自组装制备而成的生物纳米材料，及其制备方法和用途。

Description

一类能够利用废水进行微生物自组装合成纳米颗粒的高效好氧除磷菌 技术领域
本发明涉及废水磷处理及纳米材料制备技术领域，特别是涉及一类能够利用废水进行微生物自组装合成纳米颗粒的高效好氧除磷菌。
背景技术
磷是组成生命体的一种重要元素，同时也是生物生长必不可少的营养物质，然而，如果磷过度排放到水体中则会引起水体富营养化和赤潮等危害，影响水体质量与生态平衡，给我国的旅游业、工业、农业及水产业都带来极大的危害，因此有效降低废水中磷浓度是一个急需解决的水污染问题。
目前，国内外污水除磷技术主要有吸附法、化学沉淀法和生物法。其中，吸附法是利用某些多孔或大比表面积的固体物质对水中磷酸根离子的亲和力来实现除磷的过程。但吸附法除磷在吸附剂的抗干扰性、溶解损失和再生等方面还存在诸多问题。由于传统吸附剂吸附容量较低，吸附法作为单独除磷手段目前尚未得到广泛应用，经常作为辅助手段与其它除磷方法结合使用。化学沉淀法是利用金属阳离子与磷酸根结合生成沉淀的过程，但此方法会产生大量化学污泥造成二次污染，同时药剂费用较高导致处理成本较高，残留的金属离子浓度也较高，并且化学沉淀法不适用于低磷废水的处理。
与吸附法和化学沉淀法相比，生物除磷方法具有效率高，成本低及环境友好等优点。依赖于聚磷菌作用的强化生物除磷系统是目前应用最广泛的生物除磷方法。其原理是利用聚磷菌在厌氧条件下释磷、好养条件下过量摄磷，最后通过排泥的方式达到除磷的目的。但该方法只是将磷储存在了细胞内，仍需要进一步的厌氧消解、化学沉淀才能达到磷的稳定回收。而且当处理高盐度废水如海水冲厕废水时，高盐环境会对微生物会产生抑制作用，即使废水被稀释到盐度为1％时处理也很困难，且相比较硝化菌和反硝化菌而言，除磷菌对于盐度更加敏感，有文献报道当盐度从0％上升到0.4％时，盐度对氮的去除没有影响，而磷的去除率从85％下降到25％。因此传统的生物除磷方法在处理高盐废水时受到极大的限制，同时生物污泥又具有驯化周期长、启动困难等缺陷，因此筛选利用生长在高盐环境中具有耐盐性能的除磷菌株，探索其在磷的进一步去除中的应用具有重要的理论及现实意义。
纳米材料是指在三维材料中至少有一维处于纳米尺度范围或由它们作为基本单元构成的材料。纳米材料具有表面效应、小尺寸效应、量子效应等一些独特的特性，从而被广泛应用于能源、催化、生物传感器、生物医学等行业。传统的物理化学合成纳米材料的方法主要有两相法、反相微乳法、光化学合成法、电极电解法以及加热法、超声法等等，但是这些传统 的合成方法具有种种难以克服的缺陷，如原料价格高，耗能高，反应条件苛刻或者难于规模化生产，同时化学合成方法往往需要前驱体，然而这些前驱体价格昂贵且可能具有毒性，并且化学合成方法都需要在高饱和度的溶液中进行，需要投加大量化学试剂导致成本较高，这些缺陷都极大的限制了化学方法的应用。与之相比，生物合成纳米材料具有清洁、反应条件温和、成本低、操作简单等优势，且生物合成的纳米材料具有良好的分散性、稳定性、生物相容性及可调节性等优点，现已成为国内外研究热点。目前已发现的具有合成纳米材料的微生物种类很有限，主要包括原核生物和真核生物，如细菌、酵母菌、某些病毒离子、真菌及植物等具有胞外、胞内合成或纳米自组装的能力，同时也有个别利用植物提取物以及天然多糖海洋多糖等合成纳米材料的报道。但是已报道的应用生物方法合成的纳米材料主要集中于贵金属包括及金属硫化物纳米材料方面，如金(Au)、银(Ag)、铂(Pt)及硫化镉(CdS)、硒化镉(CdSe)等。
另一方面，磷是一种有限的重要资源，而磷过度排放到水体中既会引起水体富营养化和赤潮等危害，又会造成磷资源的浪费。同时，陆地上已探明的可用磷资源在未来几十年内将被开采殆尽。因此，磷的循环利用，尤其是从废水中回收磷越来越受到人们的重视。纳米羟基磷灰石作为一种回收磷的有效手段，并将其作为产品应用到环境、生物医药等领域越来越受到人们的关注。含磷纳米材料的化学人工合成都在超饱和磷酸根离子液体中、前驱体作用下进行，常温条件、中性pH、低于4000μM的磷酸盐化学沉淀还没有报导。关于生物合成钙磷纳米颗粒的菌株报道也很少，有文献报道一株沙雷氏菌Serratia sp.能够在不同的培养条件下可以合成不同粒径和特性的羟基磷灰石纳米颗粒，该沙雷氏菌Serratia sp.只能在高饱和的溶液(P：5mM)以及生物缓冲液中产生钙磷纳米颗粒，并且此类钙磷纳米颗粒的产生并不是严格意义上的生物合成，而是一种生物降解过程，该沙雷氏菌Serratia sp.通过产生一种非典型性酸性磷酸酶来分解基质中的甘油磷酸钠从而释放出大量无机磷酸根离子，高浓度的磷酸根离子与钙离子在细胞表面或者胞外聚合物中形成羟基磷灰石纳米颗粒，然后将之应用于水溶液中放射性核素的去除。然而其形成纳米颗粒的条件仍较为苛刻。如何通过简单的方法从废水中有效的获得钙磷纳米材料，仍是本领域内面临的技术难题。
将纳米材料作为微纳结构单元组装成具有等级结构的宏观尺度材料会使其产生更优异的整体的协同性质，是提髙纳米材料实际应用能力的有效途径。近年来，己发展了多种组装策略，如电化学沉积法，表面功能化法及微压印技术等。但存在设备要求高、反应条件苛刻、易造成二次污染和成本高等缺点。因此，发展一种高效、低成本、环境友好的组装纳米单元的技术来制备具有一定结构和功能的材料对于解决纳米材料在实际应用中的问题具有重大意 义。
因此，开发既能够降解废水中的污染物，又能在低浓度条件下合成纳米羟基磷灰石并进行自组装的生物合成方法具有十分重要的研究和应用价值。
发明内容
针对上述现有技术的不足，本发明的目的在于提供一类能够利用废水进行微生物自组装合成纳米颗粒的高效好氧除磷菌。
本发明的另一目的在于提供上述菌株在制备自组装生物材料中的应用。
为实现上述目的，本发明采用如下技术方案：
本发明的第一方面，提供一类能够利用废水进行微生物自组装合成纳米颗粒的高效好氧除磷菌。
本发明的能够利用废水进行微生物自组装合成纳米颗粒的高效好氧除磷菌，包括：希瓦氏菌(Shewanella sp.)CF8-6、海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403和柠檬色赤杆菌(Erythrobacter citreus)X3-1411。
所述希瓦氏菌(Shewanella sp.)CF8-6已于2016年3月29日保藏于中国典型培养物保藏中心，保藏地址为：湖北省武汉市武昌珞珈山，保藏号为：CCTCC M 2016154。
该菌株可在温度5-35℃，pH5.8-9.8，盐度0-12％、严格好氧的培养条件下生长，且具有较好的磷去除效果，菌体形态特征为革兰氏染色呈阴性，电子显微镜下观察为杆菌，有荚膜及鞭毛，菌株固体培养24小时菌落特征为圆形，乳白色。
所述海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403已于2016年3月29日保藏于中国典型培养物保藏中心，地址：武汉市武昌珞珈山，保藏号：CCTCC M 2016155。
本发明所述的菌株海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403可在15～30℃，培养基pH值7～8、盐度为0～12％(最佳1％～5％)培养条件下生长，菌体形态特征为革兰氏染色呈阴性，电子显微镜下观察为球菌或短杆菌，单独、成双或聚集成团，有荚膜、无鞭毛。菌株固体LB培养基培养24h菌落特征为圆形，光滑，奶油色。
柠檬色赤杆菌(Erythrobacter citreus)X3-1411已于2016年3月29日保藏于中国典型培养物保藏中心，地址：武汉市武昌珞珈山，保藏号：CCTCC M 2016156。
该菌株可在15～30℃，pH值7～8，盐度为0～12％(最佳1％～5％)培养条件下生长，菌株的形态学特征为：革兰氏染色呈阴性，电子显微镜下观察为杆菌，单独、成双或短链状，有荚膜、无鞭毛。菌株固体LB培养24h菌落特征为圆形，光滑，黄色。
本发明的第二方面，提供一种菌剂，该菌剂的活性成分为上述的希瓦氏菌(Shewanella sp.) CF8-6、海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403和柠檬色赤杆菌(Erythrobacter citreus)X3-1411的至少一种。
进一步的，所述菌剂还可包括载体；所述载体可以为固体载体或液体载体。所述固体载体或液体载体均为常规的载体材料，其中，固体载体可以选自粘土、滑石、高岭土、蒙脱石、白碳、沸石、硅石、玉米粉、豆粉、聚乙烯醇和/或聚二醇；液体载体可以为植物油、矿物油或水。
上述菌剂中，所述活性成分可以以被培养的活细胞、活细胞的发酵液、细胞培养物的滤液或细胞与滤液的混合物的形式存在。
所述菌剂的剂型可以为液剂、悬浮剂、粉剂、颗粒剂、可湿性粉剂或水分散粒剂等多种剂型。
本发明的第三方面，提供一种含有上述的希瓦氏菌(Shewanella sp.)CF8-6、海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403和柠檬色赤杆菌(Erythrobacter citreus)X3-1411的至少一种菌株的生物膜或生物膜反应器。
该生物膜具体是由人工填料或天然材料作为载体，柠檬色赤杆菌(Erythrobacter citreus)X3-1411附着絮凝其表面形成的膜状物。
本发明的第四方面，提供上述的菌株、菌剂、生物膜或生物膜反应器在废水除磷中的应用。
本发明所述菌株或菌剂可用于含盐废水或不含盐废水中磷去除，尤其是，本发明所述菌株或菌剂对于高盐度废水处理极为有效，比如海水冲厕废水，本发明所述高盐度废水中盐度可达15％，优选的，本发明所述菌株或菌剂用于盐度0％-10％的废水中磷的处理。
本发明的第五方面，提供一种含盐废水除磷的方法，步骤如下：
将上述菌株接种在LB培养基中，经活化培养后，制得活化菌液，将活化菌液按8-12％的体积分数加入到待处理的废水中；
或将上述菌株的菌剂按5-20mg/L的加入量加入到待处理的废水中。
上述菌株和/或菌剂在制备污水处理剂中的应用也是本发明的保护范围。
本发明的第六方面，提供上述能够利用废水进行微生物自组装合成纳米颗粒的高效好氧除磷菌或保藏编号为CCTCC M2013652的假交替单胞菌(Pseudoalteromonas sp.)DSBS在制备纳米材料中的应用，特别是在低磷条件下制备自组装纳米材料中的应用。
上述高效好氧除磷菌，能够利用废水中不同浓度的磷(包括高磷条件和低磷条件)进行微生物自组装合成纳米材料；特别是低磷条件下仍能进行微生物自组装合成纳米颗粒。
所述低磷条件是指磷的浓度为低饱和或者不饱和。
上述保藏编号为CCTCC M2013652的假交替单胞菌(Pseudoalteromonas sp.)DSBS已记载在发明人的另一项专利申请“一种高效去除废水中镉和磷的假交替单胞菌及其应用”(CN103740623A)中，在此基础上，发明人进行了一系列技术扩展研究，发现该菌株不仅能高效去除水体中的镉和磷，还能在μM级或nM级镉磷不饱和的低盐和高盐废水中生长，形成纳米颗粒。
本发明的第七方面，提供一种由上述菌株合成并自组装的生物纳米材料，该生物纳米材料由上述能够利用废水进行微生物自组装合成纳米颗粒的高效好氧除磷菌或保藏编号为CCTCC M2013652的假交替单胞菌(Pseudoalteromonas sp.)DSBS在含磷废水中合成并经自组装制备而成。
所述含磷废水中，磷元素的浓度为0.3mM-1.3mM。
本发明的第八方面，提供上述的自组装的生物纳米材料的制备方法，包括菌株活化的步骤，以及将活化后的菌株在含磷废水中进行培养及自组装的步骤。
上述制备方法中，所述菌株活化的步骤包括：将上述菌株接种于LB培养基中，180～220rpm，20-30℃，活化培养18-30h；优选的，活化培养条件为200rpm，25℃，活化培养24h。
所述LB培养基的配方为：1％蛋白胨，0.3％酵母粉，陈海水配制。
上述制备方法中，所述培养及自组装的步骤包括：将活化后的菌株接种于含磷废水中，180～220rpm，15～30℃，培养42-54h；优选的，200rpm，25℃，培养48h。
所述活化后的菌株的接种量为8-12％(v/v)。
上述培养及自组装的步骤，还包括：将培养后的培养液离心，去除上清液，得到含纳米材料的菌体，即为生物纳米材料。
所述离心的转速为5000rpm，离心时间为10min。
本发明中，所述含磷废水可以为海水冲厕废水或生活废水或含镉和磷的不/低饱和体系；所述含镉和磷的不/低饱和体系中，镉和磷的浓度为μM级至nM级。
本发明的第九方面，提供所述生物纳米材料的应用，其应用主要包括环境领域和生物医药领域的应用。
环境领域：去除水中的氟、吸附苯酚、去除铅镉或其它重金属和放射性废物。
其中，重金属元素可以与氨基酸侧链上的硫原子、氮原子发生作用，具有很高的毒性。随着环境污染事件的发生，环境重金属污染以及修复问题受到广泛关注。环境重金属污染现 有修复方法主要有物理修复、化学修复及生物修复的方法。其中化学修复需要向污染环境如土壤、水体等额外投加化学药剂使重金属离子发生吸附、氧化还原反应、沉淀等作用，此方法尽管操作简单效果明显但是易产生二次污染且费用较高。利用含磷材料对环境重金属污染进行修复是一种有效的方法。本发明的生物纳米材料包括由该菌体生成的纳米羟基磷灰石，所以其可以应用于环境重金属污染的修复，具有操作简单及成本较低的优势；并且，该生物纳米材料还包括活性菌体，菌体对重金属做进一步的吸附效果。
生物医药领域：本发明中去除有机物的纳米生物材料(去除有机物后保留纳米羟基磷灰石)在制备药物载体、抗肿瘤药物、硬组织修复材料、人工骨骼、人工牙齿中的应用。
纳米材料在生物医学药学、人类健康等生命科学领域有重大应用，如利用纳米颗粒作为载体输送药物至病灶部位、利用纳米材料做为生物医学检测诊断用材料等。磷酸钙盐如羟基磷灰石是动物及人体骨骼及牙齿的主要无机矿物成分，具有良好的活性与生物相容性，如羟基磷灰石陶瓷，是一种很有前景的人工骨及人工口腔材料。生物合成的纳米磷酸钙材料既具有纳米材料的特性，又具有更好的生物相容性和适应性，如纳米羟基磷灰石在生物医学领域具有广泛的应用前景。
本发明的第十方面，提供一种纳米羟基磷灰石的制备方法，将上述生物纳米材料提纯分离得到纳米羟基磷灰石，所述提纯分离的具体方法可以采用灼烧上述生物纳米材料，将其中的有机物去除，即可得到纳米羟基磷灰石。
采用上述方法得到的纳米羟基磷灰石材料的粒径分布均匀，通过控制制备方法的条件可以制备得到所需要的粒径大小和形貌尺寸，并且本发明的菌株采用自组装方式得到的纳米羟基磷灰石的成膜性较好，在制备薄膜材料具有较好的应用。
本发明的有益效果：
(1)本发明所述的菌株对环境条件有很高的适应性，可以在不含盐、高盐以及广pH、温度、营养范围内生长，而且可以高效去除废水中的磷，使废水中磷降至0.5mg/L以下，达到排放标准，尤其是本发明所述菌株对高盐度废水如海水冲厕废水具有良好的除磷净化作用，对于解决淡水资源短缺以及建立有效的海水冲厕废水利用体系，具有良好的利用价值。
(2)本发明所述菌株在去除磷的过程中只需要活化后接种到含磷废水中培养就可以，步骤简单，操作容易，成本低廉；
(3)本发明所述的菌株可以在单一好氧条件下实现磷的去除，简化了磷的去除过程，提高了除磷过程的可操作性，为生物除磷提供了新途径；
(4)本发明所述菌株是在不/低饱和废水体系中以金属磷酸盐(本发明体系中为钙磷沉 淀)的沉淀的形式实现磷的去除的。
(5)本发明的菌株具有自絮凝和自组装的功能，菌株在降解废水中污染物的同时，还可以利用废水中的原料在低浓度条件下合成钙磷纳米颗粒，且具有纳米自组装的能力，不需要额外投加化学试剂，环境友好成本低，实现了磷资源的循环利用。
(6)本发明的生物纳米材料的制备方法，条件温和，操作简单，清洁无污染，成本低廉，效率高且能大规模推广应用。
(7)本发明制备的生物纳米材料，纳米颗粒分布于细菌细胞表面及周围，利用该生物纳米材料去除水中的氟，吸附苯酚，去除铅镉等重金属和放射性废物的清理时，去除效果得到显著的提高。将生物纳米材料的菌体去除后，可以形成多孔纳米材料，可以作为药物载体等方面的应用。
附图说明：
图1：菌株CF8-6的系统发育树；
图2：海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403革兰氏染色结果图；
图3：海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403菌体形态AFM图；
图4：柠檬色赤杆菌(Erythrobacter citreus)X3-1411革兰氏染色结果图；
图5：柠檬色赤杆菌(Erythrobacter citreus)X3-1411菌体形态AFM图；
图6a：CF8-6在不同盐度下的生长曲线；
图6b：CF8-6在不同盐度下的磷去除率；
图7：Psychrobacter aquimaris X3-1403在模拟海水冲厕废水中对TP、COD、NH4 +-N和TN的去除效果；
图8：柠檬色赤杆菌(Erythrobacter citreus)X3-1411在模拟海水冲厕废水中对TP、COD、NH4 +-N和TN的去除效果；
图9：菌株CF8-6在废水配方(一)条件下合成的纳米材料图片(AFM)；
图10：菌株CF8-6在废水配方(二)条件下合成的纳米材料图片，图10a:原子力显微镜图片(AFM)，图10b、图10c:纳米颗粒透射电镜图片(TEM)，图10d、图10e、图10f:纳米颗粒自组装透射电镜；
图11：菌体Psychrobacter aquimaris X3-1403在模拟海水冲厕废水中的自絮凝的电镜照片；
图12：菌体Psychrobacter aquimaris X3-1403在模拟海水冲厕废水中的自组装及合成纳米材料的电镜照片；
图13：菌体柠檬色赤杆菌(Erythrobacter citreus)X3-1411在模拟海水冲厕废水中的自絮凝电镜照片；
图14：菌体柠檬色赤杆菌(Erythrobacter citreus)X3-1411在模拟海水冲厕废水中的合成纳米材料的透射电镜照片。
图15：菌株假交替单胞菌(Pseudoalteromonas sp.)DSBS在低盐废水中形成纳米颗粒的原子力显微镜图像(A)、扫描电镜图像(B、C、D)和能谱分析(E)。
图16：菌株假交替单胞菌(Pseudoalteromonas sp.)DSBS在高盐废水中的磷去除情况。
图17：菌株在高盐废水中形成纳米颗粒的原子力显微镜图像(A)、扫描电镜图像(B)和能谱分析(C)。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本申请提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本申请的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案，以下将结合具体的实施例详细说明本申请的技术方案。
本发明实施例中所用的试验材料均为本领域常规的试验材料，均可通过商业渠道购买得到。
实施例1：能够在低磷条件下进行微生物自组装的高效除磷菌的分离及鉴定
1.菌株的分离
(1)将取自于中国南海的464株菌株在海水LB液体培养基中培养24h(200rpm、25℃)。然后静置15min，观察菌体是否能自絮凝。
(2)将能自絮凝的菌株在5000rpm下离心10min，弃去上清液，用去离子水清洗两遍菌体，然后在透射电镜下观察菌体表面是否有纳米级颗粒产生。
(3)筛选得到的能够自絮凝且菌体表面产生纳米级颗粒的菌株，将筛选的能够自絮凝且菌体表面产生纳米级颗粒的菌株按10％的接种量接种至模拟海水冲厕废水或模拟高盐生活废水中，按时间点取样测定菌株对废水中TP和COD的去除效果，选择除磷速度和除磷率均较 优的菌株作为目标菌株。
上述分离方法中，海水LB液体培养基的配方为：1％蛋白胨，0.3％酵母粉，陈海水配制。
模拟海水冲厕废水的配方组成如表1所示。
表1：上述模拟海水冲厕废水配方为(陈海水配制):
Figure PCTCN2017075497-appb-000001
模拟高盐生活废水成分如下：
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。
所有培养基在使用之前均121℃高温灭菌20min。接种在洁净工作台中进行。菌种保存在1.5mL离心管中(含600uL菌液与300uL甘油)，在超低温冰箱中-80℃长期保存。
经筛选分离，得到3株既能够自絮凝且菌体表面产生纳米级颗粒、还具有较好的除磷速度和除磷率的菌株，菌株编号分别为：菌株CF8-6、菌株X3-1403、菌株X3-1411。
2.菌株的鉴定
对上述筛选分离得到的3株菌进行鉴定，具体如下：
2.1菌株CF8-6的鉴定
2.1.1生理生化表征：
该菌株的生理生化表征：菌株CF8-6可在温度5-35℃，pH5.8-9.8，盐度0-12％、严格好氧的培养条件下生长，且具有较好的磷去除效果，菌体形态特征为革兰氏染色呈阴性，电子显微镜下观察为杆菌，有荚膜及鞭毛。菌株固体培养24小时菌落特征为圆形，乳白色。
2.1.2分子生物学鉴定：
该菌株的分子生物学鉴定：
使用试剂盒提取菌株CF8-6的DNA，通过PCR进行扩增16S rDNA序列，得出菌株CF8-6的16S rDNA序列，如序列表SEQ ID NO:1所示。用BLAST程序对菌株CF8-6的16S rDNA序列和GenBank中已登录的16S rNDA序列进行核苷酸同源性比较，得知菌株CF8-6属于希瓦氏菌属(Shewanella)，因此将该菌命名为Shewanella sp.CF8-6，该菌株的系统发育树如图1所示。
该希瓦氏菌(Shewanella sp.)CF8-6，于2016年3月29日保藏于中国典型培养物保藏中心，保藏地址为：湖北省武汉市武昌珞珈山，保藏号为：CCTCC M 2016154。
2.2菌株X3-1403的鉴定
2.2.1生理生化表征：
该菌株的生理生化表征：菌株X3-1403可在15～30℃，pH值7～8，盐度为0～12％(最佳1％～5％)培养条件下生长，菌体形态特征为革兰氏染色呈阴性，如图2所示，电子显微镜下观察为球菌或短杆菌，单独、成双或聚集成团，有荚膜、无鞭毛。菌株固体LB培养24h菌落特征为圆形，光滑，奶油色，如图3所示。
2.2.2分子生物学鉴定：
16S rDNA序列分析
菌株X3-1403的16s rDNA的序列如SEQ ID NO.2所示。将所测16S rDNA核苷酸序列与输入到NCBI网站的GenBank数据库中的进行相似性序列比对后，结果表明：海洋嗜冷杆菌(Psychrobacter aquimaris)X3-1403与Psychrobacter同处于一个最小分支上，其16S rDNA序列与Psychrobacter aquimaris的相似性达99.64％。结合菌落形态和16S rDNA序列分析，鉴定为海洋嗜冷杆菌(Psychrobacter aquimaris)。
通过菌株X3-1403的分子鉴定结果，进一步确认菌株Psychrobacter aquimaris X3-1403为海洋嗜冷杆菌属(Psychrobacter aquimaris)，将海洋嗜冷杆菌(Psychrobacter aquimaris)X3-1403保藏于中国典型培养物保藏中心，地址：武汉市武昌珞珈山，保藏号：CCTCC M2016155。
2.3菌株X3-1411的鉴定
2.3.1生理生化表征：
菌株X3-1411的主要生物学特性为：革兰氏染色呈阴性(结果如图4所示)，电子显微镜下观察为杆菌，单独、成双或短链状，有荚膜、无鞭毛(结果如图5所示)。菌株固体LB培养24h菌落特征为圆形，光滑，黄色。
该菌株可在15～30℃，pH值7～8，盐度为0～12％(最佳1％～5％)培养条件下生长。
2.3.2分子生物学鉴定：
菌株X3-1411的16s rDNA的序列如SEQ ID NO.3所示。将该序列在GenBank数据库进行BLAS(网址：http://blast.ncbi.nlm.nih.gov/Blast.cgi)比对,结果该序列与Erythrobacter citreus菌株的相似性为99.26％。
基于菌株的生物学特性分析和16s rDNA的同源性比对结果，将菌株X3-1411鉴定为柠 檬色赤杆菌(Erythrobacter citreus)，已于2016年3月29日保藏于中国典型培养物保藏中心，地址：武汉市武昌珞珈山，保藏号：CCTCC M 2016156。
实施例2：本发明的菌株的除磷效果研究
一、菌株Shewanella sp.CF8-6的废水除磷试验
菌株Shewanella sp.CF8-6在水处理中的应用方法如下：
(1)将菌株Shewanella sp.CF8-6在海水LB液体培养基中在25℃，200rpm的条件下培养24小时，制得活化菌液。
(2)将步骤(1)得到的活化菌液以10％的比例接种到模拟废水中不同盐度的废水中(盐度范围在0％-20％)，在25℃，200rpm的条件下培养，在不同时间测定上清液中磷的浓度以及在波长600nm处测生物量，得到菌株对磷的去除效率以及菌株在不同盐度范围下的生长曲线，水处理效果数据如图6a和图6b所示。
由图6a和图6b可以看出，本发明的菌株Shewanella sp.CF8-6对于含盐废水中的磷去除效率非常高，尤其是在10％及以下盐度的废水中，10小时内磷的去除率可达99％以上；甚至对于12％、15％盐度的废水，本发明菌株Shewanella sp.CF8-6也有极佳的磷去除率。
上述模拟废水配方与筛选菌株时废水配方除盐度值外其余成分浓度相同，磷浓度(以P计)为10mg/L。
二、海洋嗜冷杆菌(Psychrobacter aquimaris)X3-1403在含磷盐水处理中的应用
将海洋嗜冷杆菌(Psychrobacter aquimaris)X3-1403在LB中培养24h后，以10％的接种量接种到模拟海水冲厕废水中，并按时间点取样测定该菌株对TP、COD、NH4 +-N和TN的去除效果。从图7中可以看到，该菌株对TP和COD具有较高的去除效果，去除率分别为70.5％和75.5％，48h的去除速率分别为0.57mg/(L·h)和18.7mg/(L·h)。而该菌对NH4 +-N和TN的去除效果较差，只有17.8％和19.4％。
三、柠檬色赤杆菌(Erythrobacter citreus)X3-1411的除磷效果研究
1.菌株X3-1411对模拟海水冲厕废水的除磷效果研究
将实施例1分离筛选的菌株柠檬色赤杆菌(Erythrobacter citreus)X3-1411在LB培养基上培养24h后，以10％的接种量接种到模拟海水冲厕废水中，25℃、200rpm进行培养，并按时间点取样，测定该菌株对TP、COD、NH4 +-N和TN的去除效果。结果如图8所示。
由图8可以看出，该菌株对TP和COD具有较高的去除效果，去除率分别为75.0％和83.6％，48h的去除速率分别为0.59mg/(L·h)和24.9mg/(L·h)。而该菌对NH4 +-N和TN的去除效果较差，只有17.2％和25.9％。
本实施例中模拟海水冲厕废水的配方组成同实施例3。
2.菌株X3-1411对模拟生活废水的除磷效果研究
表2：模拟生活废水的配方组成为(去离子水配制)：
Figure PCTCN2017075497-appb-000002
实施例5：本发明的菌株在制备自组装纳米材料中的应用
一、利用希瓦氏菌株(Shewanella sp.)CF8-6制备纳米材料
(1)将菌株Shewanella sp.CF8-6在海水LB液体培养基中在25℃，200rpm的条件下培养24小时，制得活化菌液；活化菌液离心参数：10000rpm条件下离心10min；
(2)将步骤(1)得到的菌液离心洗涤后按10％(v/v)的比例接进模拟废水中，在25℃，200rpm的条件下培养48小时，离心即得到菌体，纳米颗粒分布于细胞表面及其周围。离心转速为4000rpm，时间为10分钟；
(3)将步骤(2)得到的含有纳米颗粒的菌体用去离子水洗两遍，转速为4000rpm,时间为15分钟。然后拿去做透射电镜(TEM)。
(4)将步骤(2)得到的含有纳米颗粒的菌体处理后去做原子力显微镜，观察颗粒形状及大小。
模拟高盐废水配方(一)(低磷废水)：C6H12O6·H2O 1.5g/L，CH3COONa 0.75g/L,MgSO4·7H2O 1.18g/L,NH4Cl 0.9g/L,KH2PO4·2H2O 0.066g/L(以P 10mg/L计:),NaCl 30g/L，用自来水溶解。
模拟高盐废水配方(二)(高磷废水):C6H12O6·H2O 1.5g/L，CH3COONa 0.75g/L,MgSO4·7H2O 1.18g/L,NH4Cl 0.9g/L,甘油磷酸二钠盐(C3H6NaO7P，以P计：50mg/L),NaCl30g/L，CaCl2(以Ca计：80mg/L)，用去离子水溶解。
其制备的纳米材料图片分别见图9和图10，其中图9为菌株CF8-6在废水配方(一)条 件下合成的纳米材料图片(AFM)，图10为菌株CF8-6在废水配方(二)条件下合成的纳米材料图片，图10a:原子力显微镜图片(AFM)，图10b、图10c:纳米颗粒透射电镜图片(TEM)，图10d、图10e、图10f:纳米颗粒自组装透射电镜。
本发明的纳米材料中，钙磷纳米颗粒粒径为100-200nm。
二、海洋嗜冷杆菌(Psychrobacter aquimaris)X3-1403在制备纳米材料纳米羟基磷灰石中的应用
将海洋嗜冷杆菌(Psychrobacter aquimaris)X3-1403在LB培养基中活化24h，其培养条件为200rpm、25℃。然后①在无菌操作台中用事先灭菌的离心管取25mL活化的培养液，10000rpm离心10min；②去其上清液，然后用10mL的灭菌去离子水重悬菌液，10000rpm离心10min；③重复步骤②一次。将重悬的菌液分别接种到模拟海水冲厕废水(配方组成同表1)和模拟生活废水(配方组成同表2)中(接种量10％)，200rpm、25℃培养48h。将培养液在5000rpm下离心10min，弃去上清液，离心管底部的菌体用去离子水清洗两遍，即得含纳米材料的菌体。重悬一部分该菌体，在铜网上固定并染色，最后烘干后用于透射电镜观察。
上述LB培养基配方为：1％蛋白胨，0.3％酵母粉，陈海水配制。
菌体在模拟海水冲厕废水中的自絮凝的电镜照片如图11。菌体在模拟海水冲厕废水中合成纳米材料并进行自组装的电镜照片如图12，图11和图12显示该纳米颗粒材料呈现蜂巢状，粒径为纳米级且分布均匀，结构紧凑，易制成层状纳米材料。
三、菌株Erythrobacter citreus X3-1411在制备纳米材料中的应用
菌株Erythrobacter citreus X3-1411在LB中活化24h，其培养条件为200rpm、25℃。然后①在无菌操作台中用事先灭菌的离心管取25mL活化的培养液，10000rpm离心10min；②去其上清液，然后用10mL的灭菌去离子水重悬菌液，10000rpm离心10min；③重复步骤②一次。将重悬的菌液分别接种到模拟海水冲厕废水(配方组成同表1)和模拟生活废水(配方组成同表2)中(接种量10％)，200rpm、25℃培养48h。将培养液在5000rpm下离心10min，弃去上清液，离心管底部的菌体用去离子水清洗两遍，即得含纳米材料的菌体。重悬一部分该菌体，在铜网上固定并染色，最后烘干后用于透射电镜观察。
上述LB配方为：1％蛋白胨，0.3％酵母粉，陈海水配制。
菌体在模拟海水冲厕废水中的自絮凝的电镜照片如图13。菌体在模拟海水冲厕废水中的合成纳米材料及自组装的透射电镜照片如图14，图13和图14显示该材料的分散性好、粒径分布均匀。
四、假交替单胞菌(Pseudoalteromonas sp.)DSBS在制备纳米材料中的应用
1.假交替单胞菌(Pseudoalteromonas sp.)DSBS在低盐废水中制备纳米材料
(1)将假交替单胞菌(Pseudoalteromonas sp.)DSBS按0.9％(v/v)接种于液体LB培养基中，在25℃、200rpm恒温摇床中培养20h，得到富集的菌体。
(2)将富集的菌悬液按10％(v/v)接种于低盐废水中，在25℃、200rpm恒温摇床中培养48h，即得到含有镉-磷-硫纳米颗粒的菌悬液。
(3)将含有镉-磷-硫纳米颗粒的菌悬液用去离子水离心洗涤。离心速度为3000rpm，采用低速离心防止菌体表面颗粒物洗脱，制备得到纳米材料。
所述的液体LB培养基组分如下：
蛋白胨10g/L，酵母粉3g/L，陈海水配制，海水盐度为3.5％。
所述的低盐废水组分如下：
一水合葡萄糖5.06g/L，NaAC 1.5g/L，NaCl 3.5g/L，NH4Cl 2.6g/L，MgSO4·7H2O 2.4g/L，总镉(Cd(NO3)2·4H2O)和总磷(K2HPO4)浓度分别为8mg/L和9mg/L，初始pH 7.2，盐度0.35％。为模拟低盐废水环境，其中镉和磷浓度为一般工业含镉废水中的浓度，在该pH和离子浓度下，不足以形成无机化学镉沉淀，属于镉磷的μM级不饱和低盐环境。
为进一步表征菌体及其形成的纳米颗粒，使用原子力显微镜和扫描电镜观察去除磷镉后的菌体。原子力显微镜观察结果如图15中A所示，菌体表面均匀聚集着纳米颗粒物质，少数颗粒脱落分散在四周，颗粒直径为25-100nm。图中部分菌体有鞭毛，部分没有，可能是在培养或样品预处理过程中脱落。
扫描电镜观察菌体的预处理过程中，磷酸缓冲液洗涤的离心速度为3000rpm，采用低速离心防止菌体表面颗粒物洗脱，其他离心速度为6000rpm，结果如图15中B、C、D所示。图15B中，直径在25-60nm的较小颗粒聚集成直径为100nm的均匀球形，粘附在菌体表面。图15C中，较小颗粒聚集在胞外多糖的纤维上。图15D中，较小颗粒成堆聚集在菌体之间。这说明细菌合成的纳米颗粒有三种不同的形态：均匀分散在菌体表面的直径25-60nm的较小颗粒，粘附在菌体表面的直径100nm的较小颗粒球形聚集体，聚集体脱离细菌成堆粘附在菌体之间的多糖纤维上。同时对图中点位的能谱分析表明，颗粒主要含有C、O、Cd、P、S元素。这说明该纳米颗粒是夹杂多糖的镉-磷-硫纳米颗粒。其中Na是析出的可溶性离子，Al是样品台元素，均不是纳米颗粒中的元素。
因此，该菌在磷镉浓度不饱和的低盐废水中可实现磷镉的同时去除，并在胞外形成具有三种形态的夹杂多糖的镉-磷-硫纳米颗粒，直径为25-60nm。
2.假交替单胞菌(Pseudoalteromonas sp.)DSBS在高盐废水中制备纳米材料
(1)将假交替单胞菌(Pseudoalteromonas sp.)DSBS按0.9％(v/v)接种于液体LB培养基中，在25℃、200rpm恒温摇床中培养20h，得到富集的菌体。
(2)将富集的菌体3000rpm离心10min，弃去上清液，再用高盐废水重悬至原体积，得到洗涤一次的菌体。
(3)将洗涤后的菌悬液按0.2‰(v/v)接种于三组高盐废水中，在25℃、200rpm恒温摇床中培养48h，即得到含有镉-磷-硫纳米颗粒的菌悬液。
(4)将含有镉-磷-硫纳米颗粒的菌悬液用去离子水洗涤，离心速度为3000rpm，采用低速离心防止菌体表面颗粒物洗脱，制备得到纳米材料。
根据本发明优选的，所述的液体LB培养基组分如下：
蛋白胨10g/L，酵母粉3g/L，陈海水配制，海水盐度为3.5％。
所述的高盐废水组分如下：
NaAC 0.82g/L，NH4Cl 0.11g/L，海水素33.33g/L，初始pH 7.2，盐度3.5％。三组合成海水的总镉(Cd(NO3)2·4H2O)和总磷(K2HPO4)浓度有差异，分别为A组0.1756×10-3mg/L和0.1548mg/L，B组5.671×10-3mg/L和5mg/L，C组10.21×10-3mg/L和9mg/L。为模拟高盐废水环境，其中镉和磷浓度为海水中的浓度，属于镉的nM级不饱和高盐环境。
在三组废水中，用钼酸盐分光光度法测定上清液中总磷的浓度变化，结果如图16所示。在高盐环境中，该菌对9mg/L和5mg/L的磷分别有46.24％和72.48％的去除率。
为进一步表征菌体及其形成的纳米颗粒，同样使用原子力显微镜和扫描电镜观察菌体。原子力显微镜结果如图17A所示，菌体表面均匀分散着纳米颗粒物质，直径均在10nm以下。合成海水中磷镉的浓度在nM级，更低的镉浓度导致了纳米颗粒的粒径更小。另外，此视野中菌体无鞭毛，可能由于断裂而未观察到。
扫描电镜结果如图17B所示，菌体间有直径10nm的球形颗粒，并粘附在纤维状的胞外多糖上。同时对图中点位的能谱分析(图17C)表明，颗粒同样主要含有C、O、Cd、P、S元素。
因此，该菌在磷镉浓度不饱和的高盐废水中也可在胞外形成夹杂多糖的镉-磷-硫纳米颗粒，直径为10nm。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。
Figure PCTCN2017075497-appb-000003
Figure PCTCN2017075497-appb-000004
Figure PCTCN2017075497-appb-000005

Claims (17)

  1. 一类能够利用废水进行微生物自组装合成纳米颗粒的高效好氧除磷菌，其特征在于，菌株包括：希瓦氏菌(Shewanella sp.)CF8-6、海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403和柠檬色赤杆菌(Erythrobacter citreus)X3-1411；
    所述希瓦氏菌(Shewanella sp.)CF8-6已于2016年3月29日保藏于中国典型培养物保藏中心，保藏地址为：湖北省武汉市武昌珞珈山，保藏号为：CCTCC M 2016154；
    所述海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403已于2016年3月29日保藏于中国典型培养物保藏中心，地址：武汉市武昌珞珈山，保藏号：CCTCC M 2016155；
    所述柠檬色赤杆菌(Erythrobacter citreus)X3-1411已于2016年3月29日保藏于中国典型培养物保藏中心，地址：武汉市武昌珞珈山，保藏号：CCTCC M 2016156；
  2. 一种菌剂，该菌剂的活性成分为权利要求1所述的希瓦氏菌(Shewanella sp.)CF8-6、海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403和柠檬色赤杆菌(Erythrobacter citreus)X3-1411的至少一种。
  3. 如权利要求2所述的菌剂，其特征在于，所述菌剂还包括载体；所述载体为固体载体或液体载体。
  4. 如权利要求2所述的菌剂，其特征在于，所述活性成分以被培养的活细胞、活细胞的发酵液、细胞培养物的滤液或细胞与滤液的混合物的形式存在。
  5. 如权利要求2所述的菌剂，其特征在于，所述菌剂的剂型为液剂、悬浮剂、粉剂、颗粒剂、可湿性粉剂或水分散粒剂。
  6. 含有权利要求1所述的希瓦氏菌(Shewanella sp.)CF8-6、海水嗜冷杆菌(Psychrobacter aquimaris)X3-1403和柠檬色赤杆菌(Erythrobacter citreus)X3-1411的至少一种菌株的生物膜或生物膜反应器。
  7. 权利要求1所述的菌株、权利要求2-5任一项所述的菌剂和/或权利要求6所述的生物膜或生物膜反应器在废水除磷中的应用。
  8. 一种含盐废水除磷的方法，其特征在于，步骤如下：
    将权利要求1所述的菌株接种在LB培养基中，经活化培养后，制得活化菌液，将活化菌液按8-12％的体积分数加入到待处理的废水中；
    或将权利要求2-5任一项所述的菌剂按5-20mg/L的加入量加入到待处理的废水中。
  9. 权利要求1所述的菌株和/或权利要求2-5任一项所述的菌剂在制备污水处理剂中的应用。
  10. 权利要求1所述的菌株或保藏编号为CCTCC M2013652的假交替单胞菌 (Pseudoalteromonas sp.)DSBS在制备自组装纳米材料中的应用。
  11. 一种自组装的生物纳米材料，由权利要求1所述的菌株或保藏编号为CCTCC M2013652的假交替单胞菌(Pseudoalteromonas sp.)DSBS在含磷废水中合成并经自组装制备而成。
  12. 如权利要求11所述的自组装的生物纳米材料，其特征在于，所述含磷废水中，磷元素的浓度为0.3mM-1.3mM。
  13. 权利要求11或12所述的自组装的生物纳米材料的制备方法，其特征在于，包括菌株活化的步骤，以及将活化后的菌株在含磷废水中进行培养及自组装的步骤。
  14. 如权利要求13所述的制备方法，其特征在于，上述培养及自组装的步骤，还包括：将培养后的培养液离心，去除上清液，得到含纳米材料的菌体，即为生物纳米材料。
  15. 权利要求11或12所述微生物自组装纳米材料的用途，包括
    (1)在环境重金属污染修复中的应用；
    或(2)在制备生物医药利用的载体或材料中的应用。
  16. 一种纳米羟基磷灰石的制备方法，其特征在于，将权利要求11或12所述微生物自组装纳米材料提纯分离得到纳米羟基磷灰石，将其中的有机物去除，即得到纳米羟基磷灰石。
  17. 如权利要求16所述的制备方法，其特征在于，所述提纯分离采用的方法为灼烧所述微生物自组装纳米材料。
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