CN110029064B - Method for separating and purifying water body microorganism bacteria and sampling device - Google Patents
Method for separating and purifying water body microorganism bacteria and sampling device Download PDFInfo
- Publication number
- CN110029064B CN110029064B CN201910350968.XA CN201910350968A CN110029064B CN 110029064 B CN110029064 B CN 110029064B CN 201910350968 A CN201910350968 A CN 201910350968A CN 110029064 B CN110029064 B CN 110029064B
- Authority
- CN
- China
- Prior art keywords
- culture medium
- solution
- separating
- mixed system
- purified
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 42
- 244000005700 microbiome Species 0.000 title claims abstract description 19
- 238000005070 sampling Methods 0.000 title abstract description 14
- 241000894006 Bacteria Species 0.000 title description 27
- 239000001963 growth medium Substances 0.000 claims abstract description 136
- 239000003094 microcapsule Substances 0.000 claims abstract description 42
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims abstract description 26
- 239000000725 suspension Substances 0.000 claims abstract description 24
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000000661 sodium alginate Substances 0.000 claims abstract description 21
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 21
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 21
- 238000009630 liquid culture Methods 0.000 claims abstract description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 18
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 18
- 230000001580 bacterial effect Effects 0.000 claims abstract description 17
- 229910000019 calcium carbonate Inorganic materials 0.000 claims abstract description 13
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 10
- 239000000194 fatty acid Substances 0.000 claims abstract description 10
- 229930195729 fatty acid Natural products 0.000 claims abstract description 10
- 229940057995 liquid paraffin Drugs 0.000 claims abstract description 10
- -1 sorbitan fatty acid ester Chemical class 0.000 claims abstract description 10
- 229960000583 acetic acid Drugs 0.000 claims abstract description 9
- 239000012362 glacial acetic acid Substances 0.000 claims abstract description 9
- 239000002244 precipitate Substances 0.000 claims abstract description 7
- 238000004140 cleaning Methods 0.000 claims abstract description 3
- 239000000499 gel Substances 0.000 claims description 64
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 39
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 26
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 26
- 239000007787 solid Substances 0.000 claims description 24
- 230000001954 sterilising effect Effects 0.000 claims description 21
- 238000004659 sterilization and disinfection Methods 0.000 claims description 21
- 238000003756 stirring Methods 0.000 claims description 20
- 239000000835 fiber Substances 0.000 claims description 19
- 239000012528 membrane Substances 0.000 claims description 17
- 238000002156 mixing Methods 0.000 claims description 14
- 239000013049 sediment Substances 0.000 claims description 13
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 11
- 239000001301 oxygen Substances 0.000 claims description 10
- 229910052760 oxygen Inorganic materials 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 8
- 239000001110 calcium chloride Substances 0.000 claims description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 7
- 238000001816 cooling Methods 0.000 claims description 7
- 239000007789 gas Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000000017 hydrogel Substances 0.000 claims description 3
- 230000004083 survival effect Effects 0.000 claims description 2
- 238000000926 separation method Methods 0.000 abstract description 18
- 230000000813 microbial effect Effects 0.000 abstract description 15
- 238000004945 emulsification Methods 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 128
- 238000011065 in-situ storage Methods 0.000 description 43
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 17
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 16
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 235000015097 nutrients Nutrition 0.000 description 16
- 239000011573 trace mineral Substances 0.000 description 16
- 235000013619 trace mineral Nutrition 0.000 description 16
- 230000000694 effects Effects 0.000 description 13
- 238000000746 purification Methods 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 8
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 8
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 8
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 8
- 239000007836 KH2PO4 Substances 0.000 description 8
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 8
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 8
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 8
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 8
- 239000008272 agar Substances 0.000 description 8
- 229910052796 boron Inorganic materials 0.000 description 8
- 229940041514 candida albicans extract Drugs 0.000 description 8
- 229910052802 copper Inorganic materials 0.000 description 8
- 239000010949 copper Substances 0.000 description 8
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 8
- 229910052742 iron Inorganic materials 0.000 description 8
- 239000011777 magnesium Substances 0.000 description 8
- 229910052749 magnesium Inorganic materials 0.000 description 8
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 8
- 229910052750 molybdenum Inorganic materials 0.000 description 8
- 239000011733 molybdenum Substances 0.000 description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 8
- 235000019345 sodium thiosulphate Nutrition 0.000 description 8
- 239000012138 yeast extract Substances 0.000 description 8
- 239000011701 zinc Substances 0.000 description 8
- 229910052725 zinc Inorganic materials 0.000 description 8
- 238000010438 heat treatment Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000000576 coating method Methods 0.000 description 5
- 238000011068 loading method Methods 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- TVEXGJYMHHTVKP-UHFFFAOYSA-N 6-oxabicyclo[3.2.1]oct-3-en-7-one Chemical compound C1C2C(=O)OC1C=CC2 TVEXGJYMHHTVKP-UHFFFAOYSA-N 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 235000016709 nutrition Nutrition 0.000 description 4
- 230000035764 nutrition Effects 0.000 description 4
- 230000001376 precipitating effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000009777 vacuum freeze-drying Methods 0.000 description 4
- 238000004804 winding Methods 0.000 description 4
- 238000005273 aeration Methods 0.000 description 3
- 238000010979 pH adjustment Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 238000003723 Smelting Methods 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000005536 corrosion prevention Methods 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 239000002068 microbial inoculum Substances 0.000 description 2
- 235000010755 mineral Nutrition 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 239000010413 mother solution Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000003911 water pollution Methods 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- YNCRBFODOPHHAO-YUELXQCFSA-N Phaseic acid Natural products CC(=CC(=O)O)C=C[C@@H]1[C@@]2(C)CO[C@@]1(C)CC(=O)C2 YNCRBFODOPHHAO-YUELXQCFSA-N 0.000 description 1
- 230000009102 absorption Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 238000003320 cell separation method Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000008717 functional decline Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000007269 microbial metabolism Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- IZGYIFFQBZWOLJ-UHFFFAOYSA-N neophaseic acid Natural products C1C(=O)CC2(C)OCC1(C)C2(O)C=CC(C)=CC(O)=O IZGYIFFQBZWOLJ-UHFFFAOYSA-N 0.000 description 1
- IZGYIFFQBZWOLJ-CKAACLRMSA-N phaseic acid Chemical compound C1C(=O)C[C@@]2(C)OC[C@]1(C)[C@@]2(O)C=CC(/C)=C\C(O)=O IZGYIFFQBZWOLJ-CKAACLRMSA-N 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009103 reabsorption Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
-
- 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
- C12N1/02—Separating microorganisms from their culture media
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Sustainable Development (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for separating and purifying water body microorganisms and a sampling device, which comprises the following steps of 1, adding a sample to be separated and purified and a calcium carbonate solution into a sodium alginate solution to obtain a precipitate and a suspension; step 2, adding the suspension into an oil phase A consisting of sorbitan fatty acid ester and liquid paraffin to obtain a mixed system A; step 3, adding 6-10 ml of glacial acetic acid into the mixed system A, reacting for 8-12 min at 22-25 ℃ to obtain a mixed system B, adding a calcium chloride solution into the mixed system B to obtain microcapsules, and cleaning the microcapsules with sterilized water to obtain a microcapsule strain library; step 4, inoculating the microcapsule strain libraries into a porous plate one by one to culture to obtain a single bacterial colony, wherein the porous plate is filled with BM liquid culture medium solution of strains to be separated and purified; the invention combines the emulsification micro-balloon method and the porous plate method, and can be used for the establishment and separation of the environmental functional microbial ecological germplasm resources.
Description
Technical Field
The invention relates to the field of water body microorganism separation culture, in particular to a method for separating and purifying water body microorganisms and a sampling device.
Background
The purification of microbial strains refers to a process of separating different types of microbes mixed together to obtain a single strain required by production and research, is a key technology for the application and research of microbial industry, and is widely applied to the industrial fields and experimental research fields of microbial corrosion prevention, water pollution control, mineral smelting, functional microbial inoculum development and the like. During specific purification, the strain to be purified is prepared into a corresponding culture medium according to needs and is separated independently. In the purification of microbial strains, the existing methods mainly comprise ten times of dilution separation methods, plate scribing methods, plate coating methods, puncture separation methods, single cell separation methods, ultramicromembrane separation methods and the like, but the methods are often fine in process and complicated in procedure, and particularly require operators to have quite high operation levels, so that the purification efficiency of pure microbial strains is greatly reduced, and partial microbial strains are lost.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for separating and purifying water body microorganisms and a sampling device, which are accurate, simple, convenient, efficient and easy to operate, and can separate and purify strains in the water body microorganisms at high flux to obtain single colonies.
The invention is realized by the following technical scheme:
a method for separating and purifying water body microorganism strains comprises the following steps,
and 4, inoculating the microcapsule strain libraries into a porous plate one by one to culture to obtain a single bacterial colony, wherein the porous plate is filled with BM liquid culture medium solution of strains to be separated and purified.
Preferably, step 2 is to add the suspension into the oil phase A at a speed of 300-350 r/min while stirring, and stir for 4-8 min to obtain a mixed system A.
Preferably, the multi-well plate in step 3 is a 96-well plate.
Preferably, the sample to be separated and purified in step 1 is obtained by the following steps,
step 1a, uniformly dispersing polyvinyl alcohol fiber yarns into a NaOH solution, stirring for 8-12 hours at 50-60 ℃, and collecting the polyvinyl alcohol fiber yarns in the NaOH solution to obtain modified polyvinyl alcohol fiber yarns;
step 1b, uniformly mixing the modified polyvinyl alcohol cellosilk with BM solid culture medium solution of strains to be separated and purified to obtain a mixed system C, and carrying out high-pressure steam sterilization on the mixed system C to obtain the culture medium solution after high-pressure steam sterilization;
step 1c, cooling the culture medium solution sterilized by high-pressure steam in an anaerobic workbench at room temperature to obtain gel, keeping the gel at-80 ℃ for 15-30 min, then carrying out gradient temperature rise at-80-0 ℃ for 80-120 min to obtain a culture medium gel strip with the water content of 10% -20%, and putting the culture medium gel strip into an acquisition device with an opening in the outer wall to obtain the acquisition device containing the culture medium gel strip;
and step 1d, inserting the collection device containing the culture medium gel strip into the sediment for a set time, wherein the culture medium gel strip with the water content of 10-20% in the collection device absorbs water and expands into hydrogel in the enrichment process, and thus a sample to be separated and purified is obtained.
Further, in step 1b, N is introduced in a volume ratio of 80:202/CO2The mixed gas in the mixed system C is subjected to oxygen discharge until reaching the standard required by the survival of the strains to be separated and purified, and then high-pressure steam sterilization is carried out.
And further, step 1c, pouring the culture medium solution sterilized by high-pressure steam into two semicircular column grooves with equal radius and length, cooling at room temperature in an anaerobic workbench, keeping at-80 ℃ and carrying out gradient heating at-80-0 ℃ to obtain two culture medium gel strips with the same radius and length and the water content of 10-20%, splicing the two culture medium gel strips into a cylinder, and filling the cylinder into a collecting device.
Furthermore, the cylinder is wound with a layer of filter membrane and then is arranged in the acquisition device.
Furthermore, the filter membrane is of a micron-scale specification.
The utility model provides a collection system of water microorganism fungus separation and purification, collection system be hollow structure, constitute by two parts, the first half is the cylinder, the latter half is the circular cone, the cylinder is the same with the diameter of circular cone contact department, be provided with the opening on collection system's the outer wall.
Furthermore, the opening is arc-shaped and is arranged in parallel with the cross section of the cylindrical part of the acquisition device, the arc length is more than or equal to 70% of the circumference, and the height is 1.2-2.5 mm.
Compared with the prior art, the invention has the following beneficial technical effects:
according to the method for separating and purifying the water microbial bacteria, a sample to be separated and purified contains the collected microbial bacteria colony, a sodium alginate solution plays a role in protecting the strain colony and buffering acid and alkali, calcium ions are introduced through calcium carbonate to serve as a cross-linking agent formed by subsequent microcapsules, and the obtained suspension is a mother solution which contains the collected microbial bacteria colony and can be used for preparing subsequent capsules; then the mother liquor is mixed with an oil phase and glacial acetic acid, a multi-element cross-linking system is formed by reaction on the basis of regulating the pH value of the mixed system by the glacial acetic acid, the oil phase can enhance the mechanical strength of the microcapsules, the microcapsules with the diameter of 3-5 mm can be obtained by adding calcium chloride, 1-5 strains are wrapped in each microcapsule, a microcapsule strain library is obtained by washing with sterile water, and then the microcapsules are transferred to a porous plate one by one to be cultured, so that the efficiency of activity identification and separation can be improved, and single bacterial colonies are finally obtained; the method combines an emulsification micro-balloon method and a porous plate method, performs high-flux separation and purification of high-activity strains on microbial community, and can be used for building and separating the ecological germplasm resources of the environmental functional microorganisms.
Furthermore, the suspension is added into the oil phase A while stirring, so that the combination of the microbial community collected in the suspension and the oil phase A can be accelerated, and the obtained mixed system A is relatively uniform.
Further, when a sample to be separated and purified is obtained, firstly, the polyvinyl alcohol fiber filaments are uniformly dispersed into NaOH solution, so that the obtained polyvinyl alcohol fiber filaments are easy to disperse and difficult to agglomerate, the mechanical strength is enhanced to meet the requirement, then, the polyvinyl alcohol fiber filaments are uniformly mixed with BM solid culture medium solution of a strain to be separated and purified to obtain culture medium solution, then, high-pressure steam sterilization is carried out, the culture medium solution is cooled at room temperature in an anaerobic environment, the culture medium solution is kept at a low temperature and is subjected to gradient temperature rise at-80-0 ℃, the obtained culture medium gel has elasticity and strong adsorption capacity, water is enriched in sediments and expands into hydrogel, the obtained sample has high mechanical strength, the water absorption and the reabsorption rate are high, and a network system with a wide pore size range is formed inside the sediment.
Further, the oxygen residue in the medium and gel can be removed by oxygen removal followed by autoclaving to provide a more anaerobic environment.
Furthermore, the culture medium solution after high-pressure steam sterilization is poured into two semicircular column grooves with equal radius and length, two culture medium gel strips with the same radius and length can be finally obtained, then the two culture medium gel strips are spliced into a cylinder and are placed into the collecting device, and the resistance is small when the sample is finally taken out, so that the sample is convenient to take out.
Furthermore, the cylinder is wound with a layer of filter membrane and then is arranged in the acquisition device, so that impurities in the sediment can be filtered, and the periphery of the culture medium gel strip can be firm.
Furthermore, the micro-scale filter membrane can ensure that microorganisms can freely enter the culture medium for enrichment.
The collecting device is of a hollow structure, the upper half part is a cylinder, the lower half part is a cone, the diameter of the contact part of the cylinder and the cone is the same, the cone part can be quickly inserted into a deposit during sampling, the cylinder part can be matched with a semicircular column groove in a separation and purification method, the resistance is small when a sample is taken out finally, the sample is convenient to take out, an opening is formed in the outer wall of the collecting device, the deposit can be sampled and collected in situ from a natural environment, the obtained microbial community is not easy to generate microbial metabolism function decline, targeted separation and culture can be carried out, the occupied area of equipment is small, and in-situ sampling separation and identification can be realized.
Drawings
FIG. 1 is a side view of a semicircular cylindrical trough of the present invention.
FIG. 2 is a schematic view of an in situ collection tube of the present invention.
FIG. 3 is a schematic view of the operation of the present invention for loading a media gel strip into an in situ collection tube.
FIG. 4 is a schematic illustration of an in situ collection tube in situ enrichment operation according to the present invention.
FIG. 5 is a graph comparing the number of highly active sulfate-reducing strains obtained by different methods.
FIG. 6 is a comparison of activities of sulfate-reducing bacteria obtained by different methods, wherein strains 1-7 are obtained by a conventional sampling and separation method, strains 8-14 are obtained by in-situ enrichment and sampling and a conventional separation method, and strains 15-21 are obtained by the method of the present invention.
In the figure: a semicircular column groove 1, an in-situ collection tube 2, a permeation grid 3, a filter membrane 4 and a culture medium gel strip 5.
Detailed Description
The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.
The invention relates to in-situ enrichment, high-flux separation and purification of strict anaerobic and facultative anaerobic water microbial strains in natural sediments, which is suitable for the industrial fields and experimental research fields of microbial corrosion prevention, water pollution control, mineral smelting, functional microbial inoculum development and the like. The method combines the emulsification micro-balloon method and the porous plate method, naturally enriches and separates functional strains under the condition of no damage to microecology, takes the separation and purification of sulfate reducing bacteria as an example for description, and concretely comprises the following steps,
adding 1.0g/L of NH4Cl, 0.3g/L of K2HPO40.3g/L KH2PO4KCl 0.1g/L, CaCl 0.1g/L2·2H2Mixing O, 30g/L NaCl, 0.1g/L yeast extract, 0.5g/L cysteine, 3.16g/L sodium thiosulfate and 1ml of trace element nutrient solution to obtain a culture medium solution, wherein the trace element nutrient solution contains copper, manganese, zinc, iron, magnesium, molybdenum and boron, adjusting the pH of the culture medium solution to 7-7.6 by using 10M or KOH solution with other concentrations, adding 0.5% of agar powder according to the mass gram of the agar powder and the volume milliliter of the culture medium solution after the pH adjustment to prepare a solution, and carrying out moist heat sterilization to obtain a BM solid culture medium solution;
uniformly dispersing 5-10 mm long polyvinyl alcohol fiber yarns into a NaOH solution, wherein the dispersion effect is not greatly influenced by the amount of NaOH generally, in the NaOH solution, the mass of NaOH is 5% of that of the NaOH solution, then stirring at 50-60 ℃ for 8-12 h, collecting the modified polyvinyl alcohol fiber yarns, and drying to obtain the modified polyvinyl alcohol fiber yarns;
step 4, pouring the culture medium solution sterilized by high-pressure steam into two semicircular column grooves 1 with equal radius and length, wherein the inner diameter of each semicircular column groove 1 is 1-2.5 cm, the length of each semicircular column groove is 8-15 cm, the side view of each semicircular column groove 1 is shown in figure 1, cooling at room temperature in an anaerobic workbench to form gel, then placing the gel into a vacuum freeze-drying system for processing, keeping the operation parameters at-80 ℃ for 15-30 min, and then carrying out gradient temperature rise at-80-0 ℃ for 80-120 min to obtain a culture medium gel strip 5 with the water content of 10-20%, wherein the culture medium gel strip obtained by the method has elasticity and strong adsorption capacity, can be combined into a whole circle, has small resistance when being sampled, and is convenient to take out;
step 5, winding a layer of culture medium gel strip 5 with the water content of 10% -20% by using a filter membrane 4, then according to the step shown in figure 3, loading the culture medium gel strip wound with the filter membrane 4 into an in-situ collection tube 2 shown in figure 2, wherein the 'in-situ' in the in-situ collection tube 2 refers to in-situ sampling and collection of sediments from a natural environment, and at the moment, the filter membrane 4 forms an inner sealing sleeve of the in-situ collection tube 2, so that impurities can be filtered, and the periphery of the culture medium gel strip can be firm; in this example, the filter 4 has a size of 4 μm; the in-situ collection tube 2 can be made of stainless steel, plastic or acrylic, the in-situ collection tube 2 is composed of two parts, the upper half part is a cylinder, the lower half part is a cone, the diameter of the contact part of the cylinder and the cone is the same, the height ratio of the cylinder to the cone is more than or equal to 5:1, the in-situ collection tube 2 is used for protecting the culture medium gel strip and improving the hardness of the culture medium gel strip, so that the culture medium gel strip can be inserted into the sediment and used for in-situ enrichment of sulfate reducing bacteria in the sediment; the cylindrical part is provided with a plurality of permeation grids 3 which are parallel up and down, the permeation grids 3 are arc-shaped openings, the height of each permeation grid 3 is 1.2-2.5 mm, the arc length of each permeation grid 3 is more than or equal to 70% of the perimeter of the shell, the permeation grids 3 are uniformly arranged up and down, the interval between each permeation grid 3 is 2.5-3.5 mm, the permeation grids are used for sampling in a layered mode, and a sampling cutter is specifically used for slicing and taking out a sample part in each opening layer; as shown in fig. 4, the in-situ collection tube 2 can be used in a water phase, a water body and a sediment, for the sediment, the insertion depth of the in-situ collection tube 2 in the sediment is determined according to needs, generally at least exceeds the tube length of the in-situ collection tube 2, the culture medium gel strip 5 with the water content of 10% -20% absorbs water and expands into water-containing gel in the enrichment process, the water-containing gel is a mixture at the moment, simultaneously various bacteria in the sediment are enriched, sulfate reducing bacteria are enriched in situ under the nutrition of a BM solid culture medium solution, and the enrichment time is generally 36-72 hours;
step 6, separating by an emulsification micro-balloon method,
pouring 1-5 g of the water-containing gel in the in-situ collection tube 2 after in-situ enrichment in the step 5 into 50-250 ml of sodium alginate solution, adding 50-250 ml of calcium carbonate solution with the concentration of 10g/L, uniformly mixing, precipitating, and separating the precipitate to obtain a suspension, wherein the mass ratio of sodium alginate to water in the sodium alginate solution is 5-30%, the added calcium carbonate can react and crosslink with the sodium alginate and the water-containing gel, and the obtained suspension is a mother solution containing the collected sulfate reducing bacteria community and can be used for subsequent capsule preparation;
and 7, adding 0.1-0.3 percent of sorbitan fatty acid ester into the liquid paraffin as an oil phase according to the volume percentage of the sorbitan fatty acid ester to the liquid paraffin, and stirring at the room temperature of 300-350 r/min at a stirring speed of 1: (2-4) adding the suspension obtained in the step (6) into an oil phase, stirring at room temperature for 4-8 min, adding 6-10 ml of glacial acetic acid, reacting at 22-25 ℃, namely reacting at room temperature for 8-12 min, adding a calcium chloride solution into a reaction system by using an injector according to the rate of 200 mu l per drop, and obtaining microcapsules with the diameter of 3-5 mm by using the principle of cross-linking polymerization, wherein the mass ratio of calcium chloride to water in the calcium chloride solution is 2% -5%, each microcapsule is coated with 1-5 strains, and then slowly settling the microcapsules, separating the microcapsules, and washing with sterilized water for 1-2 times to obtain a microcapsule strain library;
in the step 8, the step of performing the step,
step 8a, adding 1.0g/L of NH4Cl, 0.3g/L of K2HPO40.3g/L KH2PO4KCl 0.1g/L, CaCl 0.1g/L2·2H2Mixing O, 30g/L NaCl, 0.1g/L yeast extract, 0.5g/L cysteine, 3.16g/L sodium thiosulfate and 1ml trace element nutrient solution to obtain culture medium solution, wherein the trace element nutrient solution contains copper, manganese, zinc, iron, magnesium, molybdenum and boron, and dissolving the culture medium solution in 10M KOHAdjusting the pH value to 7-7.6, and performing moist heat sterilization to obtain a BM liquid culture medium solution;
step 8b, adding resazurin with the concentration of 0.1-0.15% according to the mass percentage of the resazurin to the BM liquid culture medium solution to obtain the BM liquid culture medium solution containing the resazurin;
step 8c, transferring the microcapsule bacterial libraries obtained in the step 7 one by one in an anaerobic workbench and inoculating the microcapsule bacterial libraries into a porous plate of a 96-well plate for culture, wherein the microcapsule bacterial libraries are semitransparent and faint yellow at the initial culture stage, BM liquid culture medium solution containing resazurin is filled in the porous plate, and the microcapsule bacterial libraries are cultured for 48-72 hours at the temperature of 22-25 ℃; and then coating the culture medium with the color changed from brown to black in the porous plate on a BM plate culture medium for culture, continuing to perform plate transfer and streak culture at least twice after obtaining the single colony, and finally obtaining the single colony which is the high-activity sulfate reducing bacteria pure culture.
Example 1
step 4, pouring the culture medium solution sterilized by high-pressure steam into two semicircular column grooves 1 with equal radius and length, wherein the inner diameter of each semicircular column groove 1 is 2 cm, the length of each semicircular column groove 1 is 10 cm, cooling the semicircular column grooves in an anaerobic workbench at room temperature to form gel, then placing the semicircular column grooves into a vacuum freeze drying system for treatment, keeping the semicircular column grooves at the operating parameter of-80 ℃ for 30min, and then carrying out gradient heating at the temperature of-80-0 ℃ for 120min to obtain a culture medium gel strip with the water content of 20%;
step 5, winding a layer of culture medium gel strip with the water content of 20% by using a filter membrane 4, then loading the culture medium gel strip wound with the filter membrane 4 into an in-situ collection tube 2, wherein the insertion depth of the in-situ collection tube 2 in a deposit exceeds the tube length of the in-situ collection tube 2, and a culture medium gel strip with the water content of 20% absorbs water and expands into water-containing gel in the enrichment process, wherein the water-containing gel is a mixture and simultaneously enriches various bacteria in the deposit, sulfate reducing bacteria are enriched in situ under the nutrition of BM solid culture medium solution, and the enrichment time is 48 h;
step 6, pouring 5g of water-containing gel in the in-situ collection tube 2 after in-situ enrichment in the step 5 into 250ml of sodium alginate solution, adding 250ml of calcium carbonate with the concentration of 10g/L, uniformly mixing, precipitating, and separating the precipitate to obtain suspension, wherein the mass ratio of sodium alginate to water in the sodium alginate solution is 30%;
and 7, adding sorbitan fatty acid ester with the volume percentage of 0.1 percent into the liquid paraffin as an oil phase, and stirring at the room temperature of 350r/min at a stirring speed of 1: 3, slowly adding the suspension into the oil phase, stirring for 4min, then dropwise adding glacial acetic acid, dropwise adding 10ml, reacting for 10min at 25 ℃, dropwise adding 200 mu l of calcium chloride solution into the reaction system, wherein the mass ratio of calcium chloride to water in the calcium chloride solution is 5%, slowly settling the microcapsules, separating the microcapsules, and washing for 2 times by using sterilized water to obtain a microcapsule strain library;
in the step 8, the step of performing the step,
step 8a, adding 1.0g/L of NH4Cl, 0.3g/L of K2HPO40.3g/L KH2PO4KCl 0.1g/L, CaCl 0.1g/L2·2H2Mixing O, 30g/L NaCl, 0.1g/L yeast extract, 0.5g/L cysteine, 3.16g/L sodium thiosulfate and 1ml of trace element nutrient solution to obtain 1L of culture medium solution, wherein the trace element nutrient solution contains copper, manganese, zinc, iron, magnesium, molybdenum and boron, adjusting the pH of the culture medium solution to 7.6 by using 10M KOH solution, and performing moist heat sterilization to obtain BM liquid culture medium solution;
step 8b, adding resazurin with the concentration of 0.1 percent according to the mass gram of the resazurin and the volume milliliter percent of the BM liquid culture medium solution to obtain the BM liquid culture medium solution containing the resazurin;
step 8c, transferring the microcapsule bacterial libraries obtained in the step 7 one by one in an anaerobic workbench and inoculating the microcapsule bacterial libraries into a porous plate of a 96-well plate for culture, wherein the initial culture stage is semitransparent and faint yellow, the porous plate is filled with a BM liquid culture medium solution containing resazurin and cultured for 72 hours at room temperature; and then coating the culture medium with the color changed from brown to black in the porous plate on a BM plate culture medium for culture, continuing to turn the plate to perform streak culture twice after obtaining a single bacterial colony, wherein the finally obtained single bacterial colony is a pure culture of the high-activity sulfate reducing bacteria, the strain number of the single bacterial colony is shown in figure 5, compared with the traditional sampling separation method and the in-situ enrichment sampling and traditional separation method of the invention, the strain number of the high-activity sulfate reducing bacteria is obviously increased, only 16 strains are shared in the total 123 strains, 23 and 53 are shared with other two methods, and the activity diagram of the sulfate reducing bacteria is shown in figure 6, wherein the higher the growth rate coefficient and the sulfate reduction rate coefficient are higher, the higher the growth rate and the higher the activity of the reducing bacteria are shown, so the sulfate reducing bacteria obtained by the invention have high activity.
Example 2
step 4, pouring the culture medium solution sterilized by high-pressure steam into two semicircular column grooves 1 with equal radius and length, wherein the inner diameter of each semicircular column groove 1 is 1 cm, the length of each semicircular column groove is 8 cm, cooling the semicircular column grooves in an anaerobic workbench at room temperature to form gel, then placing the semicircular column grooves into a vacuum freeze drying system for treatment, keeping the semicircular column grooves at the operating parameter of-80 ℃ for 15min, and then carrying out gradient heating at the temperature of-80-0 ℃ for 100min to obtain culture medium gel strips with the water content of 10%;
step 5, winding a layer of culture medium gel strip with the water content of 10% by using a filter membrane 4, then loading the culture medium gel strip wound with the filter membrane 4 into an in-situ collection tube 2, wherein the insertion depth of the in-situ collection tube 2 in a deposit exceeds the tube length of the in-situ collection tube 2, and a culture medium gel strip with the water content of 10% absorbs water and expands into water-containing gel in the enrichment process, wherein the water-containing gel is a mixture and simultaneously enriches various bacteria in the deposit, sulfate reducing bacteria are enriched in situ under the nutrition of BM solid culture medium solution, and the enrichment time is 36 h;
step 6, pouring 1g of the water-containing gel in the in-situ collection tube 2 after in-situ enrichment in the step 5 into 150ml of sodium alginate solution, adding 150ml of calcium carbonate with the concentration of 10g/L, uniformly mixing, precipitating, and separating the precipitate to obtain a suspension, wherein the mass ratio of sodium alginate to water in the sodium alginate solution is 15%;
step 7, adding 0.3 volume percent of sorbitan fatty acid ester serving as an oil phase into liquid paraffin, slowly adding the suspension into the oil phase according to the volume ratio of a water phase to the oil phase being 1: 2 at the room-temperature stirring speed of 300r/min, stirring for 8min, then dropwise adding glacial acetic acid, dropwise adding 8ml, reacting for 12min at 22 ℃, dropwise adding 200 microliter of calcium chloride solution into a reaction system, wherein the mass ratio of calcium chloride to water in the calcium chloride solution is 3%, slowly settling the microcapsules, separating the microcapsules, and washing for 2 times with sterilized water to obtain a microcapsule strain library;
in the step 8, the step of performing the step,
step 8a, adding 1.0g/L of NH4Cl, 0.3g/L of K2HPO40.3g/L KH2PO4KCl 0.1g/L, CaCl 0.1g/L2·2H2Mixing O, 30g/L NaCl, 0.1g/L yeast extract, 0.5g/L cysteine, 3.16g/L sodium thiosulfate and 1ml of trace element nutrient solution to obtain 1L of culture medium solution, wherein the trace element nutrient solution contains copper, manganese, zinc, iron, magnesium, molybdenum and boron, adjusting the pH of the culture medium solution to 7 by using 10M KOH solution, and carrying out moist heat sterilization to obtain BM liquid culture medium solution;
step 8b, adding resazurin with the concentration of 0.15 percent according to the mass gram of the resazurin and the volume milliliter percent of the BM liquid culture medium solution to obtain the BM liquid culture medium solution containing the resazurin;
step 8c, transferring the microcapsule bacterial libraries obtained in the step 7 one by one in an anaerobic workbench and inoculating the microcapsule bacterial libraries into a porous plate of a 96-well plate for culture, wherein the initial culture stage is semitransparent and faint yellow, the porous plate is filled with a BM liquid culture medium solution containing resazurin and cultured for 48h at room temperature; and then coating the culture medium with the color changed from brown to black in the porous plate on a BM plate culture medium for culture, continuing to perform plate transfer and streak culture for three times after obtaining the single colony, and finally obtaining the single colony which is the high-activity sulfate reducing bacteria pure culture.
Example 3
step 4, pouring the culture medium solution sterilized by high-pressure steam into two semicircular column grooves 1 with equal radius and length, wherein the inner diameter of each semicircular column groove 1 is 2.5 cm, the length of each semicircular column groove is 15 cm, cooling the semicircular column grooves in an anaerobic workbench at room temperature to form gel, then placing the semicircular column grooves into a vacuum freeze drying system for treatment, keeping the semicircular column grooves at the operating parameter of-80 ℃ for 20min, and then carrying out gradient heating at the temperature of-80-0 ℃ for 80min to obtain a culture medium gel strip with the water content of 15%;
step 5, winding a layer of culture medium gel strip with the water content of 15% by using a filter membrane 4, then loading the culture medium gel strip wound with the filter membrane 4 into an in-situ collection tube 2, wherein the insertion depth of the in-situ collection tube 2 in a deposit exceeds the tube length of the in-situ collection tube 2, and a culture medium gel strip with the water content of 15% absorbs water and expands into water-containing gel in the enrichment process, wherein the water-containing gel is a mixture and simultaneously enriches various bacteria in the deposit, sulfate reducing bacteria are enriched in situ under the nutrition of BM solid culture medium solution, and the enrichment time is 72 h;
step 6, pouring 3g of water-containing gel in the in-situ collection tube 2 after in-situ enrichment in the step 5 into 50ml of sodium alginate solution, adding 50ml of calcium carbonate with the concentration of 10g/L, uniformly mixing, precipitating, and separating the precipitate to obtain suspension, wherein the mass ratio of sodium alginate to water in the sodium alginate solution is 5%;
step 7, adding sorbitan fatty acid ester with volume percentage content of 0.2% into liquid paraffin as an oil phase, slowly adding the suspension into the oil phase according to the volume ratio of water phase to oil phase of 1: 4 at the room temperature stirring speed of 320r/min, stirring for 6min, then dropwise adding glacial acetic acid, dropwise adding 6ml, reacting for 8min at 24 ℃, dropwise adding 200 microliter of calcium chloride solution into the reaction system, wherein the mass ratio of calcium chloride to water in the calcium chloride solution is 2%, slowly settling the microcapsules, separating the microcapsules, and washing for 1 time by using sterilized water to obtain a microcapsule strain library;
in the step 8, the step of performing the step,
step 8a, adding 1.0g/L of NH4Cl, 0.3g/L of K2HPO40.3g/L KH2PO4KCl 0.1g/L, CaCl 0.1g/L2·2H2Mixing O, 30g/L NaCl, 0.1g/L yeast extract, 0.5g/L cysteine, 3.16g/L sodium thiosulfate and 1ml of trace element nutrient solution to obtain 1L of culture medium solution, wherein the trace element nutrient solution contains copper, manganese, zinc, iron, magnesium, molybdenum and boron, adjusting the pH of the culture medium solution to 7.3 by using 10M KOH solution, and performing moist heat sterilization to obtain BM liquid culture medium solution;
step 8b, adding resazurin with the concentration of 0.12 percent according to the mass gram of the resazurin and the volume milliliter percent of the BM liquid culture medium solution to obtain the BM liquid culture medium solution containing the resazurin;
step 8c, transferring the microcapsule bacterial libraries obtained in the step 7 one by one in an anaerobic workbench and inoculating the microcapsule bacterial libraries into a porous plate of a 96-well plate for culture, wherein the initial culture stage is semitransparent and faint yellow, the porous plate is filled with a BM liquid culture medium solution containing resazurin and cultured for 60 hours at room temperature; and then coating the culture medium with the color changed from brown to black in the porous plate on a BM plate culture medium for culture, continuing to perform plate transfer and streak culture for four times after obtaining the single colony, wherein the single colony obtained finally is the high-activity sulfate reducing bacteria pure culture.
According to the invention, BM solid culture medium culture and BM liquid culture medium culture required by strains to be separated and purified are prepared, so that a large amount of pure sulfate reducing bacteria culture can be obtained by separation under the condition of original inhabitation environment, and the sulfate reducing bacteria can be separated under the micro-ecological condition to the greatest extent.
Claims (8)
1. A method for separating and purifying water body microorganism strains is characterized by comprising the following steps,
step 1, adding a sample to be separated and purified and a calcium carbonate solution into a sodium alginate solution to obtain a precipitate and a suspension, wherein the mass ratio of sodium alginate in the sodium alginate solution to calcium carbonate in the calcium carbonate solution is (2.5-75): (0.5 to 2.5);
step 2, adding suspension into an oil phase A consisting of sorbitan fatty acid ester and liquid paraffin to obtain a mixed system A, wherein when the oil phase A is formed, the volume of the sorbitan fatty acid ester is 0.1-0.3% of the volume of the liquid paraffin, and when the suspension is added, the volume ratio of the suspension to the oil phase is 1: (2-4);
step 3, adding 6-10 ml of glacial acetic acid into the mixed system A, reacting for 8-12 min at 22-25 ℃ to obtain a mixed system B, adding a calcium chloride solution into the mixed system B, wherein the mass of calcium chloride added each time is 0.004-0.01 g, obtaining microcapsules with the diameter of 3-5 mm, and cleaning the microcapsules with the diameter of 3-5 mm by using sterilized water to obtain a microcapsule strain library;
and 4, inoculating the microcapsule strain libraries into a porous plate one by one to culture to obtain a single bacterial colony, wherein the porous plate is filled with BM liquid culture medium solution of strains to be separated and purified.
2. The method for separating and purifying water microorganisms according to claim 1, wherein the suspension is added into the oil phase A in the step 2 at a speed of 300-350 r/min while stirring, and the mixture is stirred for 4-8 min to obtain a mixed system A.
3. The method for separating and purifying water microorganisms according to claim 1, wherein the multi-well plate in step 4 is a 96-well plate.
4. The method for separating and purifying water microorganisms according to claim 1, wherein the sample to be separated and purified in step 1 is obtained by the following steps,
step 1a, uniformly dispersing polyvinyl alcohol fiber yarns into a NaOH solution, stirring for 8-12 hours at 50-60 ℃, and collecting the polyvinyl alcohol fiber yarns in the NaOH solution to obtain modified polyvinyl alcohol fiber yarns;
step 1b, uniformly mixing the modified polyvinyl alcohol cellosilk with BM solid culture medium solution of strains to be separated and purified to obtain a mixed system C, and carrying out high-pressure steam sterilization on the mixed system C to obtain the culture medium solution after high-pressure steam sterilization;
step 1c, cooling the culture medium solution sterilized by high-pressure steam in an anaerobic workbench at room temperature to obtain gel, keeping the gel at-80 ℃ for 15-30 min, then carrying out gradient temperature rise at-80-0 ℃ for 80-120 min to obtain a culture medium gel strip with the water content of 10% -20%, and putting the culture medium gel strip into an acquisition device with an opening in the outer wall to obtain the acquisition device containing the culture medium gel strip;
step 1d, inserting a collection device containing a culture medium gel strip into the sediment for 36-72 hours, wherein the culture medium gel strip with the water content of 10% -20% in the collection device absorbs water and expands into hydrogel in the enrichment process, and a sample to be separated and purified is obtained;
the collecting device is of a hollow structure and comprises two parts, wherein the upper half part is a cylinder, the lower half part is a cone, the diameter of the contact part of the cylinder and the cone is the same, an arc-shaped opening is formed in the outer wall of the collecting device and is parallel to the cross section of the cylindrical part of the collecting device, the arc length is larger than or equal to 70% of the circumference, and the height is 1.2-2.5 mm.
5. The method for separating and purifying water microorganisms as claimed in claim 4, wherein N is introduced into the step 1b according to a volume ratio of 80:202/CO2The mixed gas in the mixed system C is subjected to oxygen discharge until reaching the standard required by the survival of the strains to be separated and purified, and then high-pressure steam sterilization is carried out.
6. The method for separating and purifying water microorganisms according to claim 4, wherein in step 1c, the culture medium solution sterilized by high pressure steam is poured into two semicircular column grooves with equal radius and length, then cooled at room temperature in an anaerobic workbench, kept at-80 ℃ and subjected to gradient temperature rise at-80 ℃ to 0 ℃ to obtain two culture medium gel strips with the same radius and length and the water content of 10-20%, then the two culture medium gel strips are spliced into a cylinder, and the cylinder is placed into a collecting device.
7. The method for separating and purifying water microorganisms as claimed in claim 6, wherein the cylinder is wound with a layer of filter membrane and then loaded into the collecting device.
8. The method for separating and purifying water microorganisms according to claim 7, wherein the filter membrane is a micron-sized filter membrane.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910350968.XA CN110029064B (en) | 2019-04-28 | 2019-04-28 | Method for separating and purifying water body microorganism bacteria and sampling device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910350968.XA CN110029064B (en) | 2019-04-28 | 2019-04-28 | Method for separating and purifying water body microorganism bacteria and sampling device |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110029064A CN110029064A (en) | 2019-07-19 |
| CN110029064B true CN110029064B (en) | 2021-11-23 |
Family
ID=67240587
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910350968.XA Active CN110029064B (en) | 2019-04-28 | 2019-04-28 | Method for separating and purifying water body microorganism bacteria and sampling device |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110029064B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114515555A (en) * | 2022-03-16 | 2022-05-20 | 中南大学 | Preparation method and application of sulfate reducing microorganism capsule |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008035372A2 (en) * | 2006-07-06 | 2008-03-27 | Reliance Life Sciences Pvt Ltd | Efficient process for purification of high molecular weight hyaluronic acid |
| CN103275962A (en) * | 2013-06-04 | 2013-09-04 | 国家粮食局科学研究院 | Method for preparing coated microcapsules before microbial fermentation |
| CN208791609U (en) * | 2018-06-07 | 2019-04-26 | 南方科技大学 | Deep-Sea Microorganisms water body in-situ culture apparatus |
-
2019
- 2019-04-28 CN CN201910350968.XA patent/CN110029064B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008035372A2 (en) * | 2006-07-06 | 2008-03-27 | Reliance Life Sciences Pvt Ltd | Efficient process for purification of high molecular weight hyaluronic acid |
| CN103275962A (en) * | 2013-06-04 | 2013-09-04 | 国家粮食局科学研究院 | Method for preparing coated microcapsules before microbial fermentation |
| CN208791609U (en) * | 2018-06-07 | 2019-04-26 | 南方科技大学 | Deep-Sea Microorganisms water body in-situ culture apparatus |
Non-Patent Citations (2)
| Title |
|---|
| Protection of lactoperoxidase activity with sugars during lyophilization and evaluation of its antibacterial properties;Shariat SS等;《Res Pharm Sci》;20150430;152-160 * |
| 微胶囊复合菌剂净水技术及其应用;王超 等;《绿色科技》;20160930;53-56 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110029064A (en) | 2019-07-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chartrain et al. | Microbial ecophysiology of whey biomethanation: characterization of bacterial trophic populations and prevalent species in continuous culture | |
| Ma et al. | Removal of environmental estrogens by bacterial cell immobilization technique | |
| CN109956563A (en) | A kind of preparation method and applications of high-efficiency aerobic Denitrifying Phosphate Accumulating Organisms immobilized spherule | |
| CN110029064B (en) | Method for separating and purifying water body microorganism bacteria and sampling device | |
| Jiefeng et al. | Repeated-batch cultivation of encapsulated Monascus purpureus by polyelectrolyte complex for natural pigment production | |
| CN103710287A (en) | Culturing method for ammonia oxidizing bacteria | |
| CN115725468A (en) | Heterotrophic nitrification-aerobic denitrification composite microbial agent and preparation method and application thereof | |
| WO2022179073A1 (en) | Aneurinibacillus sp. and use thereof in decolorization of dye wastewater | |
| CN114807110A (en) | Microorganism immobilized particle for water treatment and preparation method thereof | |
| CN102199561A (en) | Aerobic denitrifying bacterium treating nitrite as nitrogen source and screening method thereof | |
| Dubourguier et al. | Characterization of two strains of Pelobacter carbinolicus isolated from anaerobic digesters | |
| CN110438059A (en) | A kind of superior microorganism microbial inoculum of water pollution control and preparation method thereof | |
| Chartrain et al. | Microbial ecophysiology of whey biomethanation: comparison of carbon transformation parameters, species composition, and starter culture performance in continuous culture | |
| CN111826292B (en) | Yeast RY-6 and application thereof | |
| Bhatti et al. | Treatment performance and microbial structure of a granular consortium handling methanolic waste | |
| CN110982717B (en) | Honey yeast and method for producing single-cell protein by treating high-ammonia-nitrogen biogas slurry with same | |
| CN102897923A (en) | Bioleaching method for promoting deep dehydration of water-blooming cyanobacteria | |
| CN105754904B (en) | One plant of ocean Psychrobacter strain and its application in water body dephosphorized | |
| CN112574921A (en) | Method for preparing aerobic denitrification composite microbial inoculum by utilizing kitchen waste and application thereof | |
| Lepistö et al. | Conversion of volatile fatty acids in an extreme thermophilic (76–80° C) upflow anaerobic sludgeblanket reactor | |
| CN114783714B (en) | Method for promoting anaerobic fermentation by using magnetic straw biochar | |
| Li et al. | Activity enhancement and the anammox mechanism under low temperature via PVA-SA and nano Fe2O3-PVA-SA entrapped beads | |
| Tomei et al. | Interactions in syntrophic associations of endospore-forming, butyrate-degrading bacteria and H2-consuming bacteria | |
| CN112694987B (en) | Method for enriching and domesticating perchlorate reducing flora by using conductive material | |
| CN115304170A (en) | Treatment process for degrading kitchen wastewater by using immobilized microorganism pellets |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240409 Address after: Room 9099, 2nd Floor, Building 4, Free Trade Industrial Park, No. 2168 Zhenghe Fourth Road, Fengdong New City, Xi'an City, Shaanxi Province, China Patentee after: Shaanxi Jiaruide Biotechnology Co.,Ltd. Country or region after: China Address before: No. 1, Weiyang District university garden, Xi'an, Shaanxi Province, Shaanxi Patentee before: SHAANXI University OF SCIENCE & TECHNOLOGY Country or region before: China |
|
| TR01 | Transfer of patent right |