CN111440725B - Method for enriching and domesticating functional microorganisms in undisturbed state of water sediment - Google Patents
Method for enriching and domesticating functional microorganisms in undisturbed state of water sediment Download PDFInfo
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- CN111440725B CN111440725B CN202010304483.XA CN202010304483A CN111440725B CN 111440725 B CN111440725 B CN 111440725B CN 202010304483 A CN202010304483 A CN 202010304483A CN 111440725 B CN111440725 B CN 111440725B
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- water
- enrichment
- domestication
- culture
- sediment
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title claims abstract description 180
- 239000013049 sediment Substances 0.000 title claims abstract description 102
- 244000005700 microbiome Species 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 39
- 239000001963 growth medium Substances 0.000 claims abstract description 49
- 238000005273 aeration Methods 0.000 claims abstract description 25
- 230000001105 regulatory effect Effects 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 38
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 claims description 25
- 239000004021 humic acid Substances 0.000 claims description 25
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 16
- 230000002572 peristaltic effect Effects 0.000 claims description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 12
- 239000008346 aqueous phase Substances 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 11
- 238000005070 sampling Methods 0.000 claims description 11
- 239000006004 Quartz sand Substances 0.000 claims description 10
- 239000012071 phase Substances 0.000 claims description 10
- 238000012216 screening Methods 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 239000004323 potassium nitrate Substances 0.000 claims description 6
- 235000010333 potassium nitrate Nutrition 0.000 claims description 6
- 239000000741 silica gel Substances 0.000 claims description 6
- 229910002027 silica gel Inorganic materials 0.000 claims description 6
- 229920001971 elastomer Polymers 0.000 claims description 5
- 239000007791 liquid phase Substances 0.000 claims description 4
- 230000002503 metabolic effect Effects 0.000 claims description 4
- 230000002906 microbiologic effect Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 54
- BBEAQIROQSPTKN-UHFFFAOYSA-N pyrene Chemical compound C1=CC=C2C=CC3=CC=CC4=CC=C1C2=C43 BBEAQIROQSPTKN-UHFFFAOYSA-N 0.000 description 28
- 241000894006 Bacteria Species 0.000 description 16
- GVEPBJHOBDJJJI-UHFFFAOYSA-N fluoranthrene Natural products C1=CC(C2=CC=CC=C22)=C3C2=CC=CC3=C1 GVEPBJHOBDJJJI-UHFFFAOYSA-N 0.000 description 14
- 230000015556 catabolic process Effects 0.000 description 13
- 238000006731 degradation reaction Methods 0.000 description 13
- RLJMLMKIBZAXJO-UHFFFAOYSA-N lead nitrate Chemical compound [O-][N+](=O)O[Pb]O[N+]([O-])=O RLJMLMKIBZAXJO-UHFFFAOYSA-N 0.000 description 13
- 239000007789 gas Substances 0.000 description 12
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 12
- FMMWHPNWAFZXNH-UHFFFAOYSA-N Benz[a]pyrene Chemical compound C1=C2C3=CC=CC=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 FMMWHPNWAFZXNH-UHFFFAOYSA-N 0.000 description 11
- 239000010410 layer Substances 0.000 description 11
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 10
- 239000000725 suspension Substances 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 241001057811 Paracoccus <mealybug> Species 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 230000000593 degrading effect Effects 0.000 description 6
- 239000004317 sodium nitrate Substances 0.000 description 6
- 235000010344 sodium nitrate Nutrition 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000001276 controlling effect Effects 0.000 description 4
- 239000012531 culture fluid Substances 0.000 description 4
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 4
- RVPVRDXYQKGNMQ-UHFFFAOYSA-N lead(2+) Chemical compound [Pb+2] RVPVRDXYQKGNMQ-UHFFFAOYSA-N 0.000 description 4
- 239000002344 surface layer Substances 0.000 description 4
- 229940088594 vitamin Drugs 0.000 description 4
- 229930003231 vitamin Natural products 0.000 description 4
- 235000013343 vitamin Nutrition 0.000 description 4
- 239000011782 vitamin Substances 0.000 description 4
- 150000003722 vitamin derivatives Chemical class 0.000 description 4
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 4
- 229910002651 NO3 Inorganic materials 0.000 description 3
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 3
- UZYLFCGUYBDKPH-UHFFFAOYSA-N benzo[e]pyrene propan-2-one Chemical compound CC(=O)C.C1=CC=C2C=CC=3C=CC=C4C5=C(C1=C2C43)C=CC=C5 UZYLFCGUYBDKPH-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000002352 surface water Substances 0.000 description 3
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- QWMFKVNJIYNWII-UHFFFAOYSA-N 5-bromo-2-(2,5-dimethylpyrrol-1-yl)pyridine Chemical compound CC1=CC=C(C)N1C1=CC=C(Br)C=N1 QWMFKVNJIYNWII-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 2
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
- 229930003451 Vitamin B1 Natural products 0.000 description 2
- 229930003779 Vitamin B12 Natural products 0.000 description 2
- 229930003471 Vitamin B2 Natural products 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 229960004050 aminobenzoic acid Drugs 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 238000009412 basement excavation Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 description 2
- 229940052299 calcium chloride dihydrate Drugs 0.000 description 2
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 description 2
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- WGFWACSXWYYVAA-UHFFFAOYSA-L copper(II) chloride pentahydrate Chemical compound O.O.O.O.O.Cl[Cu]Cl WGFWACSXWYYVAA-UHFFFAOYSA-L 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000003344 environmental pollutant Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000004744 fabric Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 229960000304 folic acid Drugs 0.000 description 2
- 235000019152 folic acid Nutrition 0.000 description 2
- 239000011724 folic acid Substances 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- AGBQKNBQESQNJD-UHFFFAOYSA-M lipoate Chemical compound [O-]C(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-M 0.000 description 2
- 235000019136 lipoic acid Nutrition 0.000 description 2
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- UGNANEGDDBXEAS-UHFFFAOYSA-L nickel(2+);dichloride;dihydrate Chemical compound O.O.Cl[Ni]Cl UGNANEGDDBXEAS-UHFFFAOYSA-L 0.000 description 2
- 229960003512 nicotinic acid Drugs 0.000 description 2
- 239000011664 nicotinic acid Substances 0.000 description 2
- 235000001968 nicotinic acid Nutrition 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 229940055726 pantothenic acid Drugs 0.000 description 2
- 235000019161 pantothenic acid Nutrition 0.000 description 2
- 239000011713 pantothenic acid Substances 0.000 description 2
- 231100000719 pollutant Toxicity 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 229960002477 riboflavin Drugs 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000010802 sludge Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 229960003495 thiamine Drugs 0.000 description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 2
- 229960002663 thioctic acid Drugs 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 235000010374 vitamin B1 Nutrition 0.000 description 2
- 239000011691 vitamin B1 Substances 0.000 description 2
- 235000019163 vitamin B12 Nutrition 0.000 description 2
- 239000011715 vitamin B12 Substances 0.000 description 2
- 235000019164 vitamin B2 Nutrition 0.000 description 2
- 239000011716 vitamin B2 Substances 0.000 description 2
- 239000011592 zinc chloride Substances 0.000 description 2
- 235000005074 zinc chloride Nutrition 0.000 description 2
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 244000179970 Monarda didyma Species 0.000 description 1
- 235000010672 Monarda didyma Nutrition 0.000 description 1
- 241001478310 Paracoccus aminovorans Species 0.000 description 1
- 241001478304 Paracoccus versutus Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000003673 groundwater Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- JCXJVPUVTGWSNB-UHFFFAOYSA-N nitrogen dioxide Inorganic materials O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
-
- 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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
-
- 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
- C12N1/36—Adaptation or attenuation of cells
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Sustainable Development (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for enriching and domesticating functional microorganisms in undisturbed water sediments. The invention utilizes a water sediment undisturbed enrichment and domestication device to perform domestication culture to obtain functional microorganisms, wherein the water sediment undisturbed enrichment and domestication device comprises an enrichment and domestication bin, a water distribution part, a water phase circulation part, a culture solution supply part and an aeration part. The method enriches functional microorganisms on the premise of not damaging the original structure of the sediment, and improves the probability of separating the habitat sensitive microorganisms; the culture condition of the sediment is controlled by the gas phase environment, and simultaneously, the selective culture medium is continuously and slowly provided for the interior or the surface of the sediment, so that the succession of the microorganisms for regulating the sediment to the target direction is achieved, and the method can be used as a powerful tool for researching the microbiological ecology of the sediment.
Description
Technical Field
The invention belongs to the field of microbial resource excavation and utilization, and particularly relates to a method for enriching and domesticating functional microorganisms in undisturbed water sediment.
Background
The surface water body is accompanied with the development of human beings, plays roles of water transportation, flood discharge, landscapes, even sewage receiving and the like, and makes an important contribution to the development of human society. In recent decades, particularly in developing countries and regions, rapid economic development and urban population proliferation have been accompanied by lagged environmental management, causing surface waters to suffer serious pollution and losing their own ecological regulation function. In particular, contaminants tend to combine with the particulates and settle to the substrate after entering the body of water, and the sediment enriched with a large amount of contaminants becomes an endogenous source of contamination.
The microorganism strengthening method is an important method for repairing water, and is widely applied to repairing refractory pollutants of saturated media (groundwater layers and sediments) due to the characteristics of environmental friendliness and low energy consumption mainly by applying high-efficiency functional microorganisms to accelerate the degradation and detoxification of pollutants. The functional microorganism is used as the core of environmental biological treatment, and the excavation and the breeding of functional strain resources are important.
There are two main methods of microorganism enrichment and domestication. The first method is sampling culture, namely, adding the collected sample into a selective culture medium for domestication and enrichment culture, and then carrying out multiple passages to enrich microorganisms capable of growing in the selective culture medium, so that the subsequent separation and purification are facilitated. The second method is in-situ enrichment, namely, placing an enrichment device with attachments (such as filler and culture medium) in a target environment, allowing microorganisms to grow on the attachments with functional selectivity for enrichment, and retrieving the device to obtain enriched microorganisms. The first method is a conventional method, and has strong applicability, so that people can excavate a large amount of microbial resources through the method; however, this method has destroyed the spatial structure of the sample during sampling and culturing, and the habitat of microorganism growth has changed greatly; microorganisms that are low in abundance or cannot accommodate a sudden change in habitat will be eliminated at the beginning of the culture and therefore it is difficult to enrich for such habitat sensitive microorganisms. The second approach solves the problem of sudden changes in habitat, however, it belongs to passive enrichment and cannot effectively control the conditions of enrichment and domestication.
Disclosure of Invention
The invention aims to solve the defects of the two methods in the prior art, and aims at the characteristics of spatial heterogeneity of water sediment, maintains the original structure of the sediment, and achieves the aim of enriching and domesticating target functional microorganisms in the sediment by controlling microorganism metabolic factors in a continuous flow device; therefore, the invention provides a method for enriching and domesticating functional microorganisms in the undisturbed state of water sediments.
The method of the invention can avoid the extinction of the habitat sensitive microorganisms without damaging the natural deposition structure of the sediment, and can regulate and control the metabolic factors, so that the target microorganisms with low natural abundance can be domesticated and proliferated in the original environment, thereby being beneficial to the subsequent separation and purification and having great advantages in the aspect of excavating the functional microorganisms in the saturated medium.
The method for the undisturbed enrichment and domestication of the functional microorganisms of the water sediment utilizes the undisturbed enrichment and domestication device of the water sediment to carry out undisturbed enrichment and domestication culture to obtain the functional microorganisms;
the water sediment undisturbed enrichment and domestication device comprises an enrichment domestication bin, a water distribution part, an aqueous phase circulation part, a culture solution supply part and an aeration part, wherein the top of the enrichment domestication bin is provided with an air inlet hole and an air outlet hole, the aeration part is connected with the air inlet hole, the bottom of the enrichment domestication bin is provided with a water inlet hole, the water distribution part is inserted into a cavity of the enrichment domestication bin through the water inlet hole, one end of the water distribution part, which is left outside the enrichment domestication bin, is connected to a liquid outlet of the culture solution supply part through one set of aqueous phase circulation part, the side wall of the enrichment domestication bin is provided with a water outlet hole, and the water outlet hole is connected to a liquid inlet of the culture solution supply part through the other set of aqueous phase circulation part;
the undisturbed enrichment and domestication culture are as follows: collecting undisturbed water sediment in an acclimation and enrichment bin, injecting a crude water body of sampling water sediment into the acclimation and enrichment bin, adding a culture solution for enriching and acclimating target functional microorganisms into a culture solution supply part, starting an aqueous phase circulation part to input the culture solution into the surface or the inside of the water sediment through a water distribution part, and timely adjusting the components of the culture solution to complete undisturbed enrichment and acclimation culture.
Preferably, the method for undisturbed enrichment and domestication of functional microorganisms in water sediment further comprises the step of liquid phase enrichment culture after undisturbed enrichment and domestication culture are completed: and (3) placing the domesticated and cultured water sediment in an enrichment culture medium for culture and subculturing for a plurality of times to obtain the target functional microbial flora.
Preferably, the culture solution in the undisturbed enrichment and domestication culture step is humic acid culture medium, and each liter contains: 0.2g of magnesium sulfate, 0.25g of potassium nitrate, 0.2-6g of humic acid and the balance of water; wherein, every 5 days, the concentration of humic acid is increased from 0.2g/L to 0.4g/L, 1.6g/L and 6g/L in sequence; the enrichment culture medium in the liquid phase enrichment culture step is an enrichment humic acid culture medium, and each liter of the enrichment culture medium contains: 0.2g of magnesium sulfate, 0.25g of potassium nitrate, 2g of humic acid and the balance of water; the multiple subcultures are 5 subcultures.
Preferably, the method for undisturbed enrichment and domestication of functional microorganisms in water sediment further comprises the step of screening and culturing after undisturbed enrichment and domestication culturing are completed: and (3) placing the domesticated and cultured water sediment in a target function screening culture medium for culture, and separating to obtain the target function microorganism strain.
Preferably, the water distribution part comprises a water inlet pipe and a water distribution disk, the water inlet pipe is of a hollow structure, the side surface of the water inlet pipe is sealed and is used for conveying the culture solution to the water distribution disk, the length of the water inlet pipe is greater than the thickness of water sediment, and the water distribution disk is made of quartz sand.
Preferably, the water distribution part is a hollow water inlet pipe made of quartz sand, the top of the water inlet pipe is sealed, and the length of the water inlet pipe is not more than the thickness of water sediment.
Preferably, the water phase circulating part comprises a hose and peristaltic pumps, one ends of the two peristaltic pumps are respectively connected with the water outlet hole and the water inlet hole through two hoses, and the other ends of the two peristaltic pumps are respectively connected with the liquid outlet and the liquid inlet of the culture liquid supplying part.
Preferably, the culture solution supply part comprises a container and a cover body arranged at the top of the container, wherein the cover body is axially provided with a liquid outlet and a liquid inlet, the edges of the holes of the liquid outlet and the liquid inlet are provided with silica gel buffer rubber rings, the liquid outlet is provided with a liquid outlet pipe for extracting culture solution from the container, and the liquid inlet is provided with a liquid inlet pipe for inputting sediment upper layer liquid extracted from the enrichment domestication bin to the container.
Preferably, the aeration component comprises a gas cylinder, a gas control valve, an aeration pipe and an aeration head which are sequentially connected, wherein the aeration pipe is inserted into the cavity of the enrichment and domestication bin through the gas inlet, and the aeration head is hoisted in the cavity of the enrichment and domestication bin and is immersed in the sediment upper layer liquid.
The beneficial effects of the invention are as follows:
(1) On the premise of not damaging the original structure of the sediment, functional microorganisms are enriched, so that the probability of separating the habitat sensitive microorganisms is improved;
(2) The culture condition of the sediment is controlled by the gas phase environment, and simultaneously, the selective culture medium is continuously and slowly provided for the interior or the surface of the sediment, so that the succession of the microorganisms for regulating the sediment to the target direction is achieved, and the method can be used as a powerful tool for researching the microbiological ecology of the sediment.
Drawings
FIG. 1 is a schematic diagram 1 of an apparatus for undisturbed enrichment and acclimation of water sediments.
FIG. 2 is a schematic diagram 2 of an apparatus for undisturbed enrichment and acclimation of water sediments.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
The method for enriching and domesticating functional microorganisms in the undisturbed water sediment needs to utilize the undisturbed water sediment enriching and domesticating device to complete domestication culture.
According to the invention, as shown in figures 1 and 2, the undisturbed enrichment and domestication device for the water sediment comprises an enrichment domestication bin 1, a water distribution part 2, an aqueous phase circulation part 3, a culture solution supply part 4 and an aeration part 5, wherein the top of the enrichment domestication bin 1 is provided with an air inlet hole 111 and an air outlet hole 112, the aeration part 5 is connected with the air inlet hole 111, the bottom of the enrichment domestication bin 1 is provided with a water inlet hole 131, the water distribution part 2 is inserted into a cavity of the enrichment domestication bin 1 through the water inlet hole 131, one end of the water distribution part 2, which is left outside the enrichment domestication bin 1, is connected to a liquid outlet of the culture solution supply part 4 through one group of aqueous phase circulation parts 3, the side wall of the enrichment domestication bin 1 is provided with an water outlet hole 121, and the water outlet hole 121 is connected to a liquid inlet of the culture solution supply part 4 through the other group of aqueous phase circulation part 3.
The device is provided with the enrichment domestication bin for containing the water sediment, so that the original structure of the water sediment is not damaged, the sediment keeps stable in space structure in the sampling and enrichment culture processes, and the probability of separating the habitat sensitive microorganisms is improved; the aeration component is arranged to regulate and control the gas phase environment condition of sediment culture, the culture fluid supply component and the water phase circulation component are arranged to realize continuous upflow nutrition supply and provide the culture fluid for the sediment, the water distribution component can continuously and slowly provide the culture fluid to the interior or the surface of the sediment, the enrichment condition is controlled by adjusting the components of the culture fluid, and the target functional microorganisms are actively enriched in situ, so that the aim of regulating succession of the microorganisms of the sediment to the target direction is fulfilled.
More specifically, the enriching domestication bin 1 comprises an upper cover 11, a main body 12 and a lower cover 13, wherein the upper cover 11 and the lower cover 13 are respectively arranged at the upper end and the lower end of the main body 12, the upper cover 11 is axially provided with an air inlet hole 111 and an air outlet hole 112, the lower cover 13 is axially provided with a water inlet hole 131, and the upper half part of the main body 12 is radially provided with a water outlet hole 122.
The connection modes of the main body 12 and the upper cover 11 and the lower cover 13 can be various, the sealing connection between the main body 12 and the upper cover 11 and the lower cover 13 can be realized, and the lower cover 13 can firmly support the bottom mud and the upper liquid after being connected with the main body 12; for example, the main body 12 and the lower cover 13 are made of acrylic material, the upper cover 11 may be a silica gel or a soft plastic plug, and in order to conveniently connect the main body 12 to the columnar sediment sampler for use, an embodiment is preferred in the present invention, that is, two ends of the main body 12 are provided with threads, and the main body 12 is fixedly connected with the upper cover 11 and the lower cover 13 through the threads. The main body 12 of the enrichment and domestication bin 1 is connected to the bottom end of a columnar sediment sampler in advance during sampling, water sediment can be kept in the main body 12 of the enrichment and domestication bin in the original state during sampling, the main body 12 of the enrichment and domestication bin can be directly detached to serve as an independent culture bin after sampling, and metabolic factors are regulated and controlled through the water phase circulating component and the culture solution supply component on the premise of not damaging the original structure of the water sediment.
The hole edges of the air inlet hole 111, the air outlet hole 112 and the water inlet hole 131 are provided with silica gel buffer rubber rings, and the silica gel buffer rubber rings play a role in protecting pipelines. The main function of the air outlet 112 is to adjust the air pressure in the enrichment domestication bin 1 and keep the air pressure at the standard atmospheric pressure.
When water distribution on the surface of the water sediment is needed, the following method is adopted: the water distribution part 2 comprises a water inlet pipe 21 and a water distribution disc 22, wherein the water inlet pipe 21 is of a hollow structure, the side surface of the water inlet pipe 21 is sealed and is used for conveying the culture solution to the water distribution disc 22, the length of the water inlet pipe 21 is larger than the thickness of the water sediment, and the water distribution disc 22 is made of quartz sand (a quartz sand filter disc with micropores of 100-160 mu m in the embodiment) and is used for filtering the culture solution out of the sediment surface. The upper end of the water inlet pipe 21 is provided with an opening closely combined with the lower end of the water distribution plate 22, the culture solution is dispersed on the surface of the water sediment through the outer surface of the water distribution plate 22, the length of the water sediment protruding from the water inlet pipe 21 is preferably within 1cm, the culture solution conveyed from the culture solution supply part 4 can reach the surface of the water sediment quickly, and nutrition is provided for microorganisms attached on the surface of the water sediment.
When water distribution in the water sediment is needed, the following method is adopted: the water distribution part 2 is a hollow water inlet pipe 21 which is an integral body made of quartz sand material and is used for filtering out culture solution from the sediment; unlike the above surface water distribution mode, the top of the water inlet pipe 21 in this embodiment is sealed, and the length of the water inlet pipe 21 is not greater than the thickness of the water sediment. The culture solution is dispersed into the interior of the water sediment through the quartz sand gap of the water inlet pipe 21, so as to provide nutrition for microorganisms attached to the interior of the water sediment.
The water phase circulation part 3 comprises a hose 31 and peristaltic pumps 32, one ends of the two peristaltic pumps 32 are respectively connected with the water outlet 121 and the water inlet 131 through the two hoses 31, and the other ends of the two peristaltic pumps 32 are respectively connected with the liquid outlet and the liquid inlet of the culture liquid supply part. The two sets of water phase circulation components 3 control the flow rate through peristaltic pumps 32, the culture solution stored in the culture solution supply component 4 is input into the enrichment and domestication bin 1, and meanwhile, the upper layer liquid (the original river water of the water sediment or the culture solution or the mixed solution of the two) of the enrichment and domestication bin 1 is input into the culture solution supply component 4 to form circulation.
The culture solution supply part 4 comprises a container 41 and a cover body 42 arranged at the top of the container 41, wherein the cover body 42 is axially provided with a solution outlet and a solution inlet, the edges of the holes of the solution outlet and the solution inlet are provided with silica gel buffer rubber rings, the solution outlet is provided with a liquid outlet pipe for extracting culture solution from the container 41, and the solution inlet is provided with a liquid inlet pipe for inputting sediment upper layer liquid (raw water river water of water sediment or culture solution or mixture of the two) extracted from the enrichment domestication bin 1 into the container 41. The container 41 is preferably an upright cylindrical acrylic cup. Depending on the screening purpose, a selective medium is added to vessel 41.
The aeration component 5 comprises a gas cylinder 51, a gas control valve 52, an aeration pipe 53 and an aeration head 54 which are sequentially connected, the aeration pipe 53 is inserted into the cavity of the enrichment and domestication bin 1 through an air inlet hole 111, the aeration head 54 is hoisted in the cavity of the enrichment and domestication bin 1, and the aeration head 54 is immersed in sediment upper layer liquid. The aeration means 5 serves to dissolve the gas in the gas cylinder 51 into the liquid on the upper layer of the sediment, and control the culture environment. When different environments need to be created, for example, oxygen or air is aerated in an aerobic environment, nitrogen is aerated in an anaerobic environment, and a mixed gas of nitrogen and carbon dioxide is aerated in a fermentation environment, a gas cylinder 51 storing corresponding gas is selected and used according to different strains.
In the following examples, the above-mentioned device for enriching and domesticating water sediments was used to perform domestication culture of microorganisms with different target functions.
Example 1: enrichment screening test of denitrifying pyrene degrading bacteria in sediment
A cylindrical sampler with an enrichment and domestication bin main body 12 at the bottom end of a wharf in a Guangzhou river channel of a Zhujiang river is used for collecting undisturbed river sediment with the thickness of about 5cm on the surface layer in the enrichment and domestication bin main body 12 (with the diameter of 8.5cm and the height of 25 cm), a lower cover 13 is arranged at the bottom of the enrichment and domestication bin main body 12, the enrichment and domestication bin is taken down from the cylindrical sampler, and the lower end of the lower cover 13 is inserted into a quartz sand cloth water pipe; uniformly and randomly dripping 10mL of pyrene solution (0.06 mg/mL, n-hexane as solvent) on the surface of the sediment by using a glass needle tube, standing for 30min (waiting for volatilization of n-hexane on the surface), slowly injecting river water into the enrichment and domestication bin main body 12 to form a water layer with the height of about 12cm, and covering the enrichment and domestication bin main body 12 with an upper cover 11; 600mL of denitrification medium was added to the culture solution supply unit 4; a water inlet and outlet hose 31 is connected, and a peristaltic pump 32 is used for controlling the flow rate (1.5 mL/min); domestication culture is carried out at 28 ℃ for 45 days, the denitrification culture medium in the culture solution supply part is replaced every 5 days, and the concentration of nitrate is gradually increased in the process. The method comprises the following steps: the denitrification medium used contained per liter: 1.0g of ammonium chloride, 1.0g of monopotassium phosphate, 0.1g of magnesium chloride, 10mL of trace element solution, 10mL of vitamin solution, 0.17-0.68g of sodium nitrate and the balance of water; wherein, every 15 days, the concentration of sodium nitrate is increased from 0.17g/L to 0.425g/L and 0.68g/L in sequence. The microelement solution contains 30mg of cobalt chloride hexahydrate, 0.15mg of copper chloride pentahydrate, 5.7mg of boric acid, 20mg of manganese chloride tetrahydrate, 25mg of sodium tungstate dihydrate, 1.5mg of nickel chloride dihydrate, 2.1mg of zinc chloride, 5mg of calcium chloride dihydrate and the balance of water. The vitamin solution contains 50mg of vitamin B1, 50mg of vitamin B2, nicotinic acid, pantothenic acid, 50mg of aminobenzoic acid, 20mg of lipoic acid, 20mg of folic acid, 1mg of vitamin B12 and the balance of water.
1g of sediment with the surface layer of 3cm is randomly taken from the enrichment and domestication bin main body 12 after domestication is finished, and is placed in 9mL of sterile water, and is vibrated and uniformly mixed to obtain mud suspension with 10 times of dilution; then 1mL of mud suspension is put into 9mL of new sterile water to obtain 100 times of diluted mud suspension, and the steps are repeated to obtain 10 3 Multiple of 10 4 Multiple of 10 5 Double sum 10 6 Multiple dilutions of the mud suspension; take 10 3 Multiple of 10 4 Multiple of 10 5 Double sum 10 6 The sludge suspension with the double dilution degree is respectively 0.5mL, the sludge suspension is coated on a pyrene-containing solid denitrification culture medium, the culture is carried out in an anaerobic incubator at 28 ℃, after bacterial colonies grow out for about 5 days, single bacteria are separated out by streaking on a new pyrene-containing solid denitrification culture medium for several times. The preparation method of the pyrene-containing solid denitrification culture medium comprises the steps of adding agar with the final concentration of 20g/L according to the formula of the denitrification culture medium (wherein the concentration of sodium nitrate is 425 mg/L), pouring the agar into a flat plate after sterilization, adding 0.2mL of n-hexane solution of pyrene (with the concentration of 0.15 mg/mL) into a glass needle tube after the culture medium is solidified, and forming a light white pyrene solid thin layer on the surface of the culture medium after n-hexane volatilizes in a sterile environment (around an alcohol lamp), thereby obtaining the pyrene-containing solid denitrification culture medium.
Verifying the effect of denitrifying pyrene degrading bacteria, selecting the single bacteria, placing the single bacteria in a denitrification culture medium (wherein the concentration of sodium nitrate is 425 mg/L) for activation for 2 days, taking 2mL of activated bacteria liquid, adding the activated bacteria liquid into 18mL of a pyrene-containing (liquid) denitrification culture system (wherein the concentration of pyrene is 30g/L, the concentration of sodium nitrate is 425mg/L,3 repeated samples), culturing for 12 days at the anaerobic 28 ℃, adding 10mL of n-hexane for extraction, and determining the concentration of pyrene by using an organic phase and the concentration of nitrate by using a water phase; a high-efficiency denitrifying degradation bacterium is screened out through the pyrene degradation rate, identified as Paracoccus sp, and the pyrene can be degraded from 30mg/L to 13.83+/-0.24 mg/L, the degradation rate reaches 53.9%, and the nitrate is synchronously reduced by 72.6%.
Example 2: enrichment screening of Metal-resistant benzopyrene degrading bacteria in sediment
A cylindrical sampler with an enrichment and domestication bin main body 12 at the bottom end of a wharf in a Guangzhou river channel of a Zhujiang river is used for collecting undisturbed river sediment with the thickness of about 5cm on the surface layer in the enrichment and domestication bin main body 12 (with the diameter of 8.5cm and the height of 25 cm), a lower cover 13 is arranged at the bottom of the enrichment and domestication bin main body 12, the enrichment and domestication bin is taken down from the cylindrical sampler, and the lower end of the lower cover 13 is inserted into a quartz sand cloth water pipe; uniformly and randomly dripping 10mL of benzopyrene solution (0.04 mg/mL, acetone as solvent) on the surface of the sediment by using a glass needle tube, standing for 30min (waiting for volatilization of surface acetone), slowly injecting river water into the enrichment and domestication bin main body 12 to form a water layer with the height of about 12cm, and covering the enrichment and domestication bin main body 12 with an upper cover 11; 600mL of lead nitrate medium was added to the medium supply unit 4; a water inlet and outlet hose 31 is connected, and a peristaltic pump 32 is used for controlling the flow rate (1 mL/min); the culture medium was acclimatized at 30℃for 60 days, and the lead nitrate medium in the medium supplying member was changed every 15 days. The lead nitrate medium used contained per liter: 320mg of lead nitrate (i.e. containing 200mg of lead ions), 2g of sodium nitrate, 0.31g of ammonium chloride, 2.7722g of sodium dihydrogen phosphate dihydrate, 0.13g of potassium chloride, 1.5420g of disodium hydrogen phosphate dodecahydrate, 12.5mL of trace element solution, 5mL of vitamin solution and the balance of water. The microelement solution contains 30mg of cobalt chloride hexahydrate, 0.15mg of copper chloride pentahydrate, 5.7mg of boric acid, 20mg of manganese chloride tetrahydrate, 25mg of sodium tungstate dihydrate, 1.5mg of nickel chloride dihydrate, 2.1mg of zinc chloride, 5mg of calcium chloride dihydrate and the balance of water. The vitamin solution contains 50mg of vitamin B1, 50mg of vitamin B2, nicotinic acid, pantothenic acid, 50mg of aminobenzoic acid, 20mg of lipoic acid, 20mg of folic acid, 1mg of vitamin B12 and the balance of water.
And after the acclimation is finished, randomly taking 120g of sediment from the enrichment acclimation bin main body 12, placing the sediment into a conical flask filled with 200mL of lead nitrate culture medium, dropwise adding 1mL of benzopyrene acetone solution (0.1 mg/mL), introducing nitrogen under the liquid surface for 10min, sealing by a bottle cap, culturing at 30 ℃ in a dark place for 7 days, and vibrating to obtain acclimation mud suspension. 10mL of domesticated mud suspension is taken and added into a conical flask filled with 90mL of lead nitrate culture medium, 1mL of benzopyrene acetone solution (0.4 mg/mL) is added dropwise, and after light-shielding culture is carried out for 7 days at 30 ℃, the transfer is repeated for 5 times, thus obtaining bacterial suspension. Dilution 10 was obtained by gradient dilution 3 Multiple of 10 4 Multiple of 10 5 Double sum 10 6 The bacterial suspension is diluted by times, coated on a solid lead nitrate culture medium, cultured in an anaerobic incubator at 30 ℃ for about 5 days to grow bacterial colonies, single bacteria are streaked on a new solid lead nitrate culture medium for several times, and 16S rDNA sequences are taken and aligned in GenBank, and the similarity with paracoccus ammoniaphaeovorans (Paracoccus aminovorans) reaches 100 percent, which is called paracoccus ammoniaphaeovorus F2. The solid lead nitrate culture medium is prepared by adding 2% agar into the above lead nitrate culture medium, sterilizing, pouring into a culture dish, cooling, uniformly dripping 100 μl of benzopyrene acetone solution (1 mg/mL) onto the surface of the coagulated agar culture medium, standing in sterile environment, and volatilizing acetone thoroughly.
Paracoccus ammoniaphagosides F2 was taken to verify benzopyrene degradation in lead-containing and lead-free media, and another strain of Paracoccus aerobics (Paracoccus versutus) A1, which was screened in a lead-ion-free environment, was used as a control. Taking the single bacteria, placing the single bacteria in a liquid culture medium for activation for 2 days, taking 2mL of activated bacteria liquid, respectively adding the activated bacteria liquid into 18mL of culture medium containing lead (lead ion concentration is 200mg/L,3 repeated samples) and not containing lead (lead ion concentration is 0mg/L,3 repeated samples) (the formula is the same as that of the lead nitrate culture medium, but the concentration of lead nitrate is different from that of the lead nitrate culture medium, wherein the concentration of benzopyrene is 4 mg/L), culturing for 12 days at the anaerobic 30 ℃, adding 10mL of acetone for extraction, and using an organic phase for measuring the concentration of benzopyrene; the results show that: (1) The strain F2 degrades benzopyrene from 3.715 +/-0.167 mg/L to 1.663 +/-0.236 mg/L in a lead-free culture medium, and the degradation rate is 55.2%; (2) The strain A1 degrades benzopyrene from 3.697 +/-0.183 mg/L to 1.632 +/-0.151 mg/L in a lead-free culture medium, and the degradation rate is 55.9%; (3) The benzopyrene of the strain F2 is degraded from 3.700+/-0.114 mg/L to 1.720 +/-0.116 mg/L in a lead-containing culture medium, and the degradation rate is 53.5%; (4) The strain A1 degrades benzopyrene from 3.686 +/-0.181 mg/L to 2.246+/-0.130 mg/L in a lead-containing culture medium, and the degradation rate is 39.1%; the strain F2 still maintains a higher degradation rate of benzopyrene relative to the strain A1 under the influence of lead ion pressure, which shows that the paracoccus ammoniaphaeovorans F2 obtained by domestication of the undisturbed sediment of the method is a benzopyrene degradation bacterium with lead ion resistance.
Example 3: enrichment of Total organic carbon degrading flora in sediment
A cylindrical sampler with an enrichment and domestication bin main body 12 is connected to the bottom end of a black and odorous river in the city of bergamot of Guangdong, a raw river sediment with the thickness of about 5cm is collected on the surface layer of the enrichment and domestication bin main body 12 (the diameter is 8.5cm and the height is 25 cm), a lower cover 13 is arranged at the bottom of the enrichment and domestication bin main body 12, the enrichment and domestication bin is taken down from the cylindrical sampler, a water inlet pipe 21 and a water distribution disc 22 are inserted into the lower end of the lower cover 13, river water is slowly injected into the enrichment and domestication bin main body 12 to form a water layer with the height of about 12cm, and the enrichment and domestication bin main body 12 is covered with an upper cover 11; 600mL of humic acid medium was added to the culture medium supply unit 4; a water inlet and outlet hose 31 is connected, and a peristaltic pump 32 is used for controlling the flow rate (1.5 mL/min); domestication culture is carried out for 20 days at normal temperature (20-25 ℃), humic acid culture medium in a culture solution supply part is replaced every 5 days, and the concentration of humic acid is gradually increased in the process. The method comprises the following steps: the humic acid medium used contains per liter: 0.2g of magnesium sulfate, 0.25g of potassium nitrate, 0.2-6g of humic acid and the balance of water; wherein, every 5 days, the concentration of humic acid is increased from 0.2g/L to 0.4g/L, 1.6g/L and 6g/L in sequence.
And after the domestication is finished, 0.5g of sediment is taken from the enrichment domestication bin main body 12 and placed in 9mL of freshly prepared enrichment humic acid culture medium (enrichment humic acid culture medium, each liter of the enrichment humic acid culture medium comprises 0.2g of magnesium sulfate, 0.25g of potassium nitrate and 2g of humic acid, and the balance is water), 1mL of humic acid culture solution is taken after 3 days of culture, and is added into 9mL of freshly prepared enrichment humic acid culture medium, and the obtained humic acid culture solution is the humic acid degrading flora after 5 passages.
1mL of humic acid degrading bacteria are put into 5mL of black and odorous river sediment (the water content is 50%, the total organic carbon content is 140.03 +/-6.12 mg/g) and cultured for 14 days at 25 ℃, and 3 repeated treatments are carried out; taking out the sediment, freeze-drying, and measuring the total organic carbon; the result shows that the total organic carbon content is reduced to 103.80+/-3.90 g/g, and the organic carbon degradation rate reaches 25.8%, which shows that the sediment organic matter degradation flora is successfully enriched.
Claims (7)
1. A method for enriching and domesticating functional microorganisms in undisturbed water sediments is characterized in that an undisturbed enrichment and domestication device is utilized for carrying out undisturbed enrichment and domestication culture on the undisturbed water sediments to obtain the functional microorganisms;
the water sediment undisturbed enrichment and domestication device comprises an enrichment domestication bin, a water distribution part, an aqueous phase circulation part, a culture solution supply part and an aeration part, wherein the top of the enrichment domestication bin is provided with an air inlet hole and an air outlet hole, the aeration part is connected with the air inlet hole, the bottom of the enrichment domestication bin is provided with a water inlet hole, the water distribution part is inserted into a cavity of the enrichment domestication bin through the water inlet hole, one end of the water distribution part, which is left outside the enrichment domestication bin, is connected to a liquid outlet of the culture solution supply part through one set of aqueous phase circulation part, the side wall of the enrichment domestication bin is provided with a water outlet hole, and the water outlet hole is connected to a liquid inlet of the culture solution supply part through the other set of aqueous phase circulation part; the enrichment domestication bin comprises an upper cover, a main body and a lower cover, wherein the upper cover and the lower cover are respectively arranged at the upper end and the lower end of the main body, the upper cover is axially provided with an air inlet hole and an air outlet hole, the lower cover is axially provided with a water inlet hole, and the upper half part of the main body is radially provided with a water outlet hole; threads are arranged at two ends of the main body, and the main body is fixedly connected with the upper cover and the lower cover through the threads; the main body of the enrichment and domestication bin is connected to the bottom end of a columnar sediment sampler in advance during sampling, water sediment can be kept in the main body of the enrichment and domestication bin in the original state during sampling, the main body of the enrichment and domestication bin can be directly detached to serve as an independent culture bin after sampling, and metabolic factors are regulated and controlled through the water phase circulating component and the culture solution supply component on the premise of not damaging the original structure of the water sediment;
the undisturbed enrichment and domestication culture are as follows: collecting undisturbed water sediment in an acclimation and enrichment bin, injecting a crude water body of sampling water sediment into the acclimation and enrichment bin, adding a culture solution for enriching and acclimating target functional microorganisms into a culture solution supply part, starting an aqueous phase circulation part to input the culture solution into the surface or the inside of the water sediment through a water distribution part, and timely adjusting the components of the culture solution to finish undisturbed enrichment and acclimation culture;
after the undisturbed enrichment and domestication culture are completed, the method further comprises the step of liquid phase enrichment culture: placing the domesticated and cultured water sediment in an enrichment medium for culture and subculturing for a plurality of times to obtain a target functional microorganism flora;
after the undisturbed enrichment and domestication culture is completed, the method further comprises the step of screening culture: and (3) placing the domesticated and cultured water sediment in a target function screening culture medium for culture, and separating to obtain the target function microorganism strain.
2. The method according to claim 1, wherein the culture medium in the undisturbed enrichment and acclimation culture step is humic acid culture medium, and each liter of the culture medium comprises: 0.2 Magnesium sulfate, 0.25-g potassium nitrate, 0.2-6-g humic acid and the balance of water; wherein, every 5 days, the concentration of humic acid is increased from 0.2g/L to 0.4g/L, 1.6g/L and 6g/L in sequence; the enrichment culture medium in the liquid phase enrichment culture step is an enrichment humic acid culture medium, and each liter of the enrichment culture medium contains: 0.2 Magnesium sulfate, 0.25g potassium nitrate, 2g humic acid and the balance of water; the multiple subcultures are 5 subcultures.
3. The method according to claim 1, wherein the water distribution part comprises a water inlet pipe and a water distribution disc, the water inlet pipe is of a hollow structure, the side surface of the water inlet pipe is sealed and is used for conveying the culture solution to the water distribution disc, the length of the water inlet pipe is greater than the thickness of the water sediment, and the water distribution disc is made of quartz sand.
4. The method of claim 1, wherein the water distribution member is a hollow water inlet pipe made of quartz sand, the top of the water inlet pipe is sealed, and the length of the water inlet pipe is not greater than the thickness of the water sediment.
5. The method according to claim 1, wherein the water phase circulation part comprises a hose and peristaltic pumps, one ends of the peristaltic pumps are respectively connected with the water outlet hole and the water inlet hole through two hoses, and the other ends of the peristaltic pumps are respectively connected with the water outlet and the water inlet of the culture solution supply part.
6. The method according to claim 1, wherein the culture solution supply unit comprises a container and a cover body arranged at the top of the container, the cover body is axially provided with a solution outlet and a solution inlet, the edges of the holes of the solution outlet and the solution inlet are provided with silica gel buffer rubber rings, the solution outlet is provided with a liquid outlet pipe for extracting culture solution from the container, and the solution inlet is provided with a solution inlet pipe for inputting sediment upper layer liquid extracted from the enrichment domestication bin into the container.
7. The method of claim 1, wherein the aeration component comprises a gas cylinder, a gas control valve, an aeration pipe and an aeration head which are connected in sequence, the aeration pipe is inserted into the cavity of the enrichment and domestication bin through the gas inlet hole, and the aeration head is hoisted in the cavity of the enrichment and domestication bin and is immersed in the upper layer liquid of the sediment.
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