CN114958696A - 一种产甲烷菌培养基及培养方法 - Google Patents
一种产甲烷菌培养基及培养方法 Download PDFInfo
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- CN114958696A CN114958696A CN202210849580.6A CN202210849580A CN114958696A CN 114958696 A CN114958696 A CN 114958696A CN 202210849580 A CN202210849580 A CN 202210849580A CN 114958696 A CN114958696 A CN 114958696A
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- methanogens
- culture medium
- methanogen
- culture
- sodium
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- 239000001963 growth medium Substances 0.000 title claims abstract description 47
- 238000012136 culture method Methods 0.000 title abstract description 8
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000004280 Sodium formate Substances 0.000 claims abstract description 21
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 21
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 21
- HLBBKKJFGFRGMU-UHFFFAOYSA-M sodium formate Chemical compound [Na+].[O-]C=O HLBBKKJFGFRGMU-UHFFFAOYSA-M 0.000 claims abstract description 21
- 235000019254 sodium formate Nutrition 0.000 claims abstract description 21
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 12
- 239000007789 gas Substances 0.000 claims abstract description 12
- 239000001632 sodium acetate Substances 0.000 claims abstract description 12
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000002609 medium Substances 0.000 claims description 31
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims description 26
- 230000012010 growth Effects 0.000 claims description 25
- 241000202974 Methanobacterium Species 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 19
- 238000012258 culturing Methods 0.000 claims description 14
- 229940088594 vitamin Drugs 0.000 claims description 14
- 229930003231 vitamin Natural products 0.000 claims description 14
- 235000013343 vitamin Nutrition 0.000 claims description 14
- 239000011782 vitamin Substances 0.000 claims description 14
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 12
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 claims description 12
- 229910021654 trace metal Inorganic materials 0.000 claims description 11
- 108090000698 Formate Dehydrogenases Proteins 0.000 claims description 10
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 9
- 239000011734 sodium Substances 0.000 claims description 9
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 claims description 8
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 8
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 7
- 241000203353 Methanococcus Species 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 7
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 claims description 6
- 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 claims description 6
- 229930003779 Vitamin B12 Natural products 0.000 claims description 6
- 229960004050 aminobenzoic acid Drugs 0.000 claims description 6
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 claims description 6
- 229960002079 calcium pantothenate Drugs 0.000 claims description 6
- 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 claims description 6
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 6
- 229960004172 pyridoxine hydrochloride Drugs 0.000 claims description 6
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 claims description 6
- 239000011764 pyridoxine hydrochloride Substances 0.000 claims description 6
- 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 claims description 6
- 235000019190 thiamine hydrochloride Nutrition 0.000 claims description 6
- 239000011747 thiamine hydrochloride Substances 0.000 claims description 6
- 235000019163 vitamin B12 Nutrition 0.000 claims description 6
- 239000011715 vitamin B12 Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 5
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims description 5
- 238000011109 contamination Methods 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 229910052708 sodium Inorganic materials 0.000 claims description 5
- 229960000344 thiamine hydrochloride Drugs 0.000 claims description 5
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 4
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 4
- 241000193751 Methanoculleus Species 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 4
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 4
- 229960002685 biotin Drugs 0.000 claims description 4
- 235000020958 biotin Nutrition 0.000 claims description 4
- 239000011616 biotin Substances 0.000 claims description 4
- 229910017052 cobalt Inorganic materials 0.000 claims description 4
- 239000010941 cobalt Substances 0.000 claims description 4
- 229910052802 copper Inorganic materials 0.000 claims description 4
- 239000010949 copper Substances 0.000 claims description 4
- 229910052742 iron Inorganic materials 0.000 claims description 4
- 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 claims description 4
- 229910052759 nickel Inorganic materials 0.000 claims description 4
- 229960003512 nicotinic acid Drugs 0.000 claims description 4
- 235000001968 nicotinic acid Nutrition 0.000 claims description 4
- 239000011664 nicotinic acid Substances 0.000 claims description 4
- 229910052725 zinc Inorganic materials 0.000 claims description 4
- 239000011701 zinc Substances 0.000 claims description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 3
- 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 claims description 3
- 241000205265 Methanospirillum Species 0.000 claims description 3
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 claims description 3
- 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 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 3
- 229910052782 aluminium Inorganic materials 0.000 claims description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims description 3
- 239000011575 calcium Substances 0.000 claims description 3
- 229910052791 calcium Inorganic materials 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 3
- 235000018417 cysteine Nutrition 0.000 claims description 3
- 229910001873 dinitrogen Inorganic materials 0.000 claims description 3
- 229960000304 folic acid Drugs 0.000 claims description 3
- 235000019152 folic acid Nutrition 0.000 claims description 3
- 239000011724 folic acid Substances 0.000 claims description 3
- AGBQKNBQESQNJD-UHFFFAOYSA-M lipoate Chemical compound [O-]C(=O)CCCCC1CCSS1 AGBQKNBQESQNJD-UHFFFAOYSA-M 0.000 claims description 3
- 235000019136 lipoic acid Nutrition 0.000 claims description 3
- 229910052750 molybdenum Inorganic materials 0.000 claims description 3
- 239000011733 molybdenum Substances 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 239000011591 potassium Substances 0.000 claims description 3
- 229910052700 potassium Inorganic materials 0.000 claims description 3
- 229960002477 riboflavin Drugs 0.000 claims description 3
- 239000002151 riboflavin Substances 0.000 claims description 3
- 235000019192 riboflavin Nutrition 0.000 claims description 3
- 229910052979 sodium sulfide Inorganic materials 0.000 claims description 3
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 claims description 3
- 241000894007 species Species 0.000 claims description 3
- 229960002663 thioctic acid Drugs 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 claims description 2
- 229940038032 nitrogen 20 % Drugs 0.000 claims 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 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 23
- 239000001257 hydrogen Substances 0.000 abstract description 22
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 abstract description 20
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 description 36
- 239000000243 solution Substances 0.000 description 21
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 20
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 15
- 230000000696 methanogenic effect Effects 0.000 description 13
- 239000000758 substrate Substances 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 10
- 241000894006 Bacteria Species 0.000 description 10
- 235000019253 formic acid Nutrition 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 6
- 230000005526 G1 to G0 transition Effects 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 6
- 239000000306 component Substances 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 description 5
- 229940052299 calcium chloride dihydrate Drugs 0.000 description 5
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(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+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 5
- 239000012452 mother liquor Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 230000003698 anagen phase Effects 0.000 description 4
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 4
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 4
- 230000037353 metabolic pathway Effects 0.000 description 4
- LAIZPRYFQUWUBN-UHFFFAOYSA-L nickel chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Ni+2] LAIZPRYFQUWUBN-UHFFFAOYSA-L 0.000 description 4
- 239000011780 sodium chloride 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
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 241001529871 Methanococcus maripaludis Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 235000019270 ammonium chloride Nutrition 0.000 description 3
- 229960001040 ammonium chloride Drugs 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 239000003638 chemical reducing agent Substances 0.000 description 3
- 230000037149 energy metabolism Effects 0.000 description 3
- 150000002431 hydrogen Chemical class 0.000 description 3
- 230000001788 irregular Effects 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 3
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 235000000638 D-biotin Nutrition 0.000 description 2
- 239000011665 D-biotin Substances 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241000205263 Methanospirillum hungatei Species 0.000 description 2
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 2
- 101100001347 Mus musculus Akt1s1 gene Proteins 0.000 description 2
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 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
- 229960005069 calcium Drugs 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 150000001722 carbon compounds Chemical class 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
- 230000003750 conditioning effect Effects 0.000 description 2
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- MQLVWQSVRZVNIP-UHFFFAOYSA-L ferrous ammonium sulfate hexahydrate Chemical compound [NH4+].[NH4+].O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O MQLVWQSVRZVNIP-UHFFFAOYSA-L 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 description 2
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 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
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 229960002816 potassium chloride Drugs 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
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Abstract
本发明提供产甲烷菌的培养基及培养方法,所述培养基包括8g/L的甲酸钠和4g/L的乙酸钠。通过增加或额外添加培养基中的甲酸钠和乙酸钠至8g/L的甲酸钠和4g/L的乙酸钠,并通入80%氮气+20%二氧化碳的混合气,可在不使用氢气的条件下培养产甲烷菌,并使产甲烷菌快速大量生长。
Description
技术领域
本发明涉及一种产甲烷菌的培养基及培养方法,尤其是具有甲酸脱氢酶(FDH)的氢营养型产甲烷菌的培养基及培养方法,尤其是在实验室培养中。
背景技术
产甲烷菌是一种生存于绝对无氧环境中的古细菌。产甲烷菌的培养要求绝对无氧的还原性体系,苛求二氧化碳、甲醇、甲酸、乙酸等简单碳化合物实现能量代谢,且通常只能利用这些简单碳化合物实现能量代谢,而不能使用常见的复杂碳源如葡萄糖、丙酮酸钠、乳酸钠等,极难在实验室中培养。
甲烷产生是产甲烷菌生长的唯一能量代谢途径。产甲烷菌主要有三种产甲烷途径,分别利用氢气+二氧化碳、乙酸、甲基化合物。据此可将产甲烷菌分为氢营养型、乙酸发酵型、甲基营养型。根据产甲烷菌的代谢途径、形态、系统发育关系等特征,可将产甲烷菌分为6目、13科、33属。
一半以上的产甲烷菌为氢营养型(23属),即利用氢气作为电子供体、二氧化碳作为电子受体,通过产甲烷菌特有的产甲烷途径实现能量代谢,进而实现产甲烷菌的实验室培养。然而,用这种方式培养产甲烷菌需要在培养容器中持续通入80%氢气和20%二氧化碳混合气30分钟以上以彻底排除氧气,每次制作培养基10×50mL泄漏的氢气量可高达200L以上。易燃易爆的高浓度氢气对实验室安全造成很大隐患,也提高了产甲烷菌培养的实验室基础设施门槛。因此,改良产甲烷菌培养方式,避免使用高浓度氢气作产甲烷菌培养底物,是产甲烷菌实验室培养的重大需求。
发明内容
75%(18属)的氢营养型产甲烷菌具有甲酸脱氢酶(Formate dehydrogenase,FDH),可催化氢气、二氧化碳与甲酸的相互转化。因此理论上使用甲酸(或甲酸盐)代替氢气、二氧化碳作为具有FDH的产甲烷菌培养底物在原理上是可行的。
尽管许多氢营养型产甲烷菌被证明可以利用甲酸产甲烷,但除少数产甲烷菌(如Methanobacterium formicicum)外,大部分使用甲酸培养的产甲烷菌面临生长缓慢、生物量低等问题,难以实现产甲烷菌的大量培养。因此,当前主流微生物保藏中心(美国ATCC、德国DSMZ)给出的氢营养型产甲烷菌培养基均使用80%H2+20%CO2培养(如DSM medium 119、DSM medium 141、DSM medium 1523、ATCC medium 1405、ATCC medium2487等)。而在最近使用这些产甲烷菌纯培养物进行科学研究的论文中,也均使用氢气培养。
本发明提供一种不使用氢气的产甲烷菌的实验室培养方法。
本发明提供如下技术方案:
一方面,本发明提供产甲烷菌的培养基,其特征在于,所述培养基包括8g/L的甲酸钠和4g/L的乙酸钠。
在一些实施方案中,所述产甲烷菌为具有甲酸脱氢酶的氢营养型产甲烷菌。
在一些实施方案中,产甲烷菌选自亨氏甲烷螺菌、海沼甲烷球菌、甲酸甲烷杆菌、甲烷叶菌和巴伐利亚甲烷粒菌。
在一些实施方案中,所述培养基还包括磷酸盐、硫酸盐、氯化物、微量金属元素、刃天青指示剂、半胱氨酸、硫化钠和维生素。
在一些实施方案中,所述培养基还包括蛋白胨和酵母提取物。
在一些实施方案中,所述磷酸盐包括磷酸二氢钾、无水磷酸氢二钾中的一种或多种。
在一些实施方案中,所述磷酸盐包括为磷酸二氢钾。
在一些实施方案中,所述磷酸盐包括为无水磷酸氢二钾。
在一些实施方案中,所述硫酸盐包括七水硫酸镁、六水硫酸亚铁铵中的一种或多种。
在一些实施方案中,所述硫酸盐为七水硫酸镁。
在一些实施方案中,所述硫酸盐为七水硫酸镁和六水硫酸亚铁铵。
在一些实施方案中,所述氯化物包括氯化钠、氯化铵、二水氯化钙、氯化钾、六水氯化镁中的一种或多种。
在一些实施方案中,所述氯化物包括氯化钠、氯化铵和二水氯化钙。
在一些实施方案中,所述氯化物包括氯化铵、二水氯化钙、氯化钾和六水氯化镁。
在一些实施方案中,所述微量金属元素包括:铁、锌、锰、钴、铜、镍、钼、钠、钙、铝、钾中的一种或多种。
在一些实施方案中,所述微量金属元素包括:铁、锌、锰、钴、铜、镍、钼和钠。
在一些实施方案中,所述微量金属元素包括:铁、锌、锰、钴、铜、镍、钠、钙、铝和钾。
在一些实施方案中,所述维生素包括:维生素B12、对氨基苯甲酸、生物素、烟酸、泛酸钙、盐酸吡哆醇、盐酸硫胺素、叶酸、核黄素、硫辛酸中的一种或多种。
在一些实施方案中,所述维生素包括:维生素B12、对氨基苯甲酸、生物素、烟酸、泛酸钙、盐酸吡哆醇和盐酸硫胺素。
在一些实施方案中,所述维生素包括:维生素B12、对氨基苯甲酸、生物素、烟酸、泛酸钙、盐酸吡哆醇、盐酸硫胺素、叶酸、核黄素和硫辛酸。
在一些实施方案中,8g/L的甲酸钠和4g/L的乙酸钠在标准培养基的基础上添加或补加,所述标准培养基为DSM medium 119、DSM medium141、DSM medium 1523、ATCC medium1405或ATCC medium 2487。
在一些实施方案中,所述标准培养基为DSM medium 1523。
在一些实施方案中,所述标准培养基为DSM medium 141。
在一些实施方案中,所述标准培养基为DSM medium 141c。
另一方面,本发明提供产甲烷菌的培养方法,其特征在于,所述培养方法包括在如上所述的培养基中接种产甲烷菌,并在所述培养基中通入80%氮气+20%二氧化碳的混合气,对所述产甲烷菌进行培养。
在一些实施方案中,所述培养方法还包括监测产甲烷菌生长情况,若怀疑有污染风险,则鉴定产甲烷菌种类。
另一方面,本发明提供如上所述的培养基在培养产甲烷菌中的应用。
另一方面,本发明提供制备甲烷的方法,其特征在于,所述方法包括利用如上所述的培养基,并在所述培养基中通入80%氮气+20%二氧化碳的混合气培养产甲烷菌,并收集所述产甲烷菌产生的甲烷。
有益效果
与常规的产甲烷菌培养方法比较,本发明具有如下优点和积极效果:
1.本发明避免使用高度危险的80%氢气培养产甲烷菌,更好地保证了实验人员的安全。
2.本发明减少了使用氢气所需的防爆设施,降低了产甲烷菌的培养难度,有利于产甲烷菌培养的实验室普及。
3.甲酸钠、乙酸钠溶解后分配到不同的培养容器,因此只要是同一批分配的培养基,甲酸钠、乙酸钠浓度均相同。若使用氢气,则可能因培养容器气体体积不同,导致底物量存在差异,因此本发明保证了同一批制作的培养基碳底物浓度的一致性,有利于保证使用同一批培养基进行同一批传代的产甲烷菌生长的均一性。
附图说明
图1为氢营养型产甲烷菌甲烷产生途径的代谢过程,从分支以下开始为利用氢气+二氧化碳作底物的代谢途径,从左分支开始为利用甲酸作底物的代谢途径。箭头首尾为代谢物的化学结构,写在箭头中间的正体(代表酶)或斜体(代表基因)字为代谢途径所需的关键酶或关键基因。可以看出,甲酸脱氢酶(FDH)催化甲酸分解为二氧化碳和氢气(实际也可催化反向过程,但在产甲烷途径中只能催化正向过程),提供了氢营养型产甲烷菌所需的氢气+二氧化碳底物。因此在FDH存在下氢气+二氧化碳与甲酸均可作为代谢底物。
图2示出亨氏甲烷螺菌(DSM 864)、甲酸甲烷杆菌(DSM 1535)、海沼甲烷球菌(DSM2067)、甲烷叶菌(DSM 4140)、巴伐利亚甲烷粒菌(DSM 4179)的生长曲线。
图3示出亨氏甲烷螺菌(DSM 864)、甲酸甲烷杆菌(DSM 1535)、海沼甲烷球菌(DSM2067)、甲烷叶菌(DSM 4140)、巴伐利亚甲烷粒菌(DSM 4179)的甲烷产生量(mL)随生长时间的变化。
图4示出亨氏甲烷螺菌(DSM 864)、甲酸甲烷杆菌(DSM 1535)、海沼甲烷球菌(DSM2067)、甲烷叶菌(DSM 4140)、巴伐利亚甲烷粒菌(DSM 4179)的荧光显微镜图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,以下结合具体实施例,并参照附图,对本发明作进一步的详细说明。
甲酸钠是产甲烷代谢核心底物,甲酸钠的量决定了微生物能生长的量。不同微生物需要的乙酸钠的量不同(有的甚至不需要),但乙酸钠过量不会影响微生物生存,因此添加较高浓度的乙酸钠较好,据此本发明提供一种用甲酸钠和乙酸钠培养产甲烷菌的方法,为保证普适性,随机选择了五株不同属、形态差异大、原始存活生态环境差异大,但都具有FDH的氢营养型模式产甲烷菌(type strain),以配制10瓶(每瓶50mL)培养基为例对本发明的实施方案进行描述。具体来说选用亨氏甲烷螺菌(Methanospirillum hungatei,DSM864)(DSM 864是德国菌种保藏中心(German Collection of Microorganisms and CellCultures GmbH)赋予保藏微生物的编号,可直接从该菌种保藏中心购买微生物)、海沼甲烷球菌(Methanococcus maripaludis,DSM 2067)(DSM 2067是德国菌种保藏中心(GermanCollection of Microorganisms and Cell Cultures GmbH)赋予保藏微生物的编号,可直接从该菌种保藏中心购买微生物)、甲酸甲烷杆菌(Methanobacterium formicicum,DSM1535)(DSM 1535是德国菌种保藏中心(German Collection of Microorganisms and CellCultures GmbH)赋予保藏微生物的编号,可直接从该菌种保藏中心购买微生物)、甲烷叶菌(Methanofollis liminatans,DSM 4140)(DSM 4140是德国菌种保藏中心(GermanCollection of Microorganisms and Cell Cultures GmbH)赋予保藏微生物的编号,可直接从该菌种保藏中心购买微生物)、巴伐利亚甲烷粒菌(Methanocorpusculum bavaricum,DSM 4179)(DSM 4179是德国菌种保藏中心(German Collection of Microorganisms andCell Cultures GmbH)赋予保藏微生物的编号,可直接从该菌种保藏中心购买微生物)进行试验。
亨氏甲烷螺菌(DSM 864)是1974年分离自美国污水污泥的产甲烷菌,具有独特的螺旋形态和鞘结构,是甲烷螺菌属下的唯一种,是模式微生物。
海沼甲烷球菌(DSM 2067),1974年分离自美国南卡罗来纳州波利斯岛(SouthCarolina,Pawley's island)海洋沼泽沉积物,形态为球形,耐受高盐环境,生长迅速,是模式微生物。
甲酸甲烷杆菌(DSM 1535)是最早一批分离的产甲烷菌,1947年分离自美国伊利诺伊州厄巴纳(Illinois,Urbana)厌氧污泥消化池,形态为杆状,是模式微生物。
甲烷叶菌(DSM 4140),在1990年分离自德国美因河畔法兰克福(Frankfurt amMain)的工业废水处理厂,形态为不规则球形或叶片状,是模式微生物。
巴伐利亚甲烷粒菌(DSM 4179)是1989年分离自德国巴伐利亚(Bavaria)糖厂污水处理厂厌氧沉淀物的产甲烷菌,形态为不规则球形,是模式微生物。
实施例1培养基的配制及产甲烷菌的培养
亨氏甲烷螺菌、甲酸甲烷杆菌、甲烷叶菌、巴伐利亚甲烷粒菌标准培养基(参考DSMmedium 1523)成分如下:
备注:标准培养基甲酸钠、乙酸钠量太少,不足以实现微生物大量生长。
其中,微量金属元素溶液SL-10:
成分 | 质量或体积 |
25%盐酸(25%HCl) | 10.00mL |
四水氯化亚铁(FeCl<sub>2</sub>·4H<sub>2</sub>O) | 1.50g |
氯化锌(ZnCl<sub>2</sub>) | 70.00mg |
四水氯化锰(MnCl<sub>2</sub>·4H<sub>2</sub>O) | 100.00mg |
硼酸(H<sub>3</sub>BO<sub>3</sub>) | 6.00mg |
六水氯化钴(CoCl<sub>2</sub>·6H<sub>2</sub>O) | 190.00mg |
二水氯化铜(CuCl<sub>2</sub>·2H<sub>2</sub>O) | 2.00mg |
六水氯化镍(NiCl<sub>2</sub>·6H<sub>2</sub>O) | 24.00mg |
二水钼酸钠(Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O) | 36.00mg |
水 | 990mL |
其中,七种维生素溶液:
成分 | 质量或体积 |
维生素B12(Vitamin B12) | 100.00mg |
对氨基苯甲酸(p-Aminobenzoic acid) | 80.00mg |
D-生物素(D(+)-Biotin) | 20.00mg |
烟酸(Nicotinic acid) | 200.00mg |
泛酸钙(Calcium pantothenate) | 100.00mg |
盐酸吡哆醇(Pyridoxine hydrochloride) | 300.00mg |
盐酸硫胺素(Thiamine-HCl·2H<sub>2</sub>O) | 200.00mg |
水 | 1000.00mL |
海沼甲烷球菌标准培养基(参考DSM medium 141c)如下:
其中,微量金属元素溶液成分如下:
成分 | 质量或体积 |
氨三乙酸(Nitrilotriacetic acid) | 1.50g |
七水硫酸镁(MgSO<sub>4</sub>·7H<sub>2</sub>O) | 3.00g |
一水硫酸锰(MnSO<sub>4</sub>·H<sub>2</sub>O) | 0.50g |
氯化钠(NaCl) | 1.00g |
七水硫酸亚铁(FeSO<sub>4</sub>·7H<sub>2</sub>O) | 0.10g |
七水硫酸钴(CoSO<sub>4</sub>·7H<sub>2</sub>O) | 0.18g |
二水氯化钙(CaCl<sub>2</sub>·2H<sub>2</sub>O) | 0.10g |
七水硫酸锌(ZnSO<sub>4</sub>·7H<sub>2</sub>O) | 0.18g |
五水硫酸铜(CuSO<sub>4</sub>·5H<sub>2</sub>O) | 0.01g |
十二水硫酸铝钾(KAl(SO<sub>4</sub>)·12H<sub>2</sub>O) | 0.02g |
硼酸(H<sub>3</sub>BO<sub>3</sub>) | 0.01g |
二水钼酸钠(Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O) | 0.01g |
六水氯化镍(NiCl<sub>2</sub>·6H<sub>2</sub>O) | 0.03g |
五水硒酸钠(Na<sub>2</sub>SeO<sub>3</sub>·5H<sub>2</sub>O) | 0.30mg |
二水钨酸钠(Na<sub>2</sub>WO<sub>4</sub>·2H<sub>2</sub>O) | 0.40mg |
水 | 1000mL |
其中,维生素溶液:
由于高浓度无机盐溶液会产生沉淀,比如培养基成分中有氯化钙和硫酸镁,二者配制100×母液会产生大量硫酸钙沉淀,将标准培养基无机盐成分分为磷酸盐、硫酸盐、氯化物三种,分别配制100×母液备用。微量金属元素、刃天青指示剂配制1000×母液备用。半胱氨酸溶液(作为硫源和还原剂)、硫化钠溶液(作为还原剂)、维生素溶液(提供微生物生长必须的维生素,刺激微生物生长)配制100×母液备用。维生素溶液在4℃冰箱保存。
具体而言,量取484.00mL水,依次加入5.00mL磷酸盐100×母液、5.00mL硫酸盐100×母液、5.00mL氯化物100×母液,然后依次加入0.50mL微量金属元素、0.50mL刃天青母液。称取4.00g甲酸钠和2.00g乙酸钠加入溶液中溶解。然后称取标准培养基规定量的蛋白胨、酵母提取物、碳酸氢钠溶解。
溶液摇匀后用移液管移取50.00mL至每个培养容器中。
将一次性玻璃巴斯德管插入培养基液面以下,同时向10瓶培养基内鼓入80%氮气20%二氧化碳混合气(原培养基使用80%氢气与20%二氧化碳混合气,二氧化碳与碳酸氢钠形成缓冲对维持溶液pH,因此其浓度尽量不要改变,所以只将80%氢气更换为80%氮气),每个培养容器的气流量以1-2L/min为宜,吹气40分钟。本发明的pH无需调整,培养基配制好后pH恰好落在7.0左右(大部分产甲烷菌的最适pH)。若需调整pH,小范围(±0.5)内调整可通过增减碳酸氢钠的量实现,大范围内调整可向培养基中添加盐酸或氢氧化钠溶液。培养温度37℃。
培养容器加丁基橡胶塞、钳口固定胶塞,121℃高压灭菌20分钟。
培养基温度下降至室温后移入厌氧培养箱,用无菌0.22μm滤膜过滤加入0.50mL100×半胱氨酸溶液、100×硫化钠溶液。培养基使用前再加0.50mL 100×维生素溶液,得到完全培养基。培养基进入氧气则会导致刃天青指示剂变为红色或粉色,达到无氧状态则为无色。培养基颜色与培养基本色(黄色或黄绿色)叠加会产生浅粉色、胭脂色等一系列颜色,达到无氧状态时则都为培养基本色。因此若加入维生素后培养基变色,等待约20分钟,待培养基回到本色再进行下一步操作。
在厌氧培养箱内用注射器吸出适量生长到对数中后期的产甲烷菌(亨氏甲烷螺菌、海沼甲烷球菌、甲酸甲烷杆菌、甲烷叶菌和巴伐利亚甲烷粒菌)菌液,加入完全培养基中,37℃培养。
实施例2产甲烷菌的生长情况检测
产甲烷菌生长状况可用酶标仪测定OD600,或气相色谱测定甲烷等方法来监测。产甲烷菌可用荧光显微镜DAPI模式,或测定16S RNA等方法来鉴定。
使用上述方法培养亨氏甲烷螺菌(Methanospirillum hungatei)(DSM864),微生物的荧光显微镜形态为螺旋形,与文献报道一致,无杂菌污染。生长曲线显示0-24h为调整期,24-120h为对数生长期,120h后为稳定期。
使用上述方法培养海沼甲烷球菌(Methanococcus maripaludis)(DSM2067),微生物的荧光显微镜形态为不规则球形,与文献报道一致,无杂菌污染。生长曲线显示0-2h为调整期,2-12h为对数生长期,12h后为稳定期。
使用上述方法培养甲酸甲烷杆菌(Methanobacterium formicicum)(DSM 1535),微生物的荧光显微镜形态为杆型,与文献报道一致,无杂菌污染。生长曲线显示0-12h为调整期,12-72h为对数生长期,72h后为稳定期。
使用上述方法培养甲烷叶菌(Methanofollis liminatans)(DSM 4140),微生物的荧光显微镜形态为球型,与文献报道一致,无杂菌污染。生长曲线显示0-10h为调整期,10-42h为对数生长期,42h后为稳定期。
使用上述方法培养巴伐利亚甲烷粒菌(Methanocorpusculum bavaricum)(DSM4179)微生物的荧光显微镜形态为球型,与文献报道一致,无杂菌污染。生长曲线显示0-96h为调整期,96-240h为对数生长期,240h后为稳定期。
实施例3与使用氢气的现有培养技术的比较
使用氢气的现有培养技术及本发明所用培养技术,五株产甲烷菌到达稳定期的时间如下表:
DSMZ全称:Deutsche Sammlung von Mikroorganismen und Zellkulturen(德语)German Collection of Microorganisms and Cell Cultures GmbH(英语)。
可见,使用本发明所用培养技术,产甲烷菌生长时间均与保藏中心给出的标准值和文献报道值相当,部分产甲烷菌生长时间优于文献报道值。产甲烷菌生长时间受到传代百分比、营养丰富度、温度、pH等因素影响,而本发明实验中采用相对较低的5%传代,因此可认为产甲烷菌改用甲酸钠培养后生长状况与使用氢气+二氧化碳的培养方法无明显差异。甲烷产量(甲烷产量在菌株生长至稳定期时测量)通过标准曲线结合瓶子气体体积计算,甲酸钠利用率计算方法见表下方文字。
可见,使用本发明所用培养技术,微生物均能利用培养基中的几乎全部甲酸,实现大量生长。
理论甲烷产量=(8*0.05*22400)/(68.01*4)=32.94mL,其中8为甲酸钠浓度(8g/L),0.05为培养基溶液量(0.05L),22400为标准状况下每摩尔气体的体积(22400mL/mol),68.01为甲酸钠分子量(68.01g/mol),4表示每4摩尔甲酸能产生1摩尔甲烷,因甲烷按下式产生:
4HCOOH→CH4+3CO2+2H2O
由于不可能每次都准确将培养基甲酸钠配成8g/L的浓度,当培养基的甲酸钠浓度偏高时,甲酸钠利用率可能超过100%。
实施例4与现有培养基的比较
现有技术公开了用于产甲烷菌的改良的PRAS无氮培养基,其配方组成为:NH4Cl1g,MgCl2 0.1g,K2HPO4 0.4g,甲酸钠5g,乙酸钠5g,甲醇3.5mL,pH值为7.0,蒸馏水1000mL,1%的树脂刃天青(氧化还原指示剂)1ml,121℃灭菌20min,使用前每5mL培养基加入1%Na2S(还原剂)和5%NaHCO3及3000u/mL青霉素液(抑制剂)各0.1mL。
但是产甲烷菌能利用的底物主要有氢气+二氧化碳、甲酸、乙酸、甲醇、甲胺。前两个底物对应“氢营养型产甲烷菌”,第三个底物对应“乙酸发酵型产甲烷菌”,最后两个底物对应“甲基营养型产甲烷菌”。对氢营养型产甲烷菌而言,通常其不能利用甲醇等甲基化合物,也不能利用乙酸产甲烷。因此从现有技术公开的PRAS无氮培养基非用于实验室大量培养产甲烷菌。另外,该培养基缺乏产甲烷菌生长必须的微量金属元素(比如,Ni元素,它是产甲烷菌产甲烷关键酶Mcr的必需组成部分),还缺乏刺激产甲烷菌生长的维生素、蛋白胨等,产甲烷菌很难在这样的培养基上快速生长、大量繁殖。
以上所述的具体实施例,对本发明的目的、技术方案和有益效果进行了进一步详细说明,应理解的是,以上所述仅为本发明的具体实施例而已,并不用于限制本发明,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.产甲烷菌的培养基,其特征在于,所述培养基包括8g/L的甲酸钠和4g/L的乙酸钠。
2.根据权利要求1所述的培养基,其特征在于,所述培养基还包括磷酸盐、硫酸盐、氯化物、微量金属元素、刃天青指示剂、半胱氨酸、硫化钠和维生素。
3.根据权利要求1或2所述的培养基,其特征在于,所述培养基还包括蛋白胨和酵母提取物。
4.根据权利要求1-3中任一项所述的培养基,其特征在于,所述微量金属元素包括:铁、锌、锰、钴、铜、镍、钼、钠、钙、铝、钾中的一种或多种。
5.根据权利要求1-4中任一项所述的培养基,其特征在于,所述维生素包括:维生素B12、对氨基苯甲酸、生物素、烟酸、泛酸钙、盐酸吡哆醇、盐酸硫胺素、叶酸、核黄素、硫辛酸中的一种或多种。
6.产甲烷菌的培养方法,其特征在于,所述培养方法包括在根据权利要求1-5中任一项所述的培养基中接种产甲烷菌,并在所述培养基中通入80%氮气+20%二氧化碳的混合气,对所述产甲烷菌进行培养。
7.根据权利要求6所述的培养方法,其特征在于,所述培养方法还包括监测产甲烷菌生长情况,若怀疑有污染风险,则鉴定产甲烷菌种类。
8.根据权利要求1-5中任一项所述的培养基在培养产甲烷菌中的应用。
9.制备甲烷的方法,其特征在于,所述方法包括利用根据权利要求1-5中任一项所述的培养基,并在所述培养基中通入80%氮气+20%二氧化碳的混合气培养产甲烷菌,并收集所述产甲烷菌产生的甲烷。
10.根据权利要求9所述的方法,其特征在于,所述产甲烷菌为具有甲酸脱氢酶的氢营养型产甲烷菌,任选地,所述产甲烷菌选自亨氏甲烷螺菌、海沼甲烷球菌、甲酸甲烷杆菌、甲烷叶菌和巴伐利亚甲烷粒菌。
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