CN116836883B - 一株帕姆酒耐热梭菌及其应用 - Google Patents
一株帕姆酒耐热梭菌及其应用 Download PDFInfo
- Publication number
- CN116836883B CN116836883B CN202310926418.4A CN202310926418A CN116836883B CN 116836883 B CN116836883 B CN 116836883B CN 202310926418 A CN202310926418 A CN 202310926418A CN 116836883 B CN116836883 B CN 116836883B
- Authority
- CN
- China
- Prior art keywords
- clostridium
- clostridium thermocellum
- environment
- university
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000193403 Clostridium Species 0.000 title claims abstract description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 38
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 29
- 239000001257 hydrogen Substances 0.000 claims abstract description 29
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 29
- 239000011780 sodium chloride Substances 0.000 claims abstract description 19
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims abstract description 12
- XYHKNCXZYYTLRG-UHFFFAOYSA-N 1h-imidazole-2-carbaldehyde Chemical compound O=CC1=NC=CN1 XYHKNCXZYYTLRG-UHFFFAOYSA-N 0.000 claims abstract description 6
- GWYFCOCPABKNJV-UHFFFAOYSA-M 3-Methylbutanoic acid Natural products CC(C)CC([O-])=O GWYFCOCPABKNJV-UHFFFAOYSA-M 0.000 claims abstract description 6
- GWYFCOCPABKNJV-UHFFFAOYSA-N beta-methyl-butyric acid Natural products CC(C)CC(O)=O GWYFCOCPABKNJV-UHFFFAOYSA-N 0.000 claims abstract description 6
- 150000004666 short chain fatty acids Chemical class 0.000 claims abstract description 6
- 244000005700 microbiome Species 0.000 claims abstract description 5
- 241000193448 Ruminiclostridium thermocellum Species 0.000 claims description 23
- 239000010806 kitchen waste Substances 0.000 claims description 17
- 238000004519 manufacturing process Methods 0.000 claims description 13
- 239000001913 cellulose Substances 0.000 claims description 10
- 229920002678 cellulose Polymers 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 8
- 150000003839 salts Chemical class 0.000 claims description 8
- 229920002522 Wood fibre Polymers 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 235000021391 short chain fatty acids Nutrition 0.000 claims description 4
- 239000002025 wood fiber Substances 0.000 claims description 4
- 239000002131 composite material Substances 0.000 claims description 2
- 238000013329 compounding Methods 0.000 claims description 2
- 239000010802 sludge Substances 0.000 abstract description 5
- 238000006243 chemical reaction Methods 0.000 abstract description 4
- 238000000855 fermentation Methods 0.000 description 19
- 230000004151 fermentation Effects 0.000 description 19
- 239000002609 medium Substances 0.000 description 15
- 239000000758 substrate Substances 0.000 description 14
- 241000894006 Bacteria Species 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 7
- 241000609240 Ambelania acida Species 0.000 description 6
- 239000010905 bagasse Substances 0.000 description 6
- 230000015784 hyperosmotic salinity response Effects 0.000 description 6
- 235000010980 cellulose Nutrition 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 238000011049 filling Methods 0.000 description 4
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 description 4
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 description 4
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 4
- 239000008108 microcrystalline cellulose Substances 0.000 description 4
- 229940016286 microcrystalline cellulose Drugs 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 108010024636 Glutathione Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 3
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 description 3
- 229940052299 calcium chloride dihydrate Drugs 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- XQGPKZUNMMFTAL-UHFFFAOYSA-L dipotassium;hydrogen phosphate;trihydrate Chemical compound O.O.O.[K+].[K+].OP([O-])([O-])=O XQGPKZUNMMFTAL-UHFFFAOYSA-L 0.000 description 3
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 3
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011218 seed culture Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 229920002101 Chitin Polymers 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 241000607598 Vibrio Species 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 229960002901 sodium glycerophosphate Drugs 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000218999 Begoniaceae Species 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- PEMUISUYOHQFQH-UHFFFAOYSA-L disodium;1,3-dihydroxypropan-2-yl phosphate;pentahydrate Chemical compound O.O.O.O.O.[Na+].[Na+].OCC(CO)OP([O-])([O-])=O PEMUISUYOHQFQH-UHFFFAOYSA-L 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 150000004667 medium chain fatty acids Chemical class 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000002504 physiological saline solution Substances 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
- 239000000843 powder Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- LJRGBERXYNQPJI-UHFFFAOYSA-M sodium;3-nitrobenzenesulfonate Chemical compound [Na+].[O-][N+](=O)C1=CC=CC(S([O-])(=O)=O)=C1 LJRGBERXYNQPJI-UHFFFAOYSA-M 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000012879 subculture medium Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000011144 upstream manufacturing 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P3/00—Preparation of elements or inorganic compounds except carbon dioxide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/52—Propionic acid; Butyric acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/145—Clostridium
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明属于能源转化技术领域，特别涉及一株帕姆酒耐热梭菌及其应用。本发明从造纸污泥筛选得到一株降解木质纤维素的耐盐产氢菌，且具有产中短链脂肪酸，如丁酸和异戊酸的能力。该菌的名称为帕姆酒耐热梭菌(Clostridium Thermopalmarium) HCD，保藏号为GDMCC No：63493，于2023年5月24日保藏于位于广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所的广东省微生物菌种保藏中心。该菌可在氯化钠浓度在150g/L以下的环境下发酵产氢以及产中短链脂肪酸，对环境的适应度更广，应用潜力更大。
Description
技术领域
本发明属于能源转化技术领域，特别涉及一株帕姆酒耐热梭菌及其应用。
背景技术
氢气是一种绿色环保，热值高的清洁能源，具有广阔的市场前景。暗发酵生物制氢技术，因为周期短，成本低，单位产率高而备受科学家的青睐。目前传统的生物制氢原料多为葡萄糖，蔗糖等大宗碳源，成本相较于木质纤维素等可再生能源，并不具备竞争力，因此有必要筛选优良的木质纤维素降解产氢菌，对大量的木质纤维素废弃物原料进行循环利用。同时，餐厨垃圾因其丰富的成分组成，在废弃物回收利用方面具有极大的市场价值，受其高含盐量影响，未能很好得到利用。因此本发明的一个关注点是筛选兼具耐盐特性的木质纤维素降解菌，可以同时利用混合底物，如餐厨垃圾和木质纤维素进行暗发酵产氢的优质菌株，对目标底物进行循环利用。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一株帕姆酒耐热梭菌。
本发明的另一目的在于提供上述帕姆酒耐热梭菌的应用。
本发明的目的通过下述技术方案实现：一株帕姆酒耐热梭菌，名称为帕姆酒耐热梭菌(Clostridium thermopalmarium) HCD，保藏号为GDMCC No：63493，于2023年5月24日保藏于位于广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所的广东省微生物菌种保藏中心。
上述帕姆酒耐热梭菌在制备氢气中的应用，特别适合在含盐含纤维素环境中制备氢气。
所述的含盐含纤维素环境是指含有NaCl的环境。
所述的环境中的NaCl的浓度优选为150g/L以下；优选为0～130g/L；更优选为30～70g/L。
所述的纤维素优选为木质纤维素；更优选为甘蔗渣。
所述的含盐含纤维素环境优选为木质纤维和餐厨垃圾复配形成；更优选为木质纤维和餐厨垃圾按质量比1：1复配形成。
上述帕姆酒耐热梭菌在制备短链脂肪中的应用。
所述的短链脂肪酸包括丁酸和异戊酸。
本发明相对于现有技术具有如下的优点及效果：
（1）本发明提供的HCD单菌能在30 g/L-150 g/L的 NaCl条件下降解木质纤维质材料，能适应中高温厌氧环境（50-60℃），具有独特性。
（2）本发明提供的HCD单菌能有效利用木质纤维素原料和餐厨垃圾为底物，进行暗发酵产氢气。
（3）本发明提供的HCD单菌能有效利用木质纤维素原料和餐厨垃圾为底物，进行暗发酵产挥发性中短链脂肪酸，包括丁酸和异戊酸等。
附图说明
图1是HCD菌株的照片图；其中，（A）为扫描电镜图，放大20000倍；（B）为光学显微镜下革兰氏染色照片图，放大100 倍。
图2是本发明提供的帕姆酒耐热梭菌HCD的16S rDNA进化树图。
图3.是本发明提供的帕姆酒耐热梭菌HCD和热纤醋弧菌DSM1313在不同氯化钠浓度下的产氢情况对比结果图。
图4是本发明提供的帕姆酒耐热梭菌HCD以甘蔗渣和餐厨垃圾为混合底物时的耐盐产氢结果图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1：帕姆酒耐热梭菌HCD的分离鉴定
（1）造纸污泥的富集培养：污泥初始样本采集于广东省佛山东华造纸厂。将污泥混悬液样本和以葡萄糖为底物的富集培养基按1g：10mL的比例制备得到菌悬液。置于37℃恒温培养箱中，富集培养3天，期间每24h调节一次pH至6.5-7.0。72h后得到富集的厌氧污泥样本A。然后置于37℃恒温培养箱中，以10 g/L的微晶纤维素为碳源，30g/L NaCl为筛选压力，改进的DSM122培养基为传代培养基，以10%的接种量进行传代培养，每代培养24 h，连续3-5代（每代保持相同的碳源及NaCl添加量），得到稳定的菌群B。其中，富集培养基的成分为：酵母浸粉4.00 g/L、硫酸铵1.30 g/L、二水合氯化钙0.10 g/L、 六水合氯化镁 1.00 g/L、磷酸二氢钾1.50 g/L、L- 半胱氨酸0.50 g/L、磷酸氢二钾三水合物3.93 g/L，水为溶剂，pH6.7。改进的DSM122培养基的成分为：酵母浸粉4.50 g/L、硫酸铵1.30 g/L、二水合氯化钙0.13 g/L、六水合氯化镁2.60 g/L、磷酸二氢钾1.43 g/L、磷酸氢二钾三水合物5.50 g/L、L-还原型谷胱甘肽0.25g/L、β-甘油磷酸钠五水合物 6.0 g/L、七水硫酸亚铁1.1 mg/L、浓度为0.01％(w/v)的刃天青1mL/L，pH7.0±0.3，溶剂为水。
（2）单菌落的分离：利用0.9%生理盐水梯度稀释菌群B。选择10-5、10-6、10-7等三个梯度稀释菌液，随后接种于固体培养基。固体培养基提前置于长度为12cm的亨氏厌氧培养管中，盖上几丁质胶塞和铝盖密封，进行三次抽真空和充氮气循环以营造厌氧环境。然后于115℃、20min高压灭菌后，自然降温至70℃左右，随后无菌接入提前稀释好的菌群B菌液0.3～0.5mL，然后迅速反复滚动管身使培养基和菌体混合均匀，并均匀贴在管壁上，直至凝固成型。55 ℃静置培养72 h后观察到菌落长出，分别于无菌环境挑取具有不同颜色和外观形态的菌落进一步分离纯化。以氢气产量为指标优选出一株细菌，其菌落直径约1.5～2 mm，乳白色且边缘呈圆形，镜检观察该菌呈长度为3～5μm，宽0.5～1.2 μm的梭状，革兰氏阳性细菌，如图1所示。固体培养基组成如下：微晶纤维素10 g/L，氯化钠30 g/L，酵母膏4.5g/L、磷酸氢二钾三水合物5.50 g/L、磷酸二氢钾1.43 g/L、六水合氯化镁2.60 g/L、硫酸铵1.30g/L、二水合氯化钙0.13 g/L、L-还原型谷胱甘肽0.25g/L、β-甘油磷酸钠五水合物6.00 g/L、七水硫酸亚铁1.10 mg/L、浓度为0.01％(w/v)的刃天青1mL/L，琼脂20 g/L，pH 7.0±0.3，溶剂为水。
（3）菌株的16S rDNA鉴定：将分离得到的杆状细菌接种至种子培养基中培养后，离心获得菌体，用细菌基因组提取试剂盒（OMEGA）提取菌体的基因组DNA，然后以提取的DNA为模板进行PCR扩增，扩增引物采用细菌的16SrDNA通用引物：16 S-F (5’-AGAGTTTGATCCTGGCTCAG- 3’)和16S-R(5’-ACGGTTACCTTGTTACGACTT- 3’)。PCR反应体系为：基因组DNA 2.5 μL、上游引物1 μL、下游引物1μL、dNTPs 4 μL、高保真Taq聚合酶0.5 μL、10×Buffer 5 μL，补足dd H2O至50 μL。PCR反应条件为95 ℃预变性5 min；95 ℃变性1 min、55 ℃退火1 min、72 ℃延伸2 min，30个循环；72℃保温10 min。扩增得到的PCR产物在4 ℃保存。PCR产物经过加A和胶回收后按说明书的方法与pMD-18T进行连接，筛选阳性克隆子后送至北京睿博兴科生物技术有限公司（广州）测序部进行16S rDNA测序，结果为：
AGAAGAGCTCCTTCGGGAGTAATTCTAGCGGCGGACGGGTGAGTAACACGTGGGCAACCTGCCTTAGTGAGGGGGATAGCCTCCCGAAAGGGAGATTAATACCGCATAACATTATTCTATCGCATGATAGAATAATCAAAGGAGCAATCCGCACTAAGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCGCAATGGGGGAAACCCTGACGCAGCAACGCCGCGTGAGCGATGAAGGTCTTCGGATTGTAAAGCTCTGTCTTTAGGGACGATAATGACGGTACCTAAGGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTACTGGGCGTAAAGAGTATGTAGGCGGATATTTAAGTCAGATGTGAAATTCCCGGGCTTAACCTGGGCGCTGCATTTGATACTGGATATCTAGAGTGTGGGAGAGGAAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGAGATTAGGAAGAACACCAGTGGCGAAGGCGGCTTTCTGGACCATAACTGACGCTGAGATACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCGGGGGTATCCCCCCTCTCTGCCGCGCAGCAAACGCAATAAGTATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCAGCGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAGACTTGACATCTCCTGAATTACTCGTAATGGAGGAAGCCCTTCGGGGCAGGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCGTTAGTTGCTACCATTAAGTTGAGCACTCTAACGAGACTGCCGCGGTTAACGTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTAGGGCTACACACGTGCTACAATGGCCGGTACAACGAGATGCAAACCCGTGAGGGGGAGCCAAACTTCAAAGCCGGTCCCAGTTCGGATTGTAGGCTGAAACTCGCCTACATGAAGTCGGAGTTGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGAGAGCCGGTAACACCCGAAGTCCGTG。
（4）测序的结果在NCBI上进行比对后并用MEGA 7.0软件进行系统进化树的构建，结果如图2所示。图2的结果表明分离得到菌株HCD为帕姆酒耐热梭菌。将得到的菌株命名为帕姆酒耐热梭菌(Clostridium thermopalmarium) HCD，保藏号为GDMCC No：63493，于2023年5月24日保藏于位于广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所的广东省微生物菌种保藏中心。
实施例2
帕姆酒耐热梭菌HCD的耐盐能力评估
（1）帕姆酒耐热梭菌HCD种子液的制备：在10 mL西林瓶中加入4mL种子培养基，抽真空、充氮气后以10％（v/v）的接种量接种帕姆酒耐热梭菌HCD甘油冻存管菌液，于55℃、150 rpm条件下震荡培养12 h，得到活化的帕姆酒耐热梭菌HCD。在55 mL血清瓶中加入种子培养基，抽真空、充氮气后以10％的接种量接种活化的帕姆酒耐热梭菌HCD，于55 ℃、150rpm条件下震荡培养24 h，放大培养，得到种子液。
（2）发酵培养基（种子培养基成分与发酵培养基相同）的制备：将发酵培养基分装入55 mL血清瓶中（工作体积20mL），用几丁质胶塞和铝盖密封后重复进行抽真空和充入0.01MPa氮气三次操作以保证厌氧环境，随后进行灭菌（115℃、30min），获得发酵培养基。
种子培养基（或发酵培养基）的组成如下：微晶纤维素10 g/L、酵母膏4.5g/L、磷酸氢二钾三水合物5.5 g/L、磷酸二氢钾1.43 g/L、六水合氯化镁2.6 g/L、硫酸铵1.3 g/L、二水合氯化钙0.13 g/L、L-还原型谷胱甘肽0.25g/L、β-甘油磷酸钠五水合物6.0 g/L、七水硫酸亚铁1.1 mg/L、浓度为0.01％(w/v)的刃天青1mL/L、氯化钠10-150 g/L，pH 7.0±0.3，溶剂为水。
设置不同浓度梯度的NaCl添加量，包括0 g/L、10 g/L、30 g/L、50 g/L、70 g/L、90g/L、130 g/L、150 g/L等。通过厌氧暗发酵观察HCD的耐盐能力，55 ℃培养4天。以常见的嗜热降解纤维素产氢菌热纤醋弧菌DSM1313（德国微生物菌种保藏中心）为对照，培养条件同HCD菌相同，从而对二者进行耐盐对比。
从图3中可以看出，以10 g/L微晶纤维素为底物，HCD可以在0-150 g/L的NaCl中暗发酵产氢，相比于DSM1313具有更强的耐盐能力。其中，在30 g/L的NaCl浓度下，HCD产氢能力较未添加NaCl组区别不大，可以实现在高盐餐厨垃圾做底物的应用场景（餐厨垃圾中NaCl量在2-3%）。
实施例3：帕姆酒耐热梭菌HCD以木质纤维素和餐厨垃圾为混合底物的暗发酵厌氧产氢。
种子液和发酵培养基的制备同实施例2。发酵培养基中添加的底物改为木质纤维素和餐厨垃圾为混合底物。其中，木质纤维素材料为预处理的甘蔗渣（可参照“Chen S-J,Chen X, Zhu M-J, 2022. Xylose recovery and bioethanol production fromsugarcanebagasse pretreated by mild two-stage ultrasonic assisted diluteacid. Bioresource Technology, 345, 12646. ”制备得到），甘蔗渣和餐厨垃圾的添加比例为质量比1:1，总添加量为10 g/L。NaCl的添加量为30 g/L以及50 g/L。
对55 ℃培养4天后得到的产物进行检测，结果如下：从图4中可以看出，以10 g/L甘蔗渣和餐厨垃圾为混合底物，HCD可以在30 g/L以及50 g/L的NaCl中55℃暗发酵产氢。经过4天发酵，HCD的产氢量分别达到62.27和42.18 mM。因此，HCD在以木质纤维素和高盐餐厨垃圾为混合底物时具有较好的降解产氢的应用价值。同时，对发酵上清液进行高效液相色谱分析表明，HCD在添加30 g/L以及50 g/L的NaCl发酵培养基中暗发酵能代谢积累丁酸产量在8.5和5 g/L，异戊酸积累量在2.5 和0.5 g/L。该结果说明HCD在以甘蔗渣和餐厨垃圾为混合底物耐盐产氢的同时，能积累中短链脂肪酸，如丁酸和异戊酸等。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (9)
1.一株帕姆酒耐热梭菌，其特征在于：所述的帕姆酒耐热梭菌的名称为帕姆酒耐热梭菌(Clostridium Thermopalmarium) HCD，保藏号为GDMCC No：63493，于2023年5月24日保藏于位于广州市先烈中路100号大院59号楼5楼广东省科学院微生物研究所的广东省微生物菌种保藏中心。
2.权利要求1所述的帕姆酒耐热梭菌在制备氢气中的应用。
3.根据权利要求2所述的帕姆酒耐热梭菌在制备氢气中的应用，其特征在于：所述的帕姆酒耐热梭菌在含盐含纤维素环境中制备氢气。
4.根据权利要求3所述的帕姆酒耐热梭菌在制备氢气中的应用，其特征在于：所述的含盐含纤维素环境是含有NaCl的环境。
5.根据权利要求4所述的帕姆酒耐热梭菌在制备氢气中的应用，其特征在于：所述的环境中的NaCl的浓度为150g/L以下。
6.根据权利要求3所述的帕姆酒耐热梭菌在制备氢气中的应用，其特征在于：所述的纤维素为木质纤维素。
7.根据权利要求3～6任一项所述的帕姆酒耐热梭菌在制备氢气中的应用，其特征在于：所述的含盐含纤维素环境为木质纤维质材料和餐厨垃圾复配形成。
8.根据权利要求7所述的帕姆酒耐热梭菌在制备氢气中的应用，其特征在于：所述的含盐含纤维素环境为木质纤维质材料和餐厨垃圾按质量比1：1复配形成。
9.权利要求1所述的帕姆酒耐热梭菌在制备短链脂肪酸中的应用，其特征在于：所述的短链脂肪酸为丁酸和异戊酸。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310926418.4A CN116836883B (zh) | 2023-07-27 | 2023-07-27 | 一株帕姆酒耐热梭菌及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310926418.4A CN116836883B (zh) | 2023-07-27 | 2023-07-27 | 一株帕姆酒耐热梭菌及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116836883A CN116836883A (zh) | 2023-10-03 |
| CN116836883B true CN116836883B (zh) | 2023-11-17 |
Family
ID=88163490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310926418.4A Active CN116836883B (zh) | 2023-07-27 | 2023-07-27 | 一株帕姆酒耐热梭菌及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116836883B (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106609294A (zh) * | 2015-10-22 | 2017-05-03 | 中国科学院过程工程研究所 | 一种强化双菌发酵纤维素产氢的方法 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108060182A (zh) * | 2016-11-08 | 2018-05-22 | 鼎唐能源科技股份有限公司 | 制备含有丁酸及／或丁酸盐的饲料原料的方法 |
-
2023
- 2023-07-27 CN CN202310926418.4A patent/CN116836883B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106609294A (zh) * | 2015-10-22 | 2017-05-03 | 中国科学院过程工程研究所 | 一种强化双菌发酵纤维素产氢的方法 |
Non-Patent Citations (4)
| Title |
|---|
| Effect of key factors on hydrogen production from cellulose in a co-culture of Clostridium thermocellum and Clostridium thermopalmarium;Alei Geng 等;《Bioresource Technology》;第10卷(第11期);第4029-4033页 * |
| First Insights into the Genome Sequence of Clostridium thermopalmarium DSM 5974, a Butyrate-Producing Bacterium Isolated from Palm Wine;Anja Poehlein 等;《Genome Announc》;第6卷(第17期);摘要 * |
| One-step biohydrogen production from cassava pulp using novel enrichment of anaerobic thermophilic bacteria community;Patthra Pason 等;《Biocatalysis and Agricultural Biotechnology》;第27卷;摘要 * |
| 利用厌氧菌降解纤维素产氢的研究进展;刘艳 等;《生物产业技术》(第6期);第65-69页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116836883A (zh) | 2023-10-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103898004A (zh) | 假诺卡氏菌及其发酵生产骨化二醇的方法 | |
| CN110093285B (zh) | 一株耐酸发酵乳杆菌及其应用 | |
| CN104630098A (zh) | 一种蒙氏假单孢菌ky-05与应用 | |
| WO2017133331A1 (zh) | 一株铜绿假单胞菌及其在生产蛋白酶中的应用 | |
| CN110484471A (zh) | 一株高产细菌纤维素的耐酸菌株及其生产细菌纤维素的方法 | |
| CN114540252B (zh) | 一种转化畜禽养殖废弃物的微小杆菌p6及应用 | |
| WO2010031793A2 (en) | Thermophilic fermentative bacterium producing butanol and/or hydrogen from glycerol | |
| CN109666605B (zh) | 一种人工菌群的纤维素生物转化方法 | |
| US8148133B2 (en) | Fossil fuel-free process of lignocellulosic pretreatment with biological hydrogen production | |
| CN118185841A (zh) | 纤维弧菌菌株nn19及其应用 | |
| KR101401413B1 (ko) | 클로스트리디움 속 균주, 상기 균주를 이용한 부티르산 생산방법 및 상기 균주의 분리방법 | |
| CN110205268A (zh) | 一株微杆菌及其在转化芦苇秸秆水解物制备微生物絮凝剂中的应用 | |
| Morsy et al. | Dark and photofermentation H2 production from hydrolyzed biomass of the potent extracellular polysaccharides producing cyanobacterium Nostoc commune and intracellular polysaccharide (glycogen) enriched Anabaena variabilis NIES-2095 | |
| CN111826308B (zh) | 一株海洋沉积物来源的几丁质高效降解菌及其应用 | |
| CN106834177B (zh) | 一株瘤胃菌及其应用 | |
| CN116286560B (zh) | 一株拉乌尔菌hc6及其低温生产2,3-丁二醇的应用 | |
| CN116836883B (zh) | 一株帕姆酒耐热梭菌及其应用 | |
| CN115305226B (zh) | 一株降解烟碱并产氢的抗辐射不动杆菌zj-22及其应用 | |
| CN103468606B (zh) | 一株产酸克雷伯氏菌及其在生产蒜糖醇中的应用 | |
| CN102492634B (zh) | 一株耐高温酵母菌及其应用 | |
| CN110343643B (zh) | 一株类球红细菌及其发酵产氢方法和应用 | |
| CN111924838A (zh) | 一种高硫石油焦的生物脱硫工艺 | |
| CN114958654B (zh) | 一种源自白酒酿造窖泥的产丙酸型嗜蛋白菌及其应用 | |
| KR20140130569A (ko) | 코마가테이박터 속 ｓｆｃｂ22―18 균주 및 이를 이용한 박테리얼 셀룰로오스 생산방법 | |
| CN112852683B (zh) | 一株嗜热解糖厌氧杆菌及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |