JP2020058339A - Strain of heterotrophic bacterium klebsiella pneumonia 1-17, associate for producing microbial protein - Google Patents
Strain of heterotrophic bacterium klebsiella pneumonia 1-17, associate for producing microbial protein Download PDFInfo
- Publication number
- JP2020058339A JP2020058339A JP2019115158A JP2019115158A JP2020058339A JP 2020058339 A JP2020058339 A JP 2020058339A JP 2019115158 A JP2019115158 A JP 2019115158A JP 2019115158 A JP2019115158 A JP 2019115158A JP 2020058339 A JP2020058339 A JP 2020058339A
- Authority
- JP
- Japan
- Prior art keywords
- methane
- strain
- culture
- bacteria
- klebsiella pneumonia
- 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.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 26
- 241000588747 Klebsiella pneumoniae Species 0.000 title claims abstract description 20
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 20
- 230000000813 microbial effect Effects 0.000 title claims abstract description 13
- 241000894006 Bacteria Species 0.000 claims abstract description 35
- 230000001580 bacterial effect Effects 0.000 claims abstract description 12
- 238000004113 cell culture Methods 0.000 claims abstract description 3
- 230000001717 pathogenic effect Effects 0.000 claims description 4
- 244000052616 bacterial pathogen Species 0.000 claims description 3
- 239000004615 ingredient Substances 0.000 claims description 2
- 239000002028 Biomass Substances 0.000 abstract description 16
- 238000003501 co-culture Methods 0.000 abstract description 13
- 244000144972 livestock Species 0.000 abstract description 3
- 244000000010 microbial pathogen Species 0.000 abstract description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 88
- 239000001963 growth medium Substances 0.000 description 22
- 239000000047 product Substances 0.000 description 20
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 239000003345 natural gas Substances 0.000 description 17
- 235000018102 proteins Nutrition 0.000 description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 15
- 239000000306 component Substances 0.000 description 12
- 238000007254 oxidation reaction Methods 0.000 description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 9
- 229910052799 carbon Inorganic materials 0.000 description 9
- 238000012258 culturing Methods 0.000 description 9
- 229910052500 inorganic mineral Inorganic materials 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 244000005700 microbiome Species 0.000 description 9
- 239000011707 mineral Substances 0.000 description 9
- 235000010755 mineral Nutrition 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 201000008225 Klebsiella pneumonia Diseases 0.000 description 7
- 241000589346 Methylococcus capsulatus Species 0.000 description 7
- 206010035717 Pneumonia klebsiella Diseases 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 241000589220 Acetobacter Species 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 108020004465 16S ribosomal RNA Proteins 0.000 description 5
- 239000007789 gas Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 239000006916 nutrient agar Substances 0.000 description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 102000016911 Deoxyribonucleases Human genes 0.000 description 3
- 108010053770 Deoxyribonucleases Proteins 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 230000007918 pathogenicity Effects 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 235000019733 Fish meal Nutrition 0.000 description 2
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N Propane Chemical compound CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 2
- NBBJYMSMWIIQGU-UHFFFAOYSA-N Propionic aldehyde Chemical compound CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 239000004467 fishmeal Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 239000013630 prepared media Substances 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- 238000012070 whole genome sequencing analysis Methods 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- 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 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 208000035404 Autolysis Diseases 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 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 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- IKHGUXGNUITLKF-XPULMUKRSA-N acetaldehyde Chemical compound [14CH]([14CH3])=O IKHGUXGNUITLKF-XPULMUKRSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229910052785 arsenic Inorganic materials 0.000 description 1
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 1
- 150000001495 arsenic compounds Chemical class 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000003131 biological toxin Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000001273 butane Substances 0.000 description 1
- ZTQSAGDEMFDKMZ-UHFFFAOYSA-N butyric aldehyde Natural products CCCC=O ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003673 groundwater Substances 0.000 description 1
- 229940093920 gynecological arsenic compound Drugs 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 108010009977 methane monooxygenase Proteins 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 241000994220 methanotrophic bacterium Species 0.000 description 1
- 230000001450 methanotrophic effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical compound CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 description 1
- OFBQJSOFQDEBGM-UHFFFAOYSA-N n-pentane Natural products CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 230000028043 self proteolysis Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 108010027322 single cell proteins Proteins 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000033 toxigenic Toxicity 0.000 description 1
- 230000001551 toxigenic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000015099 wheat brans Nutrition 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
-
- 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
- C12P21/00—Preparation of peptides or proteins
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、微生物産業に関するものであって、とりわけメタン酸化細菌および従属栄養細菌を含んだ共培養によりタンパク質バイオマスを得るための従属栄養細菌Klebsiella pneumonia 1−17の菌株に関するものである。微生物タンパク質は、農業における家畜飼料として、また高度な加工のための原料としても利用できる。 The present invention relates to the microbial industry, in particular to a strain of the heterotrophic bacterium Klebsiella pneumonia 1-17 for obtaining a protein biomass by co-culturing with methanoxidizing bacteria and heterotrophic bacteria. Microbial proteins can be used as livestock feed in agriculture and as raw materials for advanced processing.
ロシアにおける今日の飼料用タンパク質の生産は、全体的に良い状況ではない。ロシア大統領が署名した、食料自給率を80〜90%にするためのドクトリンに従うと、飼料製品の不足は少なくとも年間二百万トンにおよぶ可能性がある。 Today's feed protein production in Russia is not in good overall condition. According to the doctrine signed by the Russian President to make food self-sufficiency 80 to 90%, the shortage of feed products could reach at least 2 million tons per year.
タンパク質製品の主要な原料は大豆かすである。しかしながら、我が国の自然条件は、十分な量の大豆の栽培には向いていない。したがって、この分野に従事する者は飼料用タンパク質を生産するためのその他の方法を探さなければならない。 The main source of protein products is soybean meal. However, the natural conditions in Japan are not suitable for the cultivation of a sufficient amount of soybeans. Therefore, those working in this field must look for other ways to produce feed protein.
飼料バイオマスの生産者のうち、様々な基質において生育することができる様々な分類群の微生物は知られている。 Among the producers of feed biomass, different taxa of microorganisms are known that can grow on different substrates.
酵母が質量の60%以下のタンパク質しか蓄えない一方で、細菌は質量の79%のタンパク質を蓄えるため、飼料用タンパク質の生産者として細菌を利用することは、酵母を利用することよりも効率がよい。 Using yeast as a feed protein producer is more efficient than using yeast because yeast stores only 60% or less of protein by weight, while bacteria store 79% of protein by weight. Good.
タンパク質およびバイオマスの生産者として、メタノールをもとにタンパク質を生産する細菌株およびアセトバクター属であるAcetobacter methylicum VSB−924 CMPM V−2942(ロシア国特許第116363号、C12No.15/00、1984年)(特許文献1) 、 Acetobacter methylicum VSB−867 CMPM V−1947(ロシア国特許第925112号、C12No.15/00、1982年)(特許文献2)、 Acetobacter methylovorans VSB−914 CMPM V−2479(ロシア国特許第1070916号、C12No.15/00、1983年)(特許文献3)が利用されている。これらすべての菌株は、絶対乾燥物に対するタンパク質含有率が76%までおよぶという特徴を持つ。 As a producer of protein and biomass, a bacterial strain that produces protein based on methanol and the genus Acetobacter methylicum VSB-924 CMPM V-2942 (Russian Patent No. 116363, C12 No. 15/00, 1984) ) (Patent Document 1), Acetobacter methylicum VSB-867 CMPM V-1947 (Russian Patent No. 925112, C12 No. 15/00, 1982) (Patent Document 2), Acetobacter methylovorans VSB-914 CMPM V-2479. National Patent No. 1070916, C12 No. 15/00, 1983) (Patent Document 3) is used. All these strains are characterized by a protein content of up to 76% in absolute dry matter.
これらの菌株の典型的なファージに対する感度や耐熱性は様々に異なる。Acetobacter methylicum VSB−867にとっての最適な成長温度は28℃〜30℃であり、Acetobacter methylicum VSB−924にとっては30〜36℃、Acetobacter methylovorans VSB−914にとっては36〜40℃である。 The sensitivity and thermostability of these strains to typical phages vary. The optimum growth temperature for Acetobacter methylicum VSB-867 is 28 ° C to 30 ° C, 30 to 36 ° C for Acetobacter methylicum VSB-924, and 36 to 40 ° C for Acetobacter methylovorans VSB-914.
しかし、小麦ふすまおよび/または小麦粉を用いた発酵槽内でのpH6.0以下の酸性培養基による非滅菌培養においては、実験が示すとおり、酵母および菌類による汚染が素早く起こり、そして全細胞数の30〜40%以上において生産者の置換反応が起こった。その結果、得られた製品のタンパク質含有レベルに大幅な低下が起こる。 However, in the non-sterile culture with an acidic culture medium having a pH of 6.0 or less in a fermenter using wheat bran and / or flour, as shown by the experiment, contamination with yeast and fungi occurs rapidly and the total cell number of 30 Producer substitution reaction occurred at -40% and above. The result is a significant reduction in the protein content level of the resulting product.
完全な飼料タンパク質製品を得る方法として見込みのあるものの一つに、メタン酸化細菌を利用する方法がある。メタン資化性細菌は、自身に適した条件下において天然ガスのメタンを活発に変換し、素早く増殖して、有用なタンパク質およびビタミンその他の生物活性化合物を豊富に含んだバイオマスを作る。 One of the promising ways to obtain a complete feed protein product is to utilize methane-oxidizing bacteria. Methane-utilizing bacteria actively convert methane in the natural gas under conditions suitable for them and grow rapidly to produce biomass rich in useful proteins and vitamins and other bioactive compounds.
単細胞タンパク質を得るために天然ガスのメタンを利用することは、液体炭化水素およびその他の基質と比較していくつもの優位性を持つものであり、とりわけ天然ガスの埋蔵量の多さ、可搬性の高さ、基質からさらに精製することなく飼料用タンパク質が得られるといった優位性が挙げられる。 The use of methane from natural gas to obtain single-cell proteins has several advantages over liquid hydrocarbons and other substrates, including high natural gas reserves and portability. It has the advantages of height and the ability to obtain a feed protein without further purification from the substrate.
ロシアでは地中ガスの埋蔵量が多く、複数のデータによると世界の埋蔵量の40%にもおよぶ量があるとされることを考慮すると、微生物を利用した単細胞タンパク質の生産を導入することは、ロシアの企業に経済効果をもたらすことができるだけでなく、国内の食糧の安定を確保することにも寄与する。 Considering the fact that Russia has a large amount of underground gas reserves, and according to several data, it can reach 40% of the world's reserves, it is not possible to introduce unicellular protein production using microorganisms. , Not only can bring economic effects to Russian companies, but also contribute to ensuring the stability of domestic food.
絶対メタン酸化細菌は基質特異性の高くない酵素メタンモノオキシゲナーゼを含んでおり、メタンだけでなく、天然ガスに含まれるメタンの同族体(たとえばエタン、プロパンおよびブタン)をも酸化することができる。 Absolute methane-oxidizing bacteria contain the enzyme methane monooxygenase, which is not highly substrate-specific, and is able to oxidize not only methane, but also the homologues of methane contained in natural gas (eg ethane, propane and butane).
天然ガスの成分、メタン酸化細菌の種の特性および培養条件によっては、酸化が不完全なメタンおよびメタンの同族体の生産物の中に、メタノール、ホルムアルデヒド、ギ酸、エタノール、アセトアルデヒド、アセテート、プロピオンアルデヒド、プロピオン酸、オイルアルデヒドが様々な割合で培養基中に存在することがある。これらの生産物が特定の濃度で培養基内に蓄積されると、メタン酸化培養菌およびメタンの酸化に阻害作用を及ぼす。 Depending on the components of natural gas, the characteristics of the species of methane-oxidizing bacteria, and the culture conditions, methanol, formaldehyde, formic acid, ethanol, acetaldehyde, acetate, propionaldehyde may be present in the products of incompletely oxidized methane and its analogues. , Propionic acid, oil aldehyde may be present in the culture medium in varying proportions. Accumulation of these products in culture media at specific concentrations has an inhibitory effect on methane-oxidizing cultures and the oxidation of methane.
天然ガスにおける安定的な連続培養は、メタン酸化微生物とそのコンパニオンたる従属栄養微生物の共培養によってのみ可能であり、前記従属栄養微生物はメタンの同族体の不完全酸化の生産物を利用し、また微生物細胞の自己分解による生産物を利用することもある。 Stable continuous cultivation in natural gas is only possible by co-culturing a methane-oxidizing microorganism and its companion, a heterotrophic microorganism, which utilizes the product of incomplete oxidation of a homologue of methane, and Sometimes the products of autolysis of microbial cells are used.
現在では、たとえ単一基質を使用する場合あっても、培養基に不完全酸化の生産物が蓄積される場合には、純粋培養ではなく共棲培養をする方が適切であると確立されている。共培養の際に主培養菌の生育は目立って活発となり、いくつかの物理化学的指標のストレス作用に対する主培養菌の耐性も目立って上昇する。 It is now established that co-cultivation rather than pure culture is more appropriate if the products of incomplete oxidation accumulate in the culture medium, even when using a single substrate. During co-culture, the growth of the main culture is noticeably active, and the resistance of the main culture to the stress effects of some physicochemical indicators is also noticeably increased.
デオキシリボヌクレアーゼ酵素の生産者であり、医薬品の主成分にすることのできる(ロシア国特許第2057178号)(特許文献4)Klebsiella pneumoniae GISK No.214菌株は知られている。デオキシリボヌクレアーゼは、液体培養基または高濃度培養基での菌株の培養、バイオマスの沈殿、超音波破壊、遠心分離により得られる。デオキシリボヌクレアーゼは、上澄み液から抽出される。 It is a producer of deoxyribonuclease enzyme and can be used as a main component of pharmaceuticals (Russian Patent No. 2057178) (Patent Document 4) Klebsiella pneumoniae GISK No. 214 strains are known. Deoxyribonuclease can be obtained by culturing a strain in a liquid culture medium or a high-concentration culture medium, precipitating biomass, sonication, and centrifugation. Deoxyribonuclease is extracted from the supernatant.
制限エンドヌクレアーゼの生産者であるKlebsiella azeanae VKM V−2008D菌株(ロシア国特許第2044055号)(特許文献5)は知られている。この酵素はヌクレオチドの配列5−PuGGNC1CPy−3を認識し、切断する。菌株により生産される制限酵素は、遺伝子工学の研究において利用することができる。 Klebsiella azeanae VKM V-2008D strain (Russian patent No. 2044055), which is a producer of restriction endonucleases, is known (Patent Document 5). This enzyme recognizes and cleaves the nucleotide sequence 5-PuGGNC1CPy-3. The restriction enzymes produced by the strains can be used in genetic engineering research.
毒性および非毒性微生物の識別において、微生物のカタラーゼ活性を判定するための参考株として利用されるKlebsiella pneumoniae GISK No.215菌株(ロシア国特許第2070923号)(特許文献6)は知られている。 Klebsiella pneumoniae GISK No. used as a reference strain for determining the catalase activity of microorganisms in distinguishing between toxic and non-toxic microorganisms. 215 strain (Russian Patent No. 2070923) (Patent Document 6) is known.
微生物群のヒ素化合物に対する耐性を決定する接合性プラスミドpVK1を含むKlebsiella pneumoniae VKPM V−7001菌株は知られている。前記の菌株を使用することにより、ヒ素を含んだ生体破壊細菌の組合せの菌株(ロシア国特許第2260044号)(特許文献7)が得られる。 A Klebsiella pneumoniae VKPM V-7001 strain containing a conjugative plasmid pVK1 that determines the resistance of microbial groups to arsenic compounds is known. By using the above-mentioned strains, a strain of a combination of biodestructive bacteria containing arsenic (Russian Patent No. 2260044) (Patent Document 7) can be obtained.
飼料用バイオマスの生産者であるMethylococcus capsulatus VCB−874菌株は知られている。菌株は、「全ロシア遺伝学および産業微生物品種改良研究所」の培養コレクションの中に、コレクション番号「CMPM V1743(Авт. свид.誌 ソ連 第770200号)(非特許文献1)」として保管されている。この菌株は炭素源およびエネルギー源としてメタンを利用しており、純粋なメタンであっても天然ガス中の成分としてのメタンであっても利用する。この菌株の欠点は、メタン同族体の共酸化において生成される生産物に対する感度が高いことであり、前記生産物は、大量なことも少量なこともあるが、必ず天然ガスの中に存在するものである。生成される生産物は、生産者の生育を阻害する。 The Methylococcus capsulatus VCB-874 strain, which is a producer of feed biomass, is known. The strain is stored as a collection number "CMPM V1743 (Авт. Свид. Magazine Soviet Union No. 770200) (Non-patent Document 1)" in the culture collection of "All-Russian Genetics and Industrial Microbial Breeding Research Institute". There is. This strain uses methane as a carbon source and an energy source, whether it is pure methane or methane as a component in natural gas. The drawback of this strain is that it is highly sensitive to the products produced in the co-oxidation of methane homologues, which may be large or small and are always present in natural gas. It is a thing. The products produced hinder the growth of the producer.
技術的本質および達成される結果が最も近いものが、全ロシア産業微生物集登録番号VKPM V−12549の、微生物タンパク質を得るためのメタン酸化細菌Methylococcus capsulatus GBS−15の菌株(ロシア国特許第2613365号)(特許文献8) である。 The closest technical essence and the result achieved is the strain of the methanotrophic bacterium Methylococcus capsulatus GBS-15 of the Russian Federation of Industrial Microorganisms Registry No. VKPM V-12549 (Russian Patent No. 2613365). ) (Patent Document 8).
本発明の目的は、微生物タンパク質生産者の共棲菌として従属栄養細菌が存在する際の天然ガスにおけるメタン酸化細菌の培養について、生産効率を上げ、高い生産性を得ることである。 It is an object of the present invention to increase the production efficiency and obtain high productivity of culturing methane-oxidizing bacteria in natural gas when heterotrophic bacteria are present as symbiotic bacteria of microbial protein producers.
本発明の技術的結果は、メタン酸化細菌の共棲菌であり、天然ガス中にあるメタン同族体の共酸化による生産物を利用することができ、同様に細菌の溶解過程でできるタンパク質、アミノ酸、多糖類を利用することのできる、新しい菌株を明らかにすることである。 The technical result of the present invention is a symbiotic bacterium of methane-oxidizing bacteria, which can utilize a product of co-oxidation of a methane homolog in natural gas, and also a protein, an amino acid, which can be formed in the lysis process of bacteria, The aim is to identify new strains that can utilize polysaccharides.
本発明の技術的結果は、登録番号V-8465として「オボレンスク 国家病原性細菌および病原性細胞培養集」に供託された従属栄養細菌Klebsiella pneumonia 1−17の菌株を、メタン酸化細菌との共培養成分として使用し、微生物タンパク質を得ることで達成される。 The technical result of the present invention is that the strain of the heterotrophic bacterium Klebsiella pneumonia 1-17, which has been submitted to the "Obolensk National Pathogenic Bacteria and Pathogenic Cell Culture Collection" under the registration number V-8465, is co-cultured with methanooxidizing bacteria. It is achieved by using it as an ingredient and obtaining a microbial protein.
供託者が与えられた菌株の符号が1−17である。 The code of the strain given to the depositor is 1-17.
Klebsiella pneumonia 1−17菌株は、天然ガスをもとに家畜飼料用飼料バイオマスを工業的に生産するためのメタン酸化細菌の共培養菌の一つとして、また高度な加工のための原料として利用できる。 The Klebsiella pneumonia 1-17 strain can be used as one of the co-culture bacteria of methane-oxidizing bacteria for industrially producing feed biomass for livestock feed based on natural gas, and as a raw material for advanced processing. .
菌株の抽出源:本発明の従属栄養細菌Klebsiella pneumonia 1−17の細菌株は、メタン酸化細菌Methylococcus capsulatus GBS−15を含む共培養から抽出される。成長の早い混合培養細菌を得るために、ロシア連邦内の天然ガスおよび石油産出地域の地下水から抽出された活発な蓄積培養菌の混合物を使用した。このように混合培養細菌を得た結果、天然ガスにおける工業的なメタン酸化細菌の培養環境において、様々な共培養の成分として使用できる細菌株が得られた。細菌株は、鉱物培養基において発酵の際にメタン酸化細菌の主たる生産者の共培養菌として成長し、生産者にとって最適な物理化学的環境において、天然ガス中に存在するメタン同族体による共酸化の生産物、また主たる生産者の物質代謝による生産物を必要とする。 Source of Strain Extract: The bacterial strain of the heterotrophic bacterium Klebsiella pneumonia 1-17 of the present invention is extracted from a co-culture containing the methane-oxidizing bacterium Methylococcus capsulatus GBS-15. To obtain the fast-growing mixed culture bacteria, a mixture of active accumulation cultures extracted from natural gas in the Russian Federation and groundwater in the oil-producing regions was used. As a result of obtaining the mixed culture bacteria in this manner, bacterial strains that can be used as components of various co-cultures in the industrial environment for culturing methane-oxidizing bacteria in natural gas were obtained. Bacterial strains grow as co-cultures of the main producers of methane-oxidizing bacteria during fermentation in mineral media and, in the physicochemical environment optimal for the producers, of co-oxidation by methane homologs present in natural gas. It requires a product, as well as a product of the metabolism of the main producer.
細菌株は、遺伝子の組み換えがなされていないものである。 Bacterial strains are those that have not been genetically modified.
細菌株の生物学的危険性(安全性)についての情報:Klebsiella pneumoniae種は、病原性分類第IVグループ(衛生疫学規則 SP 1.3.2322−08「病原性(危険性)分類第III−IVグループの微生物および寄生虫病の病原体を使用する作業の安全性」添付資料第1号「人間に対する伝染病をもたらす病原性微生物、原虫類、寄生虫、生物由来毒の病原性による分類」、2011年6月29日付ロシア連邦国家衛生医師長認定第86号 追加と改訂第2号)リストに載っている。 Information on the biological risk (safety) of bacterial strains: Klebsiella pneumoniae species is classified as virulence group IV (hygiene epidemiology regulation SP 1.3.3222-08 "pathogenicity (risk) classification III-). Safety of work using IV group microbial and parasitic pathogens "Attachment 1" Classification by pathogenicity of pathogenic microorganisms, protozoa, parasites and biological toxins that cause infectious diseases to humans ", It is listed on the June 29, 2011, Russian Federation State Health Doctor's Accreditation No. 86 added and revised No. 2).
病原性または毒性に関するその他の情報:Klebsiella pneumonia 1−17細菌株は、非病原性であり、毒素産生性がなく、毒性がなく、無害である。 Other information regarding pathogenicity or virulence: The Klebsiella pneumonia 1-17 bacterial strain is non-pathogenic, non-toxigenic, non-toxic and harmless.
特徴:供託者が与えられた菌株の符号はKlebsiella pneumonia 1−17である。 Characteristics: The code of the strain given to the depositor is Klebsiella pneumonia 1-17.
同定方法とその結果:質量分析計MALDI Biotyper(スコア値2.561)、VITEK 2(同定確率98%)、遺伝子配列決定16S−rRNA系統解析(99%)、全ゲノム配列決定。 Identification method and its results: mass spectrometer MALDI Biotyper (score value 2.561), VITEK 2 (identification probability 98%), gene sequencing 16S-rRNA phylogenetic analysis (99%), whole genome sequencing.
形態学的特徴:大きさ0.5〜0.9×1.0〜1.2μmのグラム陰性桿菌、非運動性、芽胞の形成なし。 Morphological features: Gram-negative bacilli of size 0.5-0.9 x 1.0-1.2 μm, non-motile, no spore formation.
培養特性:栄養寒天GRM No.1(国家予算学術施設 国家応用微生物学およびバイオテクノロジー研究センター)の培地において、直径3〜4mmの平らで半透明の粘液状コロニーを形成。 Culture characteristics: Nutrient agar GRM No. In the medium of 1 (National Budget Research Institute, National Center for Applied Microbiology and Biotechnology), a flat and translucent mucous colony with a diameter of 3 to 4 mm is formed.
生化学的特質:VITEK 2システム:06.01(bioMerieux)(表1)
Biochemical characteristics: VITEK 2 system: 06.01 (bioMerieux) (Table 1)
実験動物に対する毒性:BALB/c−3.2×108KOE系のマウスに対する毒性 LD50 Toxicity to experimental animals: Toxicity to BALB / c-3.2 × 10 8 KOE strain mice LD50
抗菌薬に対する耐性(感受性)(表2)
Resistance to antibiotics (sensitivity) (Table 2)
遺伝子の特性:プラットフォームであるイルミナ社Miseq上で完全なゲノム配列決定がなされた。取得リード数 2,087,324、分析されたヌクレオチド数 469,180,422、コンティグ数(プログラムSPAdes 3.11.1) 531、得られたコンティグの全長 ヌクレオチドペア5,627,276、カバレッジ平均深度88、GC組成57.01% Gene characterization: Complete genome sequencing was performed on the platform Illumina Miseq. Number of read reads 2,087,324, number of nucleotides analyzed 469,180,422, number of contigs (program SPAdes 3.11.1) 531, total length of obtained contigs nucleotide pairs 5,627,276, average coverage depth 88, GC composition 57.01%
コンティグの組み立てにおいて16SrRNA遺伝子が決定され、データベースBLAST Nucleotide collection (nr/nt) (http://blast.ncbi.nlm.nih.gov)において分析された。調査対象菌株の16SrRNA遺伝子と参考菌株であるKlebsiella pneumonia subsp. Pneumonia HS11286 (CP003200.1)の16SrRNA遺伝子の相同性は、99%(1526/1527)であった。 The 16S rRNA gene was determined in the assembly of contigs and analyzed in the database BLAST Nucleotide collection (nr / nt) (http://blast.ncbi.nlm.nih.gov). The 16S rRNA gene of the strain to be investigated and the reference strain Klebsiella pneumonia subsp. The homology of the 16S rRNA gene of Pneumonia HS11286 (CP003200.1) was 99% (1526/1527).
Klebsiella pneumonia 1−17菌株の16SrRNA遺伝子領域の配列: Sequence of 16S rRNA gene region of Klebsiella pneumonia 1-17 strain:
AGGTGATCCAACCGCAGGTTCCCCTACGGTTACCTTGTTACGACTTCACCCCAGTCATGAATCACAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACATACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATATGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTGTGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTTAACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAATCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATCGATGAGGTTATTAACCTTACGCCTTCCTCCCCGCTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCGTTACCCCACCTACTAGCTAATCCCATCTGGGCACATCTGATGGCATGAGGCCCGAAGGTCCCCCACTTTGGTCTTGCGACATTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCGCTCGTCACCCGAGAGCAAGCTCTCTGTGCTACCGCTCGACTTGCATGTGTTAGGCCTGCCGCCAGCGTTCAATCTGAGCCATGATCAAACTCT AGGTGATCCAACCGCAGGTTCCCCTACGGTTACCTTGTTACGACTTCACCCCAGTCATGAATCACAAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACATACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATATGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCA GATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTGTGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTT ACGCGTTAGCTCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAATCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTTGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGAATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTC CGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATCGATGAGGTTATTAACCTTACGCCTTCCTCCCCGCTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCGTTACCCCACCTACTAGCTAATCCCATCTGGGCACATCTGATGGCATGAGGCCCGAAGGTC CCCACTTTGGTCTTGCGACATTATGCGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCGCTCGTCACCCGAGAGCAAGCTCTCTGTGCTACCGCTCGACTTGCATGTGTTAGGCCTGCCGCCAGCGTTCAATCTGAGCCATGATCAAACTCT
培養条件:高濃度または液体の栄養培地GRM No.1、GRM培養液、栄養寒天または栄養液、34℃、24時間。炭素源としてメタノールまたはエタノールまたはプロパノールが加えられる鉱物培養基、40℃、36〜48時間。 Culture conditions: High-concentration or liquid nutrient medium GRM No. 1, GRM culture medium, nutrient agar or nutrient solution, 34 ° C., 24 hours. Mineral culture medium to which methanol or ethanol or propanol is added as a carbon source, 40 ° C., 36 to 48 hours.
栄養寒天 成分(調合済み培地1リットル換算):乾燥タンパク質酵素加水分解物 10.5g、発酵培地用乾燥ペプトン 10.5g、濾過ずみ自己消化酵母エキス 2.0g、塩化ナトリウム 5.0g、微生物培養寒天 12.0g。121℃で15分間殺菌する。 Nutrient agar components (converted to 1 liter of prepared medium): Dry protein enzyme hydrolyzate 10.5 g, dried peptone for fermentation medium 10.5 g, filtered self-digesting yeast extract 2.0 g, sodium chloride 5.0 g, microbial culture agar 12.0 g. Sterilize at 121 ° C for 15 minutes.
栄養液 成分(調合済み培地1リットル換算):乾燥タンパク質酵素加水分解物 9.1g、発酵培地用乾燥ペプトン 9.9g、濾過ずみ自己消化酵母エキス 4.7g、塩化ナトリウム 5.0g、炭酸ナトリウム 0.3g。121℃で15分間殺菌する。 Nutrient liquid component (converted to 1 liter of prepared medium): 9.1 g of dried protein enzyme hydrolyzate, 9.9 g of dried peptone for fermentation medium, 4.7 g of filtered self-digesting yeast extract, 5.0 g of sodium chloride, sodium carbonate 0 0.3 g. Sterilize at 121 ° C for 15 minutes.
GRM No.1(培養基成分、g/l):魚粉由来膵臓加水分解物 15.0、カゼイン膵臓加水分解物 10.0、酵母エキス 2.0、塩化ナトリウム 3.5、D−グルコース 1.0、寒天 10.0±2。 GRM No. 1 (culture medium component, g / l): fish meal-derived pancreatic hydrolyzate 15.0, casein pancreatic hydrolyzate 10.0, yeast extract 2.0, sodium chloride 3.5, D-glucose 1.0, agar 10 0.0 ± 2.
GRM栄養液(培養基成分、g/l):魚粉由来膵臓加水分解物 8.0、発酵培地用乾燥ペプトン 8.0、塩化ナトリウム 4.0。 GRM nutrient solution (culture medium component, g / l): fish meal-derived pancreatic hydrolyzate 8.0, dried peptone 8.0 for fermentation medium, sodium chloride 4.0.
鉱物培養基(培養基成分、g/l):(NH4)2SO4 0.52、MgSO4 0.02、K2SO4 0.06、NH4H2PO4 1.53。微量元素溶液(別途調合)(г/л):ZnSO4 0.43、MnSO4 0.88、CuSO4 0.78、H3BO3 0.4、Na2MoO4×2H2O 0.25、CoSO4×7H2O 0.25、FeSO4×7H2O 4.97。 Mineral culture medium (culture medium component, g / l): (NH 4 ) 2 SO 4 0.52, MgSO 4 0.02, K 2 SO 4 0.06, NH 4 H 2 PO 4 1.53. Trace element solution (prepared separately) (г / л): ZnSO 4 0.43, MnSO 4 0.88, CuSO 4 0.78, H 3 BO 3 0.4, Na 2 MoO 4 × 2H 2 O 0.25 , CoSO 4 × 7H 2 O 0.25 , FeSO 4 × 7H 2 O 4.97.
調合済みの微量元素溶液1mlを1000mlの鉱物培養基に加える。121℃で15分間殺菌する。pHを6.0〜6.2にする。 1 ml of the prepared trace element solution is added to 1000 ml of mineral culture medium. Sterilize at 121 ° C for 15 minutes. Bring the pH to 6.0-6.2.
基質(炭素源およびエネルギー源)としてメタノール(1.5体積%未満)、エタノール(2体積%未満)、プロパノール(1体積%未満)を使用することができる。培養温度 40℃、好気培養。 Methanol (less than 1.5% by volume), ethanol (less than 2% by volume), propanol (less than 1% by volume) can be used as a substrate (carbon source and energy source). Culture temperature 40 ° C, aerobic culture.
保管条件:菌株は栄養寒天上で保管(温度4±2℃)、三か月に一度移植を実施、菌株は凍結乾燥状態でも液体窒素内でも保存が可能。 Storage conditions: Strains are stored on nutrient agar (temperature 4 ± 2 ° C) and transplanted once every three months. Strains can be stored in freeze-dried state or in liquid nitrogen.
上述のように、本発明の菌株は、様々な成分の培養基において安定的かつ生産的に成長し、凍結乾燥状態で長期間活性を保つことができ、非病原性かつ毒素産生性、毒性がなく無害であるという特徴を持つ。 As described above, the strain of the present invention grows stably and productively in a culture medium of various components, can remain active for a long time in a freeze-dried state, and is non-pathogenic, toxin-producing and non-toxic. It has the characteristic of being harmless.
Klebsiella pneumonia 1−17菌株は、飼料用バイオマスの工業的生産を目的とした、天然ガスによるメタン酸化細菌の共培養において共棲菌として利用でき、また高度な加工のための原料としても利用できる。 The Klebsiella pneumonia 1-17 strain can be used as a co-resident bacterium in the co-culture of methane-oxidizing bacteria with natural gas for the purpose of industrial production of feed biomass, and also as a raw material for advanced processing.
従属栄養細菌Klebsiella pneumonia 1−17は、生成される生産物(メタノール、エタノール、プロパノール)を炭素源として利用し、それにより主たる生産者にかかる抑制作用を取り除く。この他にも、従属栄養体は細菌の溶解プロセスにおいてできるタンパク質、アミノ酸、および多糖類を利用することができる。 The heterotrophic bacterium Klebsiella pneumonia 1-17 utilizes the products produced (methanol, ethanol, propanol) as carbon sources, thereby eliminating the suppressive effect on the main producers. In addition to this, heterotrophs can utilize proteins, amino acids, and polysaccharides that are created during the bacterial lysis process.
メタン酸化細菌は天然ガスのメタンによって増殖し、共培養の他方の要素は、天然ガス中のメタン同族体の共酸化生産物によって、またメタン資化性菌の細菌活動による生産物によって増殖する。 Methane-oxidizing bacteria are grown by the natural gas methane, and the other component of the co-culture is grown by the co-oxidation products of methane homologues in natural gas and by the bacterial activity of methane-utilizing bacteria.
共培養の過程において、共培養菌の含有レベルは、最初の植え付け量に関係なく、ガス中にあるメタンのガス状同族体の共酸化の結果生み出され利用できる生産物の数により決まる。それは、鉱物培養基においては、メタンを利用しない共培養要素にとっての唯一の炭素源が、これらの生産物となるからである。培養菌がこのような自己調整を行うため、培養の最初の植え付けにおける培養細胞の割合は重大な意味を持たないが、加える非メタン酸化微生物の数は、全体の細胞数の0.1%以上15%以内にすることを推奨する。この範囲を外れた場合、第一にメタン酸化細菌の生育の停滞へとつながり、第二に起こりやすいのが従属栄養細菌の成長の停滞へとつながる。メタンを資化しない細菌を選んで加えた混合培養では、メタン酸化細菌だけの純粋培養に比べてバイオマスの生産量が3〜5倍に増大した。このように、メタン同族体の共酸化の生産物によって生育し、メタンを資化しない従属栄養体を選んでメタン酸化細菌に加えることで、メタンにおけるバイオマス生産量を増大させることができる。たとえメタンを資化しない従属栄養体を高濃度で取り入れても、培養菌の成長において得られる炭素源すなわちメタン同族体の共酸化の生産物の数によりその含有量は制限を受け、通常では全体の細胞数の1〜1.5%を超えないため、メタンを酸化しない従属栄養細菌を加えることはバイオマスの質に影響を与えない。 During the co-culturing process, the content level of the co-culture is determined by the number of products produced and available as a result of the co-oxidation of the gaseous homologues of methane in the gas, regardless of the initial inoculum. This is because in mineral culture media, these products are the only carbon source for co-culture elements that do not utilize methane. The proportion of cultured cells in the first inoculation of the culture is not significant because the cultures perform such self-regulation, but the number of non-methane oxidizing microorganisms added should be greater than 0.1% of the total cell number. It is recommended to be within 15%. If it deviates from this range, the growth of methanotrophic bacteria will be stagnated first, and secondly, the growth of heterotrophic bacteria will be stagnant. In the mixed culture in which bacteria that do not utilize methane were selected and added, the amount of biomass produced was increased by 3 to 5 times as compared with the pure culture of only methane-oxidizing bacteria. Thus, by selecting a heterotroph that grows by the co-oxidation product of a methane homolog and does not assimilate methane and adds it to a methane-oxidizing bacterium, the amount of biomass produced in methane can be increased. Even if a high concentration of a heterotroph that does not utilize methane is incorporated, its content is limited by the number of carbon sources obtained in the growth of the culture, that is, the products of co-oxidation of a methane homolog, and the content is usually whole cells. Addition of heterotrophic bacteria that do not oxidize methane does not affect the quality of the biomass as it does not exceed 1-1.5% of the number.
本発明は、以下の例によって説明することができる。 The invention can be illustrated by the following example.
例1 Klebsiella pneumonia 1−17菌株を培養する液体の鉱物培養基の成分(g/l):(NH4)2SO4 0.52、MgSO4 0.02、K2SO4 0.06、NH4H2PO4 1.53。 Example 1 Klebsiella pneumonia 1-17 components of the mineral culture medium of liquid culture of strain (g / l) :( NH 4 ) 2 SO 4 0.52, MgSO 4 0.02, K 2 SO 4 0.06, NH 4 H 2 PO 4 1.53.
培養基を121℃で15分間殺菌する。 The culture medium is sterilized at 121 ° C. for 15 minutes.
微生物溶液(培養基とは別に調合し、殺菌する)(g/l):ZnSO4 0.43、 MnSO4 0.88、CuSO4 0.78、H3BO3 0.4、Na2MoO4×2H2O 0.25、CoSO4×7H2O 0.25、FeSO4×7H2O 4.97。調合済みの微生物溶液1mlを1000mlの鉱物培養基に加える。pHを6.0〜6.2にする。基質(炭素源およびエネルギー源)として、メタノール1.0%を使用する。 Microbial solution (prepared separately from culture medium and sterilized) (g / l): ZnSO 4 0.43, MnSO 4 0.88, CuSO 4 0.78, H 3 BO 3 0.4, Na 2 MoO 4 × 2H 2 O 0.25, CoSO 4 × 7H 2 O 0.25, FeSO 4 × 7H 2 O 4.97. 1 ml of the prepared microbial solution is added to 1000 ml of mineral culture medium. Bring the pH to 6.0-6.2. As substrate (carbon source and energy source) 1.0% methanol is used.
菌株の培養は期間を定めた方法で、耐熱ガラス製のフラスコ内で、充填率0.3〜0.4の際に1リットルの容量で、恒温振とう培養機内で行われる。培養は34℃およびpH6.0において48時間行われる。培養終了時の乾燥物濃度は、1リットル当たりの絶対乾燥物8.2gとなる。得られた培養菌は、容量10リットル(実働容量7リットル)の自動発酵システムまたは容量40リットル(実働容量28リットル)の排出型発酵槽において、その後の細菌培養のための種菌として使用される。 Cultivation of the strain is carried out in a flask made of heat-resistant glass with a volume of 1 liter at a filling rate of 0.3 to 0.4 in a constant temperature shaking culture machine by a method for a fixed period. The culture is performed at 34 ° C. and pH 6.0 for 48 hours. The dry matter concentration at the end of culturing is 8.2 g of absolute dry matter per liter. The obtained culture is used as an inoculum for the subsequent bacterial culture in an automatic fermentation system having a capacity of 10 liters (7 liters working capacity) or in a discharge type fermenter having a capacity of 40 liters (28 liters working capacity).
例2 例1と同様に菌株の培養が行われるが、炭素源およびエネルギー源としてエタノール1.6%が使用される。物理化学的条件および培養時間は同じであり、乾燥物濃度は、1リットル当たりの絶対乾燥物3.0gとなる。 Example 2 Cultivation of the strain is carried out as in Example 1, but 1.6% ethanol is used as carbon source and energy source. The physicochemical conditions and culture time are the same, the dry matter concentration is 3.0 g absolute dry matter per liter.
例3 例1と同様に菌株の培養が行われるが、炭素源およびエネルギー源としてプロパノール0.5%が使用される。物理化学的条件および培養時間は同じであり、乾燥物濃度は、1リットル当たりの絶対乾燥物3.0gとなる。 Example 3 Cultivation of the strain is carried out as in Example 1, but 0.5% propanol is used as carbon and energy source. The physicochemical conditions and culture time are the same, the dry matter concentration is 3.0 g absolute dry matter per liter.
例4 メタン酸化細菌Methylococcus capsulatus GBS−15および従属栄養細菌Klebsiella pneumonia 1−17で構成される共培養を、容量10リットル(実働容量7リットル)の自動発酵システムの中で、下記の構成の鉱物培養基において行う。(単位g/l)H3PO4(80%) 17.2、 K2SO4 5.0、MgSO4×7H2O 4.0、FeSO4×7H2O 0.21、CuSO4 0.78、MnSO4×4H2O 0.38、ZnSO4×7H2O 0.06、H3BO3 0.25、Na2MoO4×2H2O 0.009、CoSO4×7H2O 0.0095 Example 4 A co-culture consisting of the methane-oxidizing bacterium Methylococcus capsulatus GBS-15 and the heterotrophic bacterium Klebsiella pneumonia 1-17 was prepared in an automatic fermentation system having a volume of 10 liters (7 liters of working volume) in a mineral culture medium having the following constitution. In. (Unit: g / l) H 3 PO 4 (80%) 17.2, K 2 SO 4 5.0, MgSO 4 × 7H 2 O 4.0, FeSO 4 × 7H 2 O 0.21, CuSO 4 0. 78, MnSO 4 × 4H 2 O 0.38, ZnSO 4 × 7H 2 O 0.06, H 3 BO 3 0.25, Na 2 MoO 4 × 2H 2 O 0.009, CoSO 4 × 7H 2 O 0.008. 0095
窒素源および滴定剤としてアンモニア水が供給される。培養プロセスは40〜45℃、pH5.4〜5.8において、メタンおよび酸素を含む混合気体を常時供給しながら進められる。バイオマス濃度が、1リットル当たりの絶対乾燥物9gに達したところで、培養基の希釈率D=0.25 h−1の連続培養プロセスに移行する。バイオマス濃度が、1リットル当たりの絶対乾燥物18.5gである際に、Methylococcus capsulatus GBS−15の支配率は93%となった。 Ammonia water is supplied as a nitrogen source and a titrant. The culturing process proceeds at 40 to 45 ° C. and pH 5.4 to 5.8 while constantly supplying a mixed gas containing methane and oxygen. When the biomass concentration reaches 9 g of absolute dry matter per liter, the continuous culture process is started at the culture medium dilution rate D = 0.25 h −1 . At a biomass concentration of 18.5 g absolute dry matter per liter, the predominance of Methylococcus capsulatus GBS-15 was 93%.
例5 メタン酸化細菌Methylococcus capsulatus GBS−15および従属栄養細菌Klebsiella pneumonia 1−17で構成される共培養を、容量40リットル(実働容量28リットル)の排出型発酵槽の中で、下記の構成の鉱物培養基において行う。(単位g/l)H3PO2(80%) 17.2、 K2SO4 5.0、MgSO4×7H2O 4.0、FeSO2×7H2O 0.21、CuSO4 0.78、MnSO4×4H2O 0.38、ZnSO4×7H2O 0.06、H3BO3 0.25、Na2MoO4×2H2O 0.009、CoSO4×7H2O 0.0095 Example 5 A co-culture composed of the methane-oxidizing bacterium Methylococcus capsulatus GBS-15 and the heterotrophic bacterium Klebsiella pneumonia 1-17 was placed in a discharge liter fermenter having a volume of 40 liters (working volume of 28 liters) and a mineral having the following constitution. Perform in culture medium. (Unit: g / l) H 3 PO 2 (80%) 17.2, K 2 SO 4 5.0, MgSO 4 × 7H 2 O 4.0, FeSO 2 × 7H 2 O 0.21, CuSO 4 0. 78, MnSO 4 × 4H 2 O 0.38, ZnSO 4 × 7H 2 O 0.06, H 3 BO 3 0.25, Na 2 MoO 4 × 2H 2 O 0.009, CoSO 4 × 7H 2 O 0.008. 0095
窒素源および滴定剤としてアンモニア水が供給される。培養プロセスは41〜45℃、pH5.4〜5.7において、メタンおよび酸素を含む混合気体を常時供給しながら進められる。バイオマス濃度が、1リットル当たりの絶対乾燥物9〜10gに達したところで、少しずつ0.8MPaまで気圧を上昇させる。気圧の上昇に伴って、微生物の栄養源である気体の供給を少しずつ増加させる。その際の培養プロセスの指標はD=0.30 h−1、バイオマス濃度は1リットル当たりの絶対乾燥物37g、メタン酸化細菌Methylococcus capsulatusGBS −15の支配率は93%であった。 Ammonia water is supplied as a nitrogen source and a titrant. The culturing process proceeds at 41 to 45 ° C. and pH 5.4 to 5.7 while constantly supplying a mixed gas containing methane and oxygen. When the biomass concentration reaches 9 to 10 g of absolute dry matter per liter, the atmospheric pressure is gradually raised to 0.8 MPa. As the atmospheric pressure increases, the supply of gas, which is a nutrient source for microorganisms, is gradually increased. The index of the culture process at that time was D = 0.30 h −1 , the biomass concentration was 37 g of absolute dry matter per liter, and the dominance of the methane-oxidizing bacterium Methylococcus capsulatus GBS-15 was 93%.
Claims (1)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
RU2018136037A RU2687137C1 (en) | 2018-10-11 | 2018-10-11 | Strain of heterotrophic bacteria klebsiella pneumonia is an associate for producing a microbial protein mass |
RU2018136037 | 2018-10-11 |
Publications (1)
Publication Number | Publication Date |
---|---|
JP2020058339A true JP2020058339A (en) | 2020-04-16 |
Family
ID=66430660
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2019115158A Pending JP2020058339A (en) | 2018-10-11 | 2019-06-21 | Strain of heterotrophic bacterium klebsiella pneumonia 1-17, associate for producing microbial protein |
Country Status (4)
Country | Link |
---|---|
JP (1) | JP2020058339A (en) |
EA (1) | EA037039B1 (en) |
RU (1) | RU2687137C1 (en) |
WO (1) | WO2020076191A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
RU2717991C1 (en) * | 2019-07-22 | 2020-03-27 | Общество с ограниченной ответственностью "ГИПРОБИОСИНТЕЗ"ООО " ГИПРОБИОСИНТЕЗ" | Protein feed supplement for farm animals and fish |
RU2720121C1 (en) * | 2019-10-29 | 2020-04-24 | Ооо "Гипробиосинтез" | Method of producing microbial protein based on hydrocarbon material |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5221591B2 (en) * | 1973-12-26 | 1977-06-11 | ||
SU1788965A3 (en) * | 1991-03-26 | 1993-01-15 | Poctobckий-Ha-Дohу Haучho-Иccлeдobateльckий Иhctиtуt Эпидemиoлoгии, Mиkpoбиoлoгии, Пapaзиtoлoгии И Гигиehы | Strain of bacteria klebsiella pneumonia usable for lipopolysaccharide preparation |
RU2083663C1 (en) * | 1994-05-19 | 1997-07-10 | Ростовский научно-исследовательский институт микробиологии и паразитологии | STRAIN OF BACTERIUM KLEBSIELLA PNEUMONIAE-232 - A PRODUCER OF β --HEMOLYSIN |
FR2729969B1 (en) * | 1995-01-26 | 1997-04-18 | Bieurope | NEW STRAIN OF KLEBSIELLA PNEUMONIAE, SUBSP. PNEUMONIAE AND PROCESS FOR PRODUCING L-FUCOSE-CONTAINING POLYSACCHARIDE |
RU2221864C2 (en) * | 2002-02-07 | 2004-01-20 | Московская государственная академия ветеринарной медицины и биотехнологии им. К.И. Скрябина | Bacterium strain klebsiella pneumoniae deposited in vgnki at = 23 mgavmib-dep for producing vaccine against klebsiellosis in agriculture young stock |
CN104774792B (en) * | 2015-04-13 | 2017-12-19 | 中国科学院天津工业生物技术研究所 | The Methyldmonas of one plant of enduring high-concentration methanol and its application |
-
2018
- 2018-10-11 RU RU2018136037A patent/RU2687137C1/en active IP Right Revival
-
2019
- 2019-03-28 EA EA201900129A patent/EA037039B1/en active IP Right Revival
- 2019-06-21 JP JP2019115158A patent/JP2020058339A/en active Pending
- 2019-10-01 WO PCT/RU2019/050163 patent/WO2020076191A1/en active Application Filing
Also Published As
Publication number | Publication date |
---|---|
EA037039B1 (en) | 2021-01-29 |
WO2020076191A1 (en) | 2020-04-16 |
EA201900129A1 (en) | 2020-04-30 |
RU2687137C1 (en) | 2019-05-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
RU2613365C1 (en) | Strain of methane-oxidizing bacteria methylococcus capsulatus gbs-15 for obtaining of microbial protein mass | |
Slyemi et al. | Characteristics of a phylogenetically ambiguous, arsenic-oxidizing Thiomonas sp., Thiomonas arsenitoxydans strain 3As T sp. nov | |
WO2021084159A1 (en) | Strains and processes for single cell protein or biomass production | |
JP6919910B2 (en) | Symbiotic Bacteria for Obtaining Microbial Proteins Heterotrophomonas acidaminifila GBS-15-2 Strain | |
Trček et al. | Phenotypic characterization and RAPD-PCR profiling of Acetobacter sp. isolated from spirit vinegar production | |
CN104745504B (en) | The Bacillus cercus in one plant of deep-sea source and its application in neutral proteinase is produced | |
Sivakumar et al. | SCREENING OF KERA SCREENING OF KERATINOLYTIC BACTERIA BACILLUS CEREUS US CEREUS US CEREUS FROM THE FEATHER DUMPING SOIL OF SIV THER DUMPING SOIL OF SIV THER DUMPING SOIL OF SIVAKASI | |
Tiwari et al. | Isolation and optimization of alkaline protease producing Bacteria from undisturbed soil of NE-region of India falling under Indo-Burma biodiversity hotspots | |
JP2020058339A (en) | Strain of heterotrophic bacterium klebsiella pneumonia 1-17, associate for producing microbial protein | |
Tiwari et al. | Diversity and molecular characterization of dominant Bacillus amyloliquefaciens (JNU-001) endophytic bacterial strains isolated from native Neem varieties of Sanganer region of Rajasthan | |
CN1157480C (en) | Butyrobetaine/crotonbetaine-L-carnitine metabolic gene and its use in producing L-carnition by microbial method | |
JP6966096B2 (en) | Symbiotic Bacteria for Obtaining Microbial Proteins Heterotrophic Bacteria Cupriavidus guilardii GBS-15-1 Strain | |
RU2745093C1 (en) | Methylococcus capsulatus bf 19-07 methane-oxidizing bacteria strain - producer for obtaining microbial protein mass | |
Salaheen et al. | Improvement of Bacillus licheniformis MZK05 by mutation for increased production of keratinase | |
Schloe et al. | Polyphasic characterization of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P (HB-co-HV)) metabolizing and denitrifying Acidovorax sp. strains | |
Kim et al. | Paracoccus jeotgali sp. nov., isolated from Korean salted and fermented shrimp | |
CN111100940B (en) | Method for rapidly detecting pseudomonas in needle mushroom culture material | |
TW202144563A (en) | Strain capable of producing butyric acid | |
Saha et al. | Proteolytic Bacillus spp. associated with tannery industries: Conventional and molecular identification | |
Yamauchi et al. | Isolation of a marine purple non-sulfur photosynthetic bacterium with a high ability of glycerol assimilation | |
Castells et al. | Evaluation of lactic acid production by different Bacillus subtilis strains isolated from theobroma cacao crops in Ecuador | |
RU2808127C1 (en) | Bacteria strain methylobacillus methanolivorans gsa, producer of feed protein | |
KR102079741B1 (en) | A formate removing composition comprising Methanobacterium congolense and a method for producing a succinate using the same | |
KR101008568B1 (en) | Novel Aquitalea sp. 5YN1-3 strain for effective denitrification | |
CN106834161B (en) | Bacillus subtilis Z12 and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20191112 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20201110 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20210204 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20210412 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20210510 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20210510 |
|
A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20210706 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20210707 |