JP6919910B2 - Symbiotic Bacteria for Obtaining Microbial Proteins Heterotrophomonas acidaminifila GBS-15-2 Strain - Google Patents
Symbiotic Bacteria for Obtaining Microbial Proteins Heterotrophomonas acidaminifila GBS-15-2 Strain Download PDFInfo
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- 241000894006 Bacteria Species 0.000 title claims description 34
- 108090000623 proteins and genes Proteins 0.000 title claims description 20
- 102000004169 proteins and genes Human genes 0.000 title claims description 20
- 230000000813 microbial effect Effects 0.000 title claims description 17
- 238000003501 co-culture Methods 0.000 claims description 14
- 230000001580 bacterial effect Effects 0.000 claims description 10
- 230000035479 physiological effects, processes and functions Effects 0.000 claims description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 86
- 239000003345 natural gas Substances 0.000 description 16
- 239000002028 Biomass Substances 0.000 description 14
- 239000001963 growth medium Substances 0.000 description 13
- 238000000034 method Methods 0.000 description 10
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 229910052799 carbon Inorganic materials 0.000 description 9
- 229910052500 inorganic mineral Inorganic materials 0.000 description 8
- 239000011707 mineral Substances 0.000 description 8
- 238000007254 oxidation reaction Methods 0.000 description 8
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- 244000005700 microbiome Species 0.000 description 7
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- 241000122971 Stenotrophomonas Species 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 241000589345 Methylococcus Species 0.000 description 5
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- 229920001817 Agar Polymers 0.000 description 4
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
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- 230000001717 pathogenic effect Effects 0.000 description 3
- 239000003208 petroleum Substances 0.000 description 3
- 239000003209 petroleum derivative Substances 0.000 description 3
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 3
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- 239000002994 raw material Substances 0.000 description 3
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- 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
- 241000122973 Stenotrophomonas maltophilia Species 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
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000002283 diesel fuel Substances 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000010705 motor oil Substances 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 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
- 208000035473 Communicable disease Diseases 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
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- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
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- 235000015103 Malus silvestris Nutrition 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 238000009825 accumulation Methods 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
- 241001148470 aerobic bacillus Species 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
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- 239000001273 butane Substances 0.000 description 1
- ZTQSAGDEMFDKMZ-UHFFFAOYSA-N butyric aldehyde Natural products CCCC=O ZTQSAGDEMFDKMZ-UHFFFAOYSA-N 0.000 description 1
- 238000006243 chemical reaction 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
- 239000002274 desiccant Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
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- 239000003673 groundwater Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
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- 239000002054 inoculum Substances 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 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
- 244000000010 microbial pathogen Species 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
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000001294 propane Substances 0.000 description 1
- 235000019260 propionic acid Nutrition 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
- 230000007281 self degradation Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 108010027322 single cell proteins Proteins 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
Classifications
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- 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
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- 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
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- Tropical Medicine & Parasitology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
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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 and, in particular, to strains of the heterotrophic bacterium Klebsiella pneumonia 1-17 for obtaining protein biomass by co-culture containing methaneoxidizing bacteria and heterotrophic bacteria. Microbial proteins can be used as livestock feed in agriculture and as a raw material 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 shape overall. According to the doctrine signed by the Russian President to bring food self-sufficiency to 80-90%, the shortage of feed products could amount to at least 2 million tonnes 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 ingredient in protein products is soybean meal. However, the natural conditions of Japan are not suitable for cultivating a sufficient amount of soybeans. Therefore, those engaged 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, various taxa of microorganisms that can grow on various substrates are known.
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 producer of feed protein is more efficient than using yeast, as yeast stores less than 60% of its protein by weight, while bacteria store 79% of its mass of protein. 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 proteins and biomass, a bacterial strain that produces proteins based on methanol and the genus Acetobacter 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-24 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 desiccant.
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 heat resistance of these strains to typical phages vary widely. 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 non-sterile cultures with acidic cultures below pH 6.0 in fermenters with wheat bran and / or wheat flour, as experiments show, yeast and fungal contamination occurs quickly and the total cell count is 30. Producer substitution reactions occurred in ~ 40% or more. As a result, the protein content level of the resulting product is significantly reduced.
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 natural gas methane under conditions suitable for them and proliferate 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 natural gas methane to obtain single-cell proteins has several advantages over liquid hydrocarbons and other substrates, especially the high reserves and portability of natural gas. It has the advantages of height and the ability to obtain 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 that Russia has a large amount of underground gas reserves, which is as high as 40% of the world's reserves according to multiple data, it is not possible to introduce the production of single-celled proteins using microorganisms. Not only can it bring economic benefits to Russian companies, but it also contributes to ensuring food stability in the country.
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 can oxidize not only methane but also methane homologues (eg, ethane, propane and butane) contained in natural gas.
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。 Methanol, formaldehyde, formic acid, ethanol, acetaldehyde, acetate, propionaldehyde, among the products of methane and its homologues that are incompletely oxidized, depending on the composition of the natural gas, the characteristics of the species of methane-oxidizing bacteria and the culture conditions. , Propionic acid, oil aldehyde may be present in the culture group in various proportions. Accumulation of these products in culture media at specific concentrations exerts an inhibitory effect on methane oxidation cultures and methane oxidation.
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 culture in natural gas is possible only by co-culture of methane-oxidizing microorganisms and their heterotrophic microorganisms, which utilize the product of incomplete oxidation of methane homologues and also. Products produced by self-degradation of microbial cells may also be 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 when incompletely oxidized products 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 action of some physicochemical indicators is also noticeably increased.
石油および石油製品(ディーゼル燃料、モーターオイル、油圧作動油、コンデンセート)を+5〜+37℃の幅広い温度帯において素早く処分する Stenotrophomonas maltophilia SNBS−3細菌株は知られている。細菌Stenotrophomonas maltophiliaの菌株は全ロシア産業微生物コレクションの中に登録番号「VKPM V−12247として寄託されており、石油および石油製品(ディーゼル燃料、モーターオイル、油圧作動油、コンデンセート)により汚染された土壌の浄化および水の浄化に利用されるものである。本発明により、+5〜+37℃(ロシア国特許第2617950号)(特許文献4)の幅広い温度帯において、石油および石油製品で汚染された土壌や水の浄化効率を向上させ、期間を短縮させることが可能となる。 Stenotrophomonas maltophilia SNBS-3 bacterial strains are known that quickly dispose of petroleum and petroleum products (diesel fuels, motor oils, hydraulic hydraulic oils, condensates) over a wide temperature range of + 5 to + 37 ° C. A strain of the bacterium Stenotrophomonas maltophilia has been deposited in the All-Russian Industrial Microbial Collection under registration number "VKPM V-12247" to purify soil contaminated with petroleum and petroleum products (diesel fuel, motor oil, hydraulic oil, condensate). And water purification. According to the present invention, soil and water contaminated with petroleum and petroleum products in a wide temperature range of + 5 to + 37 ° C. (Russian Patent No. 2617950) (Patent Document 4). It is possible to improve the purification efficiency of the product and shorten the period.
リンゴの木の病害を生態学的に防除するために利用されるStenotrophomonas acidaminiphila BJ1(中国特許第102851225号)(特許文献5)の菌株は知られている。 Strains of Stenotrophomonas acidaminifila BJ1 (Chinese Patent No. 102851225) (Patent Document 5) used for ecologically controlling diseases of apple trees are known.
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。 A strain of Methylococcus capsulatus VCB-874, which is a producer of feed biomass, is known. The strain is stored in the culture collection of the "Institute for All-Russian Genetics and Industrial Microbial Breeding" as the collection number "CMPM V1743 (Авт. свид. Magazine Soviet Union No. 770200) (Non-Patent Document 1)". There is. This strain utilizes methane as a carbon source and an energy source, whether it is pure methane or methane as a component in natural gas. The disadvantage of this strain is that it is highly sensitive to the products produced by the co-oxidation of methane homologues, which may be large or small, but are always present in natural gas. It is a thing. The product produced inhibits the growth of the grower.
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6) である。 The closest technical essence and results to be achieved are strains of the methane-oxidizing bacterium Methylococcus capsaturus GBS-15 for obtaining microbial proteins under All-Russian Industrial Microbial Collection Registration No. VKPM V-12549 (Russian Patent No. 2613365). ) (Patent Document 6).
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。 An object of the present invention is to increase the production efficiency and obtain high productivity for culturing methane-oxidizing bacteria in natural gas in the presence of heterotrophic bacteria as cohabitants 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 that methane-oxidizing bacteria co-existing bacteria that can utilize co-oxidized products of methane homologues in natural gas, as well as proteins, amino acids, produced during the bacterial lysis process. To identify new strains that can utilize polysaccharides.
本発明の技術的結果は、ロシア科学アカデミー G.K.スクリャビン記念生化学生理学研究所付属 全ロシア微生物コレクションに登録番号VKM B−3264Dとして寄託された従属栄養細菌Stenotrophomonas acidaminiphila GBS−15−2が、微生物タンパク質を得る目的で、メタン酸化細菌の共培養菌として利用される際に達成される。 The technical results of the present invention are described in Russian Academy of Sciences G.M. K. The heterotrophomas acidaminifila GBS-15-2, which was deposited under the registration number VKM B-3264D in the All-Russian Microbial Collection attached to the Skryabin Memorial Institute for Biochemical Physiology, was used as a co-culture of methane-oxidizing bacteria for the purpose of obtaining microbial proteins. Achieved when used.
供託者が与えられた菌株の符号がGBS−15−2である。 The code of the strain given by the depositor is GBS-15-2.
Stenotrophomonas acidaminiphila GBS−15−2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 Stenotrophomonas acidaminifila GBS-15-2 strain is one of the co-cultured 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. Available.
菌株の抽出源: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。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。 Strain Extraction Source: Strains are extracted from co-cultures containing the methane-oxidizing bacterium Methylococcus capsaturus GBS-15. To obtain fast-growing mixed-culture bacteria, a mixture of active stock-cultured bacteria extracted from natural gas and groundwater in oil-producing areas within the Russian Federation was used. Bacterial strains grow as co-cultures of the main producers of methane-oxidizing bacteria during fermentation in mineral culture media, and in the optimal physicochemical environment for the producers, co-oxidation by methane homologues present in natural gas. It requires products, as well as products from the material 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:Stenotrophomonas acidaminiphila種は、病原性分類第1〜4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)のリストに載っていない。 Information on the biological risk (safety) of bacterial strains: Stenotrophomonas acidaminifila species are pathogenic classification 1st to 4th groups (Health and Epidemiology Regulations SP 1.3.2322-08 "Pathogenicity (risk) classification No. 1" Safety of Work Using Pathogens of Group III-IV Microorganisms and Parasite Diseases "Attachment No. 1" Classification of pathogenic microorganisms, protozoa, parasites, and biological toxins that cause infectious diseases to humans , No. 86 Addition and Revision No. 86 of the National Health Doctor Certification of the Russian Federation dated June 29, 2011) is not on the list.
病原性または毒性に関するその他の情報:Stenotrophomonas acidaminiphila GBS−15−2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 toxicity: Stenotrophomonas acidaminifila GBS-15-2 strain is non-pathogenic, non-toxin-producing, non-toxic and harmless.
特徴:
I. 培養および形態学的特徴:グラム陰性である。栄養寒天(pH7.0〜7.2)上で、不透明、白−ベージュ色、直径2〜3mm、平坦な端部を持ち、柔らかい固体であり、寒天から容易に剥がすことのできるコロニーを形成する。成熟が進むと茶色の色素を作ることがある。細胞の形状は直線または少し曲がった棹状であり、単独または対で存在し、0.5〜1.5μmの長さを有し、芽胞を形成しない。
Characteristic:
I. Culture and morphological features : Gram-negative. On nutrient agar (pH 7.0-7.2), opaque, white-beige, 2-3 mm in diameter, with flat edges, soft solid, forming colonies that can be easily peeled off from the agar. .. As it matures, it may produce a brown pigment. The shape of the cells is straight or slightly curved rod-like, present alone or in pairs, having a length of 0.5-1.5 μm and not forming spores.
II. 生理学生化学的特徴:25〜50℃の温度帯で成長し、最適な成長温度は32〜35℃である。pH帯5.0〜8.0で成長し、最適なpHは6.8〜7.0である。絶対好気性細菌。培養条件および培養基の変動、すなわち炭素源の変更、培養基の活性反応値の上下変動、温度の上昇、流速変動(0.15〜0.42 h−1)においても自身の特徴を安定的に保持する。能力において幅広い可能性を有している。生産者菌株(GBS−15)の溶解による生産物であるポリペプチド、アミノ酸、有機酸、アルコールを成長のために利用する。特異的な成長因子を要求しない。 II. Physiological and biochemical characteristics : It grows in the temperature range of 25 to 50 ° C, and the optimum growth temperature is 32 to 35 ° C. It grows in the pH range 5.0-8.0 and the optimum pH is 6.8-7.0. Absolutely aerobic bacteria. Stable retention of its own characteristics even under fluctuations in culture conditions and culture media, that is, changes in carbon source, fluctuations in the activity reaction value of culture media, temperature rise, and flow velocity fluctuations (0.15 to 0.42 h- 1). do. It has a wide range of potential in terms of ability. Polypeptides, amino acids, organic acids and alcohols produced by lysis of the producer strain (GBS-15) are utilized for growth. Does not require specific growth factors.
培養に推奨される培養基および条件:
栄養培地の案:
案1:
鉱物培養基の成分(g/l):(NH4)2SO4 0.52、MgSO4 0.02、K2SO4 0.06、NH4H2PO4 1.53。微生物溶液(別途調合される)(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。
Recommended culture media and conditions for culturing:
Proposal of nutrient medium:
Proposal 1:
Mineral culture group components (g / l): (NH 4 ) 2 SO 4 0.52, sulfonyl 4 0.02, K 2 SO 4 0.06, NH 4 H 2 PO 4 1.53. Microbial solution (prepared separately) (g / l): ZnSO 4 0.43, MnSO 4 0.88, CuSO 4 0.78, H 3 BO 3 0.4, Na 2 MoO 4 × 2 H 2 O 0. 25, CoSO 4 × 7H 2 O 0.25, FeSO 4 × 7H 2 O 4.97. Add 1 ml of the prepared microbial solution to 1000 ml of mineral culture medium.
121℃で15分間殺菌する。pHを6.0〜6.2にする。 Sterilize at 121 ° C for 15 minutes. Set the pH to 6.0-6.2.
基質(炭素源およびエネルギー源)としてブタノール、プロパノールまたはエタノール(0.15〜1.0%)を使用することができる。培養温度 32〜35℃、好気培養。 Butanol, propanol or ethanol (0.15-1.0%) can be used as the substrate (carbon source and energy source). Culture temperature 32 to 35 ° C, aerobic culture.
案2:
栄養液または栄養寒天。
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。
Proposal 2:
Nutrient solution or nutritional agar.
Nutritional agar components (1 liter of prepared medium): 10.5 g of dried protein enzyme hydrolyzate, 10.5 g of dried peptone for fermentation medium, 2.0 g of filtered autolyzed yeast extract, 5.0 g of sodium chloride, 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 solution components (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 autolyzed yeast extract, 5.0 g of sodium chloride, 0 of sodium carbonate .3g. Sterilize at 121 ° C for 15 minutes.
推奨される保管方法および保管条件:菌株を栄養寒天上で保管(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、液体窒素の温度でも保存が可能。 Recommended storage method and storage conditions: Strains are stored on nutrient agar (temperature 4 ± 2 ° C), transplanted once every three months, and the strains can be stored in a lyophilized state or at the temperature of 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 the culture medium of various components, can maintain its activity for a long period of time in a lyophilized state, and is non-pathogenic, toxin-producing, and non-toxic. It has the characteristic of being harmless.
Stenotrophomonas acidaminiphila GBS−15−2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 Stenotrophomonas acidaminifila GBS-15-2 strain can be used as a co-cultivator in co-culture of methane-oxidizing bacteria with natural gas for the purpose of industrial production of feed biomass, and can also be used as a raw material for advanced processing. ..
従属栄養細菌Stenotrophomonas acidaminiphila GBS−15−2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 heterotrophomonas acidaminifila GBS-15-2 utilizes the products produced (methanol, ethanol, propanol) as a carbon source, thereby removing the inhibitory effect on the main producer. In addition, heterotrophs can utilize proteins, amino acids, and polysaccharides produced in 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 grow on natural gas methane, and the other element of co-culture grows on the co-oxidation products of methane homologues in natural gas and on 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。 In the process of co-culture, the content level of co-culture bacteria 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 planting amount. This is because in mineral culture groups, these products are the only carbon source for co-culture elements that do not utilize methane. Since the cultured bacteria perform such self-regulation, the proportion of cultured cells in the initial planting of the culture is not significant, but the number of non-methane-oxidizing microorganisms added is 0.1% or more of the total number of cells. It is recommended to keep it within 15%. Outside this range, it first leads to stagnation of growth of methane-oxidizing bacteria, and secondly, stagnation of growth of heterotrophic bacteria. In the mixed culture in which bacteria that do not assimilate methane were selected and added, the amount of biomass produced increased 3 to 5 times as compared with the pure culture in which only methane-oxidizing bacteria were added. Thus, the biomass production in methane can be increased by selecting heterotrophs that grow on the co-oxidation products of the methane homologue and do not assimilate methane and add them to the methane-oxidizing bacteria. Even with high concentrations of heterotrophs that do not assimilate methane, their content is limited by the number of carbon sources, or co-oxidized products of the methane homologue, obtained in the growth of the cultured bacterium, and is usually limited to the total cells. Adding 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.
例1
VKM−3264DのStenotrophomonas acidaminiphila GBS−15−2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
Liquid mineral culture group components (g / l) for culturing the Stenotrophomonas acidaminifila GBS-15-2 strain of VKM-3264D: (NH 4 ) 2 SO 4 0.52, sulfonyl 4 0.02, K 2 SO 40 . 06, NH 4 H 2 PO 4 1.53.
培養基を121℃で15分間殺菌する。 Sterilize the culture medium 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。 Microbial solution (prepared separately from the culture group 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. Add 1 ml of the prepared microbial solution to 1000 ml of mineral culture medium. Set the pH to 6.0-6.2.
基質(炭素源およびエネルギー源)として、エタノール1.0%を使用する。 Ethanol 1.0% is used as the substrate (carbon source and energy source).
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35℃およびpH6.2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7g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。 The strains are cultivated in a heat-resistant glass flask with a volume of 1 liter at a filling rate of 0.3 to 0.4 in a constant temperature shaking incubator by a method for a fixed period. Culturing is carried out at 35 ° C. and pH 6.2 for 48 hours. The dry matter concentration at the end of culturing is 7 g of absolute dry matter per liter. The obtained cultured bacteria are used as inoculum for subsequent bacterial culture in an automatic fermentation system having a capacity of 10 liters (working capacity of 7 liters) or a discharge fermenter having a capacity of 40 liters (working capacity of 28 liters).
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プロパノール0.8%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2.5gとなる。 Example 2 The strain is cultivated in the same manner as in Example 1, but 0.8% propanol is used as a carbon source and an energy source. The physicochemical conditions and culture time are the same, and the dry matter concentration is 2.5 g of 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ブタノール0.15%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0.8gとなる。 Example 3 The strain is cultivated in the same manner as in Example 1, but 0.15% butanol is used as a carbon source and an energy source. The physicochemical conditions and culture time are the same, and the dry matter concentration is 0.8 g of 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Stenotrophomonas acidaminiphila GBS−15−2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 composed of the methane-oxidizing bacterium Methylococcus capsaturus GBS-15 and the heterotrophic bacterium Stenototropomonas acidaminifila GBS-15-2 in an automatic fermentation system having a capacity of 10 liters (actual capacity of 7 liters) has the following configuration. Perform on a mineral culture medium. (Unit: g / l) H 3 PO 4 (80%) 17.2, K 2 SO 4 5.0, 0073 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 × 2 H 2 O 0.009, CoSO 4 × 7H 2 O 0. 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41〜43℃、pH5.5〜5.7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10g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16gである際に、Methylococcus capsulatus GBS−15の支配率は92%となった。 Ammonia water is supplied as a nitrogen source and a titrator. The culturing process is carried out at 41 to 43 ° C. and pH 5.5 to 5.7 while constantly supplying a mixed gas containing methane and oxygen. When the biomass concentration reaches 10 g of the absolute dry matter per liter, the process proceeds to a continuous culture process in which the dilution ratio of the culture medium is D = 0.25 h-1. When the biomass concentration was 16 g of absolute dry matter per liter, the predominance of Methylococcus capsaturus GBS-15 was 92%.
例5 メタン酸化細菌Methylococcus capsulatus GBS−15および従属栄養細菌Stenotrophomonas acidaminiphila GBS−15−2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)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 5 A co-culture composed of the methane-oxidizing bacterium Methylococcus capsulatus GBS-15 and the heterotrophonas acidaminifila GBS-15-2 is arranged in a discharge-type fermenter having a capacity of 40 liters (actual capacity of 28 liters) as follows. Performed in the mineral culture medium of. (Unit: g / l) H 3 PO 4 (80%) 17.2, K 2 SO 4 5.0, 0073 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 × 2 H 2 O 0.009, CoSO 4 × 7H 2 O 0. 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℃、pH5.5〜5.7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=0.28 h−1、バイオマス濃度は1リットル当たりの絶対乾燥物43g、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 titrator. The culturing process is carried out at 41 to 45 ° C. and pH 5.5 to 5.7 while constantly supplying a mixed gas containing methane and oxygen. When the biomass concentration reaches 10 g of absolute dry matter per liter, the atmospheric pressure is gradually increased to 0.8 MPa. As the air pressure rises, the supply of gas, which is a nutrient source for microorganisms, is gradually increased. At that time, the index of the culture process was D = 0.28 h- 1 , the biomass concentration was 43 g of absolute dry matter per liter, and the dominant rate of the methane-oxidizing bacterium Methylococcus capsaturus GBS-15 was 93%.
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