JP2020058339A - Strain of heterotrophic bacterium klebsiella pneumonia 1-17, associate for producing microbial protein - Google Patents
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- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
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- 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
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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
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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%.
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