JP6966096B2 - Symbiotic Bacteria for Obtaining Microbial Proteins Heterotrophic Bacteria Cupriavidus guilardii GBS-15-1 Strain - Google Patents
Symbiotic Bacteria for Obtaining Microbial Proteins Heterotrophic Bacteria Cupriavidus guilardii GBS-15-1 Strain Download PDFInfo
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- 241000894006 Bacteria Species 0.000 title claims description 44
- 108090000623 proteins and genes Proteins 0.000 title claims description 20
- 102000004169 proteins and genes Human genes 0.000 title claims description 20
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- 241001528480 Cupriavidus Species 0.000 title description 6
- 238000003501 co-culture Methods 0.000 claims description 11
- 241001634906 Cupriavidus gilardii Species 0.000 claims description 10
- 230000001580 bacterial effect Effects 0.000 claims description 7
- 230000001419 dependent effect Effects 0.000 claims description 3
- 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 100
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 239000002028 Biomass Substances 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 10
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- 230000009089 cytolysis Effects 0.000 description 2
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- 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
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- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
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- 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
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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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
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- 239000003102 growth factor Substances 0.000 description 1
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- 239000001257 hydrogen Substances 0.000 description 1
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- 125000004435 hydrogen atom Chemical class [H]* 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
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- 239000005014 poly(hydroxyalkanoate) Substances 0.000 description 1
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- 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
- 230000035945 sensitivity Effects 0.000 description 1
- 108010027322 single cell proteins Proteins 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
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- 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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- 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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- C12P21/00—Preparation of peptides or proteins
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- 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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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Cupriavidus gilardii GBS−15−1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 a strain of the heterotrophic bacterium Cupriavidus gilardii GBS-15-1 for obtaining protein biomass by co-culture containing a methane-oxidizing bacterium and a heterotrophic bacterium. 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 source of 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 of 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 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 natural gas reserves and portability. 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 reserve of underground gas, which is as high as 40% of the world's reserves according to multiple data, it is not possible to introduce the production of unicellular proteins using microorganisms. Not only can it bring economic benefits to Russian companies, but it also contributes 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 has low substrate specificity, 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 incompletely oxidized methane and homologues of methane, 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.
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.
ポリヒドロキシアルカノエートの生産者であるCupriavidus eutrophus VKPM V−10646(ロシア国特許第2439143号)(特許文献4)は知られている。成長基質としてグルコースまたはフルクトース、または3-酪酸、または水素、酸素および二酸化炭素の混合ガスを利用する。 Cupriavidus europhos VKPM V-10646 (Russian Patent No. 2439143) (Patent Document 4), which is a producer of polyhydroxyalkanoate, is known. Glucose or fructose, or 3-butyric acid, or a mixed gas of hydrogen, oxygen, and carbon dioxide is used as the growth substrate.
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5)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 5).
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 dependent bacterium as a symbiotic bacterium of a microbial protein producer.
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 co-existing bacterium of methane-oxidizing bacteria, which can utilize the products produced by the co-oxidation of methane homologues in natural gas, as well as proteins, amino acids, which are produced during the lysis process of the bacteria. 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−3265Dとして寄託された従属栄養細菌Cupriavidus gilardii GBS−15−1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. Cupriavidus gilardii GBS-15-1, a heterotrophic bacterium deposited under the registration number VKM B-3265D in the All-Russian Microbial Collection attached to the Skryabin Memorial Institute for Biochemical Physiology, has been 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-15-1である。 The code of the strain given by the depositor is GBS-15-1.
Cupriavidus gilardii GBS−15−1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。 Cupriavidus gilardii GBS-15-1 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.
本発明のCupriavidus gilardii GBS−15−1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。その結果、工業的環境でメタン酸化細菌を天然ガス中で培養する際の培養菌としても、また様々な共培養の構成要素としても利用できる菌株が得られた。 The Cupriavidus gilardii GBS-15-1 strain of the present invention is extracted from a co-culture 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. As a result, a strain that can be used as a culture bacterium when culturing methane-oxidizing bacteria in natural gas in an industrial environment and as a component of various co-cultures was obtained.
細菌株は、鉱物培養基における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。 Bacterial strains grow as co-cultures of the main producers of methane-oxidizing bacteria during fermentation in mineral culture media, and in the physicochemical environment optimal 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 recombined.
Cupriavidus gilardii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。 The Cupriavidus gilardii species is a work that uses pathogens of pathogenicity classifications 1 to 4 (Health and Epidemiology Regulations SP 1.3.2322-08 "Pathogenicity (risk) classification III-IV groups". Safety ”Attachment No. 1“ Classification of pathogenic microorganisms, protozoa, parasites, and biological toxins that cause infectious diseases to humans by pathogenicity ”, certified by the Chief of the National Health Doctor of the Russian Federation on June 29, 2011 No. 86 Addition and revision No. 2) is not on the list.
Cupriavidus gilardii GBS−15−1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。 The Cupriavidus guilardii GBS-15-1 strain is non-pathogenic, non-toxin-producing, non-toxic and harmless.
得られた従属栄養細菌Cupriavidus gilardii GBS−15−1の菌株は、以下のように特徴づけられる。 The resulting heterotrophic bacterium Cupriavidus guilardii GBS-15-1 strain is characterized as follows.
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、明るいクリーム色、直径2〜4mm、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。細胞の形状は0.3〜0.5×0.9〜1.2μmの長さを有する棹状であり、芽胞を形成しない。 Culture and morphological features : Gram-negative. On vegetative agar (pH 7.0-7.2), it is opaque, light cream, 2-4 mm in diameter, has flat edges, is a soft solid, and forms colonies that can be easily peeled off from the agar. The cell shape is rod-shaped with a length of 0.3-0.5 × 0.9-1.2 μm and does not form spores.
生理学生化学的特徴:菌株は30〜47℃の温度帯で成長し、最適な成長温度は32〜35℃である。生理作用の限界pHは5.0〜7.2の範囲である。最適なpH帯は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。炭素源として糖、アミノ酸、有機酸、アルコールを利用する。窒素源として硝酸塩、アンモニウム塩、アミノ酸を利用する。特異的な成長因子を要求しない。 Physiological and biochemical characteristics : The strain grows in the temperature range of 30 to 47 ° C., and the optimum growth temperature is 32 to 35 ° C. The limit pH of physiological action is in the range of 5.0 to 7.2. The optimum pH range is 6.8 to 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 rises, and flow velocity fluctuations (0.15 to 0.42 h- 1). do. It has a wide range of potential in terms of ability. Sugars, amino acids, organic acids and alcohols are used as carbon sources. Nitrate, ammonium salt and amino acids are used as nitrogen sources. Does not require specific growth factors.
培養に推奨される培養基および条件:
案1:鉱物培養基
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。調合済みの微量元素溶液1mlを1000mlの鉱物培養基に加える。
Recommended culture media and conditions for culturing:
Proposal 1: Mineral culture group Mineral culture group (culture group component, 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 .. Trace element solution (separately prepared) (г / л): 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 trace element 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。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 32−35℃、好気培養。 Sterilize at 121 ° C for 15 minutes. Set the pH to 6.0-6.2. Methanol (less than 1.5% by volume), ethanol (less than 2% by volume), and propanol (less than 1% by volume) can be used as substrates (carbon source and energy source). Culture temperature 32-35 ° C, aerobic culture.
案1:栄養寒天もしくは栄養液
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 1: Nutritional agar or nutrient solution Nutritional agar component (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, chloride 5.0 g of sodium, 12.0 g of microbial culture agar. 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。 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.
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。 The strain is stored on nutrient agar (temperature 4 ± 2 ° C) and transplanted once every three months, and the strain can be stored in lyophilized state or in liquid nitrogen.
本発明の菌株の特徴
−様々な成分の培養基において安定的かつ生産的に成長する
−凍結乾燥状態で長期間活性を保つことができる
−非病原性かつ毒素産生性や毒性がなく、無害である
Characteristics of the strain of the present invention-Stable and productive growth in culture media of various components-Can maintain activity for a long period of time in a freeze-dried state-Non-pathogenic, non-toxic, non-toxic and harmless
菌株は、天然ガス中で成長するメタン酸化細菌によりタンパク質ビタミン濃縮飼料を製造する分野において利用される。 The strain is used in the field of producing protein-vitamin concentrated feeds by methane-oxidizing bacteria that grow in natural gas.
従属栄養細菌Cupriavidus gilardii GBS−15−1は、天然ガスのメタンと鉱物培養基においてメタン酸化細菌Methylococcus capsulatus GBS−15とともに共培養され、その際にメタン酸化細菌は唯一の炭素源およびエネルギー源として天然ガスのメタンを利用する。同時にメタン酸化細菌は、メタン資化性菌の成長を阻害する生産物を生成するメタン同族体(C2、C3、C4)を共酸化する。Cupriavidus gilardii GBS−15−1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)を炭素源として使用し、それにより主たる生産者に対する阻害作用を取り除く。この他にも、従属栄養体は主たる生産者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 dependent bacterium Cupriavidus gilardii GBS-15-1 is co-cultured with the methane-oxidizing bacterium Methylococcus capsaturus GBS-15 in natural gas methane and mineral culture groups, where the methane-oxidizing bacterium is the only natural gas source of carbon and energy. Uses methane. At the same time, methane-oxidizing bacteria co-oxidize methane homologues (C 2 , C 3 , C 4 ) that produce products that inhibit the growth of methane-utilizing bacteria. Cupriavidus guilardii GBS-15-1 uses the product 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 lysis process of the main producer.
培養は、主に連続的な方法で、最適な環境であるpH5.6〜5.8および温度42〜45℃において、天然ガスと酸素含有ガスの混合気体に培養基を曝気して行われる。培養基に、全細胞数の0.1〜0.15%の数の従属栄養細菌Cupriavidus gilardii GBS−15−1を植え付ける。バイオマスの一定濃度が32g/lまでであれば、共培養菌中のメタン酸化細菌Methylococcus capsulatus GBS−15の含有割合は85〜99.9%とする。メタン酸化細菌は天然ガスのメタンにより増殖し、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の生産物およびメタン資化性菌の細菌活動による生産物により増殖する。 Culturing is carried out mainly by a continuous method, in which the culture medium is aerated with a mixed gas of natural gas and oxygen-containing gas in an optimum environment of pH 5.6 to 5.8 and a temperature of 42 to 45 ° C. The culture medium is inoculated with a heterotrophic bacterium Cupriavidus gilardii GBS-15-1, which is 0.1 to 0.15% of the total cell count. When the constant concentration of biomass is up to 32 g / l, the content ratio of the methane-oxidizing bacterium Methylococcus capsulatus GBS-15 in the co-cultured bacteria is 85 to 99.9%. Methane-oxidizing bacteria grow on natural gas methane, and the other element of co-culture grows on co-oxidation products of methane homologues in natural gas and products of bacterial activity of methane-utilizing bacteria.
メタン酸化細菌とメタンを利用しない従属栄養細菌は、種菌の培養を前提とした通常の方法で事前にそれぞれ別に採取され、同時に栄養培養基に植え付けされる。その際に植え付けられる従属栄養細菌でありメタンを酸化しない細菌の数は、全体の細胞数の0.1〜15%の範囲とする。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。 Methane-oxidizing bacteria and heterotrophic bacteria that do not utilize methane are separately collected in advance by a usual method premised on the culture of inoculum, and are simultaneously planted in a vegetative culture medium. The number of heterotrophic bacteria that are planted at this time and do not oxidize methane shall be in the range of 0.1 to 15% of the total number of cells. 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 to 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-oxidized 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 methane homologues, of co-oxidation products obtained in the growth of the cultured bacterium, and is usually limited by the total number of 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.
本発明は、以下の例で実施することができる。 The present invention can be carried out in the following examples.
例1 Cupriavidus gilardii GBS−15−1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 component (g / l) for culturing Cupriavidus gilardii GBS-15-1 strain: (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.
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。基質(炭素源およびエネルギー源)として、メタノール0.5%を使用する。 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. 0.5% methanol 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pH6.8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2-2.5g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 6.8 for 48 hours. The concentration of the dried product at the end of culturing is 2-2.5 g of the absolute dried product 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 type fermenter having a capacity of 40 liters (working capacity of 28 liters).
例2 Cupriavidus gilardii GBS−15−1菌株の培養が例1と同様に行われるが、炭素源およびエネルギー源としてエタノール0.8%が使用され、また栄養培養液として以下の成分を含む(培養液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。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4gとなる。 Example 2 The culture of the Cupriavidus gilardii GBS-15-1 strain is carried out in the same manner as in Example 1, but 0.8% of ethanol is used as a carbon source and an energy source, and the following components are contained as a nutrient culture medium (culture medium). (1 liter equivalent): 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, 0.3 g of sodium carbonate. The physicochemical conditions and culture time are the same, and the dry matter concentration is 4 g of absolute dry matter per liter.
例3 Cupriavidus gilardii GBS−15−1菌株が、全体に対する濃度が0.5%となる量のブタノールを加えた培養液で培養される。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pH6.8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2.5〜3.0gとなる。得られた懸濁液は、8000グラムの場合7分間遠心分離される。その後沈殿物を少量の生理食塩水の中で再度遠心分離し、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けるその後の細菌培養のための種菌として使用される。 Example 3 Cupriavidus guilardii GBS-15-1 strain is cultured in a culture medium supplemented with butanol in an amount such that the concentration is 0.5% with respect to the whole. 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 6.8 for 48 hours. The concentration of the dried product at the end of culturing is 2.5 to 3.0 g of the absolute dried product per liter. The resulting suspension is centrifuged at 8000 grams for 7 minutes. The precipitate is then centrifuged again in a small amount of saline and subsequent bacterial culture in an automatic fermentation system with a capacity of 10 liters (working capacity 7 liters) or a discharge fermenter with a capacity of 40 liters (working capacity 28 liters). Used as an inoculum for.
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 heterotrophonas acidaminifila GBS-15-2 in an automatic fermentation system with 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 x 4H 2 O 0.38, ZnSO 4 x 7H 2 O 0.06, H 3 BO 3 0.25, Na 2 MoO 4 x 2H 2 O 0.009, CoSO 4 x 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40〜45℃、pH5.0〜6.0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15gである際に、Methylococcus capsulatus GBS−15の支配率は90%となった。 Ammonia water is supplied as a nitrogen source and a titrator. The culturing process is carried out at 40 to 45 ° C. and pH 5.0 to 6.0 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 15 g of absolute dry matter per liter, the dominance of Methylococcus capsaturus GBS-15 was 90%.
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