JPH0757185B2 - Thermus aquaticus - Google Patents
Thermus aquaticusInfo
- Publication number
- JPH0757185B2 JPH0757185B2 JP31583692A JP31583692A JPH0757185B2 JP H0757185 B2 JPH0757185 B2 JP H0757185B2 JP 31583692 A JP31583692 A JP 31583692A JP 31583692 A JP31583692 A JP 31583692A JP H0757185 B2 JPH0757185 B2 JP H0757185B2
- Authority
- JP
- Japan
- Prior art keywords
- strain
- culture
- bacteria
- lignin
- soil
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Treatment Of Sludge (AREA)
- Soil Conditioners And Soil-Stabilizing Materials (AREA)
- Fertilizers (AREA)
Description
【0001】[0001]
【産業上の利用分野】本発明は、天然リグニンの可溶
化,木材その他の難分解性繊維物質,硬タンパク質や余
剰汚泥の分解発酵,堆・厩肥の製造,屎尿の分解等バイ
オマスの腐植熟成化を促進し、土壌の肥沃化,土壌構造
の改善に有用な新菌及びその有効な活用の技術に関す
る。TECHNICAL FIELD The present invention relates to solubilization of natural lignin, decomposition and fermentation of wood and other hardly decomposable fibrous substances, hard proteins and excess sludge, production of compost / manure, decomposition of human waste, humification and aging of biomass. The present invention relates to a new bacterium useful for promoting soil fertility and improving soil structure, and a technique for effective use thereof.
【0002】明細書でいう難分解性有機資材または難分
解性繊維物質とは、オガ屑,チップダスト,プレーナー
屑,バーク(樹皮)その他木材工業における廃材,モミ
ガラ,イナワラ,ムギワラ等の藁桿類、ダイズ,アズ
キ,落下生等の豆類の種皮や莢殻、コーヒーかす,落
葉,樹皮,ヨシやカヤ等の山野草、それにシイタケの廃
ホダ木,その他キノコの廃菌床等の炭素率40〜100
以上の難分解性の各種植物遺体をいう。これらの植物遺
体を構成している有機成分はきわめて複雑で多種多様で
あるが、一般にその主要成分は繊維素で約30〜75
%、次いでリグニンの15〜40%で、両者を合わせる
と45〜90%以上で、その大部分を占めている。その
次がヘミセルロース7〜25%の順で、そして少量では
あるが、タンパク質等の含窒素化合物,各種糖類,有機
酸,アルコール類、それに油脂,ワックス,精油等が含
まれている。The term "hardly decomposable organic material" or "hardly decomposable fiber material" as used in the specification refers to sawdust, chip dust, planer waste, bark (bark) and other waste materials in the wood industry, straw rods such as rice husks, rice straws, wheat straws, etc. , Soybean, azuki bean, fallen seeds and soybean seed coats and pods, coffee grounds, deciduous leaves, bark, mountain grasses such as reeds and kaya, as well as abolished mushrooms such as shiitake mushrooms and other mushrooms 100
The above-mentioned various persistent plant bodies. The organic components that make up these plant remains are extremely complex and diverse, but generally, the main component is fibrin, which is about 30-75.
%, And then 15 to 40% of lignin and 45 to 90% or more when both are combined, and occupy most of them. Next is hemicellulose in the order of 7 to 25%, and in a small amount, nitrogen-containing compounds such as proteins, various sugars, organic acids, alcohols, and fats, waxes, essential oils and the like are contained.
【0003】[0003]
【従来の技術】従来、微生物や酵素を使った分解剤でリ
グニンの可溶化、又は繊維素を迅速に分解させることは
非常に困難とされ、又ケラチン,コラーゲン等の硬タン
パク質も酵素作用が受け難い。従って、微生物を使った
天然の難分解性有機資材の効果的な可溶化,分解の実用
化は今だになされていない。2. Description of the Related Art Conventionally, it has been very difficult to solubilize lignin or rapidly decompose fibrin with a degrading agent using microorganisms or enzymes, and hard proteins such as keratin and collagen are also subject to enzymatic action. hard. Therefore, effective solubilization and decomposition of natural refractory organic materials using microorganisms have not yet been put into practical use.
【0004】[0004]
【発明が解決しようとする課題】本発明が解決しようと
する課題は、高いタンパク質分解能を有し且つリグニン
可溶化と繊維素分解を高めることができ難分解性の繊維
物質,硬タンパク質資材の分解肥料化,土壌改良,屎尿
の脱臭分解に有効な新菌株の提供にある。The problem to be solved by the present invention is that it has a high protein decomposing ability and is capable of enhancing lignin solubilization and fibrinolysis, and is difficult to decompose.
Decomposing fertilizers of substances and hard protein materials, soil improvement, human waste
To provide a new strain effective for deodorizing decomposition of
【0005】[0005]
【課題を解決するための手段】かかる課題を解決した本
発明の要旨は 1) 絶対好気性、生育適温が72〜76℃で、40〜
82℃の温度範囲で通常濃度の培地に生育し、作用適温
75〜85℃、作用水素イオン濃度pH=4.0〜1
1.3の高温性広域作用水素イオン濃度活性のタンパク
質分解酵素とカロチノイド系黄色色素を産生するサーマ
ス・アクアティクス菌(Thermus aquaticusbiovar SK
542,微工研条寄第3382号)にある。以下、本発明のサーマス・アクアティクス菌(Thermus
aquaticus biovar SK542,微工研条寄第3382
号)は、本発明者の発見にかかるもので、以下それぞれ
単にSK542菌株と称する。尚、本発明のSK542
菌株と共生してその効果を高める下記する好気性繊維素
分解菌クロストリジュウム・サーモセルムSK522
(Clostriduim thermocellum biovar.SK522,微工
研条寄第3459号)を以下単にSK522菌株と称す
る。 Means for Solving the Problems The gist of the present invention which has solved the above problems is as follows: 1) Absolute aerobic, optimum temperature for growth is 72-76 ° C
It grows in a medium having a normal concentration in a temperature range of 82 ° C., a suitable action temperature of 75 to 85 ° C., a action hydrogen ion concentration pH = 4.0 to 1
Thermus aquaticusbiovar SK that produces a thermolytic broad-range action hydrogen ion concentration active protease of 1.3 and a carotenoid yellow pigment
542, located in the FERM BP-3382). Hereinafter, Thermus aquaticus bacteria (Thermus of the present invention
aquaticus biovar SK542, Micro Engineering Lab. No. 3382
No.) relates to the discovery of the present inventor.
It is simply referred to as SK542 strain. The SK542 of the present invention
Aerobic fibrin described below that coexists with the strain and enhances its effect.
Decomposing bacterium Clostridium thermocellum SK522
(Clostriduim thermocellum biovar.SK522, fine work
Kenjo Yori No. 3459) is hereinafter simply referred to as SK522 strain.
It
【0006】SK542菌株の産生するタンパク質分解
酵素の理化学的性質Physicochemical properties of proteolytic enzyme produced by SK542 strain
【表8】 以上のような理化学的性質をもつ新規酵素と思われるプ
ロテアーゼAとプロテアーゼBである。 SK542菌株の産生する黄色色素の理化学的性質 本菌の産生する黄色色素は、カロチノイド系黄色色素
で、水には不溶、次のような溶剤に可溶である。メタ
ノール,エタノール,石油エーテル,ベンジン,
アセトン,クロロホルム,アセン+メタノール
(7:3)及びアセトン+エタノール(1:1)混液。
また、赤外線吸光度最大値450nm。その他430,
435,470nmに小さなピークがある。[Table 8] Protease A and Protease B, which are considered to be novel enzymes having the above physicochemical properties. Physicochemical Properties of Yellow Pigment Produced by SK542 Strain The yellow pigment produced by this strain is a carotenoid yellow pigment which is insoluble in water and soluble in the following solvents. Methanol, ethanol, petroleum ether, benzine,
A mixture of acetone, chloroform, acene + methanol (7: 3) and acetone + ethanol (1: 1).
Also, the infrared absorption maximum value is 450 nm. Other 430,
There is a small peak at 435,470 nm.
【0007】[0007]
【作用】本発明は、有用な新菌SK542菌株を提供す
る。特に本発明SK542菌株は、SK522菌株との
混合培養によって、有用性を高める。SK542菌株と
SK522菌株の共生的混合培養の相乗的効果は、リグ
ニンの顕著なる可溶化の増強と共に繊維素分解力その他
の機能も高揚する。SK542菌株は絶対好気性中等度
好熱性細菌で、高温性広域作用水素イオン濃度活性の強
力なタンパク質分解酵素とカロチノイド系黄色色素を産
生する。 The present invention provides a useful new strain SK542. In particular, the SK542 strain of the present invention is compatible with the SK522 strain.
Increased utility through mixed culture. Synergistic effects of symbiotic mixed culture of S K542 strain and SK522 strains also uplifting fibrinolysis force other features with enhanced remarkably Naru solubilization of lignin. SK542 strains absolute aerobic moderately thermophilic bacteria, that forms produce a powerful protease and carotenoid-based yellow dye thermophilic broad acting hydrogen ion concentration activity.
【0008】そして、この二つの新菌株の好熱的な共生
的混合培養は、各菌株の機能の相加的作用ではなく、相
互作用によって生ずる相乗的効果によるものであり、互
いにその機能を高め、強力かつ安定してリグニン,繊維
素,タンパク質等の有機成分を迅速かつ強力に分解す
る。本発明の混合培養物を有効主成分とすることで有機
資材の発酵分解剤を得ることができる。この有機資材の
発酵分解剤は、その用途・目的・気候・風土・土壌に応
じてSK542菌株,SK522菌株の他に有用な微生
物を混入すると更にその効果を高め、新しい有用な効果
を得ることができる。The thermophilic symbiotic mixed culture of these two new strains is not an additive action of the functions of the respective strains, but a synergistic effect produced by the interaction, and enhances the functions of each other. It strongly and stably decomposes organic components such as lignin, fibrin, and proteins quickly and strongly. By using the mixed culture of the present invention as an effective main component, a fermentative decomposition agent for organic materials can be obtained. This fermentative decomposing agent for organic materials further enhances its effect when a useful microorganism is mixed in addition to the SK542 strain, the SK522 strain depending on the use, purpose, climate, climate, soil, and a new useful effect can be obtained. it can.
【0009】[0009]
【実施例】以下、本発明及びその利用実施例について詳
しく説明する。本発明で使用する微生物は、新菌のSK
542菌株とSK522菌株である。新両菌株の諸性質
は下記の通りである。尚、以下記載の菌学的性質の試験
及び分類同定はすべてバージェーズ マニュアル オブ
デタミネイディブ バクテリオロジー 第7版(19
57),第8版(1974)及びバージェーズ マニュ
アル オブ システマテック バクテリオロジー 第1
巻(1984),第2巻(1986)(Bergey’s Manu
al ofDeterminative Bacteriology 7th. Ed.(1957),8t
h.Ed.(1974).Bergey’s Manualof Systematic Bacterio
logy Vol.1(1984), Vol.2(1986).)の記載に基づいて行
った。EXAMPLES The present invention and examples of use thereof will be described in detail below. The microorganism used in the present invention is a new SK.
542 strain and SK522 strain. Properties of both new strains are as follows. In addition, all the tests and classification and identification of mycological properties described below are carried out by the Burgers Manual of Determinative Bacteriology 7th Edition (19
57), 8th Edition (1974) and Burgers Manual of Systematic Bacteriology No. 1
Volume (1984), Volume 2 (1986) (Bergey's Manu
al of Determinative Bacteriology 7th. Ed. (1957), 8t
h.Ed. (1974) .Bergey's Manualof Systematic Bacterio
logy Vol.1 (1984), Vol.2 (1986).).
【0010】[I]SK542菌株 (Thermus aquaticus biovar SK542,微工研条寄
第3382号,FERM BP−3382) 菌学的性質 形 態 長桿状、0.4〜0.6×3.0〜5.0μ。ある条件
下、例えば培養が古くなると糸状、長さ20〜130
μ。鞭毛なし、室温懸滴標本では運動性がみられない。
内生胞子なし。グラム陰性。[I] SK542 strain (Thermus aquaticus biovar SK542, Mikoken Kenjoyori No. 3382, FERM BP-3382) Mycological properties Long rod-shaped, 0.4-0.6 × 3.0-5 0.0μ. Under certain conditions, for example, when culture becomes old, filamentous, length 20 to 130
μ. No flagella and no motility in room temperature hanging drops.
No endospores. Gram negative.
【0011】培養的性質 増殖が活発。ゼェネレーションタイムは20〜50分。 (1) 平板培養 3%寒天,60℃培養:黄色,比較的緻密,小円形コロ
ニー。 (2) 寒天穿刺培養 表面発育だけ,黄色わずかに拡張。 (3) 液体培養 表面に被膜状に生育(静置培養)。Cultural properties Proliferation is active. Generation time is 20 to 50 minutes. (1) Plate culture 3% agar, 60 ° C culture: yellow, relatively dense, small round colonies. (2) Agar stab culture Only surface growth, yellow slightly expanded. (3) Liquid culture Grows in a film on the surface (static culture).
【0012】生理生化学的性質1.酵素作用等 (1) タンパク質分解 プロテオリティック酵素 :陽性(強力) (proteolytic enzymes) ゼラチン加水分解 :陽性 ペプチダーゼ(peptidase) :陽性 (2) デンプン加水分解 :僅かに陽性 (3) ペクチン分解 :陽性(弱い) (4) 繊維素分解 :陰性 (5) リグニン分解 :陰性 繊維素及びリグニンの分解は共に陰性ではあるが、好熱
性繊維素分解菌SK522菌株との共生的混合培養によ
って、SK522菌株のリグニン可溶化能を顕著に増強
し、同時に繊維素分解力,その他の作用機能にも好影響
を与える。 (6) 脂肪分解 :陰性 (7) インベルターゼ,マルターゼ :陽性 (8) カタラーゼ,オキシダーゼ反応 :陽性 (9) 硫化水素生成 :陽性(弱い) (10) 硫酸還元反応 :陰性 2.生産物試験 (1) 糖,アルコールより生酸及びガス発生 ガス発生せず。グルコース,ガラクトース,マルトー
ス,ラクトース,グリセロールより生酸。 (2) インドール,スカトールの生成 :陰性 (3) 色素生産 黄色色素(カロチノイド系色素,吸光度最大値450n
m,その他430,435,470nmに小さなピーク
がある)を産生する。しかし、グルコース,その他の糖
類を炭素源とする合成培地ではほとんど産生されない。Physiological and biochemical properties 1. Enzyme action, etc. (1) Proteolytic proteolytic enzyme: Positive (strong) (proteolytic enzymes) Gelatin hydrolysis: Positive peptidase: Positive (2) Starch hydrolysis: Slightly positive (3) Pectin degradation: Positive (weak) ) (4) Degradation of fibrin: Negative (5) Degradation of lignin: Negative Degradation of both fibrin and lignin is negative, but lignin digestion of SK522 strain is possible by symbiotic mixed culture with thermophilic fibrinolytic SK522 strain. It significantly enhances the solubilizing ability, and at the same time has a favorable effect on the fibrin decomposing power and other action functions. (6) Lipolysis: Negative (7) Invertase, maltase: Positive (8) Catalase, oxidase reaction: Positive (9) Hydrogen sulfide production: Positive (weak) (10) Sulfate reduction reaction: Negative 2. Product test (1) Raw acid and gas generation from sugar and alcohol No gas generation. A raw acid from glucose, galactose, maltose, lactose, and glycerol. (2) Formation of indole and skatole: Negative (3) Pigment production Yellow pigment (carotenoid pigment, maximum absorbance 450n
m, others with small peaks at 430, 435, 470 nm). However, it is scarcely produced in a synthetic medium containing glucose and other sugars as a carbon source.
【0013】3.生育条件 (1) 培地濃度 サーマス属(genus Thermus)の細菌は一般に有機物濃
度に感受性がある。栄養物は少なく、濃度が低い方がよ
いといわれている。しかし、本菌株はこれらと趣を異に
し、通常の濃度においてもよく生育する。 食塩NaCl 5%以上でも生育する。至適濃度2.
0〜3.0%。一般にサーマス属(genus Thermus)の
細菌は2%以上のNaCl存在下では生育しない。 グルタミン酸ナトリウム3%以上でも生育する。 ショ糖,又はマルトース5%以上でも生育する。 2%ペプトン+1%酵母エキスの存在下でも、その生
育が阻害されない。一般にサーマス属(genus Thermu
s)の細菌はトリプトン,酵母エキス濃度,それぞれ
0.1%前後が適当で、各々1%以上になると生育しな
い。3. Growth conditions (1) Medium concentration Bacteria of the genus Thermus are generally sensitive to organic matter concentrations. It is said that the less nutrients and the lower the concentration, the better. However, this strain is different from these in that it grows well even at normal concentrations. It grows even with salt NaCl 5% or more. Optimal concentration 2.
0-3.0%. In general, Genus Thermus bacteria do not grow in the presence of more than 2% NaCl. It grows even with sodium glutamate of 3% or more. It grows on sucrose or maltose 5% or more. Its growth is not inhibited even in the presence of 2% peptone + 1% yeast extract. Generally the genus Thermu
For the bacteria of s), trypton and yeast extract concentrations of about 0.1% are suitable, and they do not grow when they exceed 1%.
【0014】(2) 生育温度 最適温度は72〜76℃,生育温度範囲40〜82℃,
40℃以下では発育できない。 (3) 水素イオン濃度 最適水素イオン濃度pH=6.0〜10.0。生育水素
イオン濃度範囲pH=4.0〜11.0。 (4) 窒素源 ゼラチン等のタンパク質,グルタミン酸塩,尿素,それ
に無機窒素源としてアンモニウム塩がよく利用される。 (5) 炭素源 グルコース,ショ糖,マルトース,ガラクトース,セロ
ビオース,ラフィノース,スタキオース,デンプン,グ
リセロール,酢酸,ラク酸,リンゴ酸,グルタミン酸塩
を利用する。 (6) 酸素との関係 絶対好気性。 (7) 微量栄養素の要求 本菌株は微量栄養素の要求が高い。ビオチン,ニコチン
酸アミド,チアミン等のビタミン類が要求されるほか
に、本菌株の良好な発育には鉄,マンガン,カルシウム
等の金属イオンを比較的高濃度に要求する。そして、こ
れらのミネラル量にも敏感である。 (8) 抗生物質に対する感受性 一般のサーマス属(genus Thermus)の細菌と同様に、
ペニシリンG,クロラムフェニコール,テトラサイクリ
ン,ストレプトマイシン,カナマイシン,その他抗生物
質に高い感受性を示す。(2) Growth temperature The optimum temperature is 72 to 76 ° C, and the growth temperature range is 40 to 82 ° C.
It cannot grow below 40 ° C. (3) Hydrogen ion concentration Optimal hydrogen ion concentration pH = 6.0 to 10.0. Growth hydrogen ion concentration range pH = 4.0 to 11.0. (4) Nitrogen source Proteins such as gelatin, glutamate, urea, and ammonium salt are often used as an inorganic nitrogen source. (5) Carbon source Glucose, sucrose, maltose, galactose, cellobiose, raffinose, stachyose, starch, glycerol, acetic acid, lactic acid, malic acid, glutamate are used. (6) Relationship with oxygen Absolutely aerobic. (7) Demand for micronutrients This strain has high demands for micronutrients. In addition to vitamins such as biotin, nicotinamide, and thiamine, a relatively high concentration of metal ions such as iron, manganese, and calcium is required for good growth of this strain. It is also sensitive to the amount of these minerals. (8) Susceptibility to antibiotics Like general Thermus genus (genus Thermus) bacteria,
It shows high sensitivity to penicillin G, chloramphenicol, tetracycline, streptomycin, kanamycin and other antibiotics.
【0015】4.DNAのG+Cの含有量 G+Cのmol%=68(Tm)と推定される。4. Content of G + C of DNA mol% of G + C = 68 (Tm) is estimated.
【0016】分類学上の位置 本菌が絶対好気性,生育至適温度72〜76℃,生育温
度範囲40〜82℃,40℃以下では生育しない。無胞
子,グラム陰性の長桿菌であること等から、その他菌学
的諸性質を勘案して、サーマス属(genus Thermus Broc
k and Freege 1967)と同定される。そして、類似する
サーマス属(genus Thermus)の細菌として、 サーマス・アクアティックス(Thermus aquaticus Broc
k and Freege 1969.:Bergey’s Manual(1984)Vol.1,P.3
37.) サーマス・サーモフィルス(Thermus thermophilus Osh
ima and Imahori 1974.:Int.J.Bacteriol.,24,102(197
4)) サーマス・フラブス(Thermus Flavus Saiki,Kimura an
d Arima 1972.:Agr.Biol.Chem.,36,2357(1972)) 等が上げられるが、現在バーゼーズマニアル(Bergey’
s Manual of SystematicBacteriology Vol.1(1984)P.33
3)のサーマス属(genus Thermus Brock and Freeg 196
7)に属する細菌種はサーマス・アクアティックス(The
rmus aquaticusBrock and Freege 1969)だけであり、
しかも下記のような既知の菌種中に見出し得ない特徴的
な性状を有するので、本菌株をサーマス・アクアティッ
クスの新菌株(strain)として、サーマス・アクアティ
クスSK542(Thermus aquaticus biovar SK54
2)と名称するのが妥当であると結論する。Taxonomic position The bacterium does not grow in absolute aerobic condition, optimum growth temperature of 72 to 76 ° C, growth temperature range of 40 to 82 ° C, 40 ° C or lower. Since it is a non-spore-free, Gram-negative long bacillus, etc., in consideration of other mycological properties, the genus Thermus (genus Thermus Broc
k and Freege 1967). And, as a similar bacterium of the genus Thermus, Thermus aquaticus Broc
k and Freege 1969.:Bergey's Manual (1984) Vol.1, P.3
37.) Thermus thermophilus Osh
ima and Imahori 1974.:Int.J.Bacteriol.,24,102(197
4)) Thermus Flavus Saiki, Kimura an
d Arima 1972.:Agr.Biol.Chem.,36,2357(1972)), etc., but currently Bersey's Manual
s Manual of Systematic Bacteriology Vol.1 (1984) P.33
3) Genus Thermus Brock and Freeg 196
Bacterial species belonging to 7) are Thermus aquatics (The
rmus aquaticusBrock and Freege 1969),
Moreover, since it has the characteristic properties that cannot be found in the known bacterial species as described below, this strain is used as a new strain (strain) of Thermus aquaticus SK542 (Thermus aquaticus biovar SK54).
We conclude that it is appropriate to name it 2).
【0017】下 記 (1) 好熱性繊維素分解菌SK522菌株との共生的混合
培養によって、そのリグニン可溶化能を顕著に増強す
る。と同時に繊維素分解力,その他の作用機能にも好影
響を与える。 (2) 広域の作用水素イオン濃度と作用温度を有する強い
タンパク質分解力を持つ。 (3) 有機物濃度の感受性が、既知のサーマス属(genus
Thermus)の細菌と異なり、非常に弱く、通常濃度の培
地においてもよく生育する。 (4) 微量栄養素の要求が強く、各種ビタミン類及び金属
イオンを要求し、それらのミネラル量にも敏感である。 (5) 黄色色素(カロチノイド系色素,吸光度最大値45
0nm,その他430,435,470nmに小さなピ
ークがある)を産生する。(1) The lignin solubilizing ability is remarkably enhanced by the symbiotic mixed culture with the thermophilic fibrinolytic bacterium SK522 strain. At the same time, it has a positive effect on the ability to decompose fibrin and other functions. (2) It has a strong proteolytic activity with a wide range of working hydrogen ion concentration and working temperature. (3) The genus Thermus (genus
(Thermus) bacteria are very weak and grow well in medium of normal concentration. (4) Strong demand for micronutrients, demand for various vitamins and metal ions, and sensitivity to their mineral contents. (5) Yellow pigment (carotenoid pigment, maximum absorbance 45)
0 nm and others have small peaks at 430, 435 and 470 nm).
【0018】微生物受託番号 本菌株の微生物受託番号は、微工研条寄第3382号
(FERMBP−3382)である。SK542菌株と
は、本菌株を標準的菌株とし、サーマス・アクアティッ
クス(Thermus aquaticus Brock and Freege 1969)に
属する細菌種中、好熱性繊維素分解菌(SK522菌
株)との共生的混合培養によって、そのリグニン可溶化
能を顕著に増強し、同時に繊維素分解力にも好影響を与
えることを特徴とし、かつSK542菌株及び自然並び
に人工的変異株を抱括する生理生化学的性状による新菌
株(strain)である。Microbial Accession Number The microorganism accession number of this strain is Microtechnology Research Institute No. 3382 (FERMBP-3382). The SK542 strain is a standard strain of this strain, and by symbiotic mixed culture with a thermophilic fibrinolytic bacterium (SK522 strain) among bacterial species belonging to Thermus aquaticus Brock and Freege 1969, A new strain with a physiological and biochemical property that significantly enhances its lignin solubilizing ability and at the same time has a favorable effect on the fibrin degrading power, and has physiobiochemical properties that embraces the SK542 strain and natural and artificial mutants ( strain).
【0019】SK542菌株のスクリーニング 分離源は九州各地の温泉源及び温泉源付近の土壌,腐植
等である。分離試料を55〜60℃で前培養した後、定
法に従って3%寒天平板培養によって単離する。これに
よって、実質的に純粋に分離できる。 分離用培地組成 (カステンホルツの培地:Castenholz,R.W.;Bacteriol.
Rev.,33,467(1969)) ニトリロ三酢酸 100mg CaSO4・2H2O 60mg MgSO4・7H2O 100mg NaCl 8mg KNO3 103mg NaNO3 689mg Na2HPO4 111mg FeCl3 0.28mg MnSO4・H2O 2.2mg ZnSO4・7H2O 0.5mg H3BO3 0.5mg CuSO4 0.016mg Na2M0O4・2H2O 0.025mg (イーストエキス 5000mg) (トリプトン 5000mg) (ショ糖 10000mg) 全量(純水で、pH=〜8) 1000ml 注:( )内の組成は発明者らが改変。Screening of SK542 strain Isolate sources are hot spring sources in various places in Kyushu, soils near the hot spring sources, humus and the like. The separated sample is precultured at 55 to 60 ° C. and then isolated by 3% agar plating according to a standard method. This allows a substantially pure separation. Separation medium composition (Castenholz medium: Castenholz, RW; Bacteriol.
Rev., 33, 467 (1969)) Nitrilotriacetic acid 100 mg CaSO 4 .2H 2 O 60 mg MgSO 4 .7H 2 O 100 mg NaCl 8 mg KNO 3 103 mg NaNO 3 689 mg Na 2 HPO 4 111 mg FeCl 3 0.28 mg MnSO 4 .H 2 O 2.2 mg ZnSO 4 .7H 2 O 0.5 mg H 3 BO 3 0.5 mg CuSO 4 0.016 mg Na 2 M 0 O 4 .2H 2 O 0.025 mg (yeast extract 5000 mg) (tryptone 5000 mg) (sucrose 10000 mg) ) Total volume (with pure water, pH = ~ 8) 1000 ml Note: The composition in () is modified by the inventors.
【0020】SK542菌株の有用性 本新菌株の有用性は、次の三つの作用機能によって特徴
づけられる。 (1) 好熱性繊維素分解菌(SK522菌株)との共生的
混合培養によって、該菌のもつリグニン分解能を顕著に
増強する。と同時に、その繊維素分解力、その他の作用
機能にも好影響を与える。 (2) 広域の作用水素イオン濃度と作用温度を有する強い
タンパク質分解力をもつ。 (3) 不溶性、カロチノイド系の黄色色素を産生する。菌
体中のこの黄色色素が他の微生物等による分解を受け水
溶性の低分子量体のものとなり、植物体に吸収されて、
果実の味、色沢、貯蔵性等の果実の品質の向上に役立
つ。又、本SK542菌株菌体を施用すると、これを基
質として繁殖する一般の従属栄養微生物や土壌有効菌の
増殖を促す。 以上のようなことが、既知のサーマス属(genus Thermu
s)の細菌と異なり有機物濃度の感受性の非常に弱いこ
とと相俟って、自然生態や農耕生態系において、多種類
の難分解性繊維物質,硬タンパク質や余剰汚泥、その他
広く各種の有機資材を利用できるようになった。更にこ
のことが、生産性向上、土壌病害や連作障害の克服、そ
して、屎尿の脱臭・分解、土壌又は植物葉面に直接撒布
する新技術の開発等、新次元の微生物利用法の発展へと
繋がる。Utility of SK542 Strain The utility of this new strain is characterized by the following three action functions. (1) By symbiotic mixed culture with a thermophilic fibrinolytic bacterium (SK522 strain), the lignin degrading ability of the bacterium is remarkably enhanced. At the same time, it also has a positive effect on its fibrin decomposing power and other action functions. (2) It has a strong proteolytic activity with a wide range of action hydrogen ion concentration and action temperature. (3) It produces an insoluble yellow carotenoid pigment. This yellow pigment in the fungus body is decomposed by other microorganisms and becomes a water-soluble low molecular weight substance, which is absorbed by the plant,
It helps improve the quality of fruits such as taste, color and storability. When the SK542 strain is applied, it promotes the growth of general heterotrophic microorganisms and soil-effective bacteria that propagate using this strain as a substrate. The above is the known genus Thermu (genus Thermu
In combination with the extremely weak sensitivity of organic matter concentration unlike bacteria of (s), in natural ecology and agricultural ecosystem, various kinds of persistent organic substances, hard proteins and surplus sludge, and various other organic materials are widely used. Is now available. Furthermore, this leads to the development of a new dimension of microbial utilization, such as productivity improvement, overcoming soil diseases and continuous crop damage, and deodorizing and decomposing human waste, and developing new technology for direct spraying on soil or plant foliage. Connect.
【0021】[II]SK522菌株 (Clostridium thermocellum biovar SK522,微工
研条寄第3459号,FERM BP−3459) 菌学的性質 随伴菌なしで、単独では生育ができない。しかし、随伴
菌は、容易に単離純粋培養されるので、単離した随伴菌
を基礎とし、これと共生培養しながら、その諸性質を試
験した結果である。 形 態 直桿状、わずかに湾曲するものもある。単独、ときどき
2連。0.3〜0.5×2.2〜4.0μ。培養が古く
なると糸状に伸延し、長連鎖状、長さ5.7〜12.8
μ。周毛、室温懸滴標本では運動性がみられない。末端
に楕円、または円形の胞子を形成して細胞を膨張し棍棒
状。グラム陰性。[II] SK522 strain (Clostridium thermocellum biovar SK522, Mikoken Kenjoyori No. 3459, FERM BP-3459) Mycological properties It cannot grow alone without a concomitant bacterium. However, since the associated bacteria are easily isolated and pure-cultured, it is the result of testing various properties of the isolated associated bacteria while co-culturing them with the isolated associated bacteria as a basis. Shape There are also straight rod shape and slightly curved shape. Alone, sometimes two. 0.3-0.5 × 2.2-4.0 μ. When the culture becomes old, it extends into filaments, long-chain, and lengths of 5.7 to 12.8.
μ. Motility is not observed in pericardium and room temperature hanging drop specimens. Oval or circular spores are formed at the ends to expand the cells and form a club. Gram negative.
【0022】培養的性質 (1) 平板培養 肉汁寒天、その他一般の常用培地による平板培養には生
育しない。しかし、ビルジョンら(Viljoen et al.)の
提示する培地テトロール(Tetrault)円形ろ紙寒天平板
培養に随伴菌と黄色斑点状のコロニーを形成するが、本
菌単独では作り得ない。 (2) 斜面培養,穿刺培養 シュワイツアー(Schweizer)の試薬処理ろ紙添加ビル
ジョンら(Viljoenet al.)培地寒天斜面培養,及び同
穿刺培養ともに生育しない。 (3) バレイショ培養,ゼラチン培養 表面,穿刺とも生育しない。 (4) 液体培養 ビルジョンらの(Viljoen et al.)培地,繊維素肉汁,
繊維素ペプトン水(生育僅少)等の繊維素を含有する培
地だけに生育が認められる。グルコース,キシロース等
の繊維素以外の物質を炭素源とすれば繊維素の分解力を
失う。Culture properties (1) Plate culture It does not grow on plate culture using broth agar or other commonly used medium. However, while the medium presented by Viljoen et al., Tetrault circular filter paper agar plate forms colonies with associated bacteria and yellow spots, it cannot be produced by this bacterium alone. (2) Slope culture and stab culture Addition of Schweizer reagent-treated filter paper to Viljoe et al. Medium Agar slope culture and stab culture do not grow. (3) It does not grow on potato culture, gelatin culture surface, or puncture. (4) Liquid culture Viljoen et al. Medium, fibrin gravy,
Growth is observed only in a medium containing fibrin such as fibrin peptone water (poor growth). If a substance other than fibrin such as glucose or xylose is used as a carbon source, the degrading power of fibrin is lost.
【0023】生理生化学的性質 1.酵素作用等 (1) 繊維素分解 本菌のもつ繊維素分解酵素は、本質的にはいくつかのβ
−1,4−グルカナーゼの複合体である。細胞外に分泌
され、作用温度80℃以上、同水素イオン濃度pH=1
0.0以上の耐熱性,耐アルカリ性の酵素が数種含まれ
ていることを確認した。ミセル構造をなす繊維素の巨大
分子の末端から切断し、グルコース,セロビオース,セ
ロオリゴ糖類等を生成する。これらの酵素を作用する基
質を主体として示すと、 ろ紙を分解する FP−アーゼ:陽性 アビセルを分解する アビセラーゼ:陽性 セロビオースを2分子のグルコースに分解する セロビアーゼ:陽性 (2) リグニン分解 イナワラリグニン可溶化 :陽性 (弱,微弱) (3) タンパク質分解 プロテオリティック酵素(Proteolytic enzymes) :陰性 ペプチダーゼ(peptidase) :陽性 (4) デンプン加水分解テスト :僅かに陽性 (5) ヘミセルロース,キシラン,ペクチンの加水分解テスト :陰性 (6) インベルターゼ,マルターゼ :陽性 (7) 脂肪分解力テスト :陰性 (8) 酸化反応 ハイドロキノン反応 :陽性 チロシン反応 :陰性 (9) 還元作用 :陽性 2.生産物試験 (1) 繊維素発酵 発酵率78〜91%,繊維素を旺盛に発酵してエタノー
ル,メタノール,アセトアルデヒド,酢酸,乳酸,ギ
酸,ラク酸,コハク酸,フマル酸,酒石酸,グルコン
酸,グルコース,セロビオース,セロオリゴ糖類,セロ
デキストリン,多量の炭酸ガス,水素,及び硫化水素等
を生成する。 (2) その他の糖,及びアルコールより生酸 グルコース,ショ糖,マルトース,セロビオースより生
酸。 (3) ガス発生試験 繊維素より猛烈にガス発生するが、グルコース,ショ
糖,マルトース,セロビオースよりガス発生は認められ
ない。 (4) ペプトン水試験 アンモニヤ :僅かに反応あり インドール :陽性 スカトール :陽性 硫化水素 :陽性 (5) 色素生産 通常,カロチノイド黄色色素生産Physiological and biochemical properties 1. Enzymatic action, etc. (1) Fibrinolysis The fibrinolytic enzyme possessed by this bacterium is essentially composed of some β
It is a complex of -1,4-glucanase. Secreted extracellularly, action temperature 80 ℃ or more, same hydrogen ion concentration pH = 1
It was confirmed that several kinds of enzymes having heat resistance and alkali resistance of 0.0 or more were contained. It cleaves from the ends of macromolecules of fibrin that form a micellar structure, producing glucose, cellobiose, cellooligosaccharides, and the like. When the substrates that act on these enzymes are mainly shown, it decomposes filter paper FP-ase: Positive Avicel is decomposed Avicelase: Positive Cellobiose is decomposed into two molecules of glucose Cellobiase: Positive (2) Lignin-decomposed Inawala lignin solubilization : Positive (weak, weak) (3) Proteolytic enzymes: Negative peptidase: Positive (4) Starch hydrolysis test: Slightly positive (5) Hemicellulose, xylan, pectin hydrolysis test : Negative (6) Invertase, Maltase: Positive (7) Lipolysis test: Negative (8) Oxidation reaction Hydroquinone reaction: Positive Tyrosine reaction: Negative (9) Reduction action: Positive 2. Product test (1) Fibrin fermentation 78-91% fermentation rate, fermenting fibrin actively and ethanol, methanol, acetaldehyde, acetic acid, lactic acid, formic acid, lactic acid, succinic acid, fumaric acid, tartaric acid, gluconic acid, It produces glucose, cellobiose, cellooligosaccharides, cellodextrin, large amounts of carbon dioxide, hydrogen, and hydrogen sulfide. (2) Produced acid from other sugars and alcohol Produced acid from glucose, sucrose, maltose, and cellobiose. (3) Gas generation test Although gas is generated more violently than fibrin, no gas generation is observed from glucose, sucrose, maltose, and cellobiose. (4) Peptone water test Ammonia: Slight reaction Indole: Positive skatole: Positive Hydrogen sulfide: Positive (5) Pigment production Usually, carotenoid yellow pigment production
【0024】3.生育条件 (1) 生育温度 至適温度は65〜72℃,温度範囲は40〜80℃,4
0℃以下では生育しない。 (2) 水素イオン濃度 至適水素イオン濃度pH=6.7〜8.0,その範囲は
5.6〜9.6。 (3) 窒素源 ペプトンが最も優れ,尿素,尿酸,アスパラギン,グル
タミン酸ナトリウム等も良好である。アンモニウム塩も
良好な無機窒素源となる。 (4) 炭素源 繊維素以外の炭水化物で継代培養を続けると、その生育
と発酵力を失う。 (5) 酸素との関係 Eh=200〜−250mVと推定され、嫌気性菌であ
る。 (6) 微量栄養素の要求 ビオチン,ピリドキサミン,ビタミンB12,p−アミノ
安息香酸等の微量栄養素を要求する。 4.DNAのG+Cの含有量 G+Cのmol%=38〜40(Tm)と推定される。3. Growth conditions (1) Growth temperature Optimum temperature is 65-72 ° C, temperature range is 40-80 ° C, 4
It does not grow below 0 ° C. (2) Hydrogen ion concentration Optimum hydrogen ion concentration pH = 6.7-8.0, the range is 5.6-9.6. (3) Nitrogen source Peptone is the best, and urea, uric acid, asparagine, sodium glutamate, etc. are also good. Ammonium salts are also good sources of inorganic nitrogen. (4) Carbon source When the subculture is continued with carbohydrates other than fibrin, its growth and fermenting power are lost. (5) Relationship with oxygen Eh = 200 to −250 mV, which is an anaerobic bacterium. (6) Requirement of micronutrients Micronutrients such as biotin, pyridoxamine, vitamin B12 and p-aminobenzoic acid are required. 4. Content of G + C of DNA mol% of G + C = 38-40 (Tm) is estimated.
【0025】分類学上の位置 生育至適温度65〜72℃,温度範囲40〜80℃とい
う高温に於いて、旺盛に繊維素を発酵する本SK522
菌株に類似するものとして、次のような好熱性繊維素分
解菌が上げられる。 クロストリジュウム・サーモセルム(Clostridium ther
mocellum Viljoen, Fred and Peterson,1926.: Jour. A
gr. Sci. (London),16,7(1926).) クロストリジュウム・ディゾルベンス(C. dissolvens
Bergey et al.1925,別名 Bacillus cellulose dessolve
ns Khouvine,1923.: Ann. Inst. Past.37,711(1923);Be
rgey’s Manual,2nd.Ed.(1925)p.344.) クロストリジュウム・セルラシウム(C.thermocellulas
eum Enebo,1951.:Bergey’s Manual,7th.Ed.(1957)p.68
9.) バチルス・サーモセルロリテックス(Bacillus thermoc
ellulolyticus Coolhaas,1928.:Cent Bakt.II,75,101
(1928),76,38(1929).) バチルス・サーモフィブリンコルス(B.thermofibrinco
lus Itano and Arakawa,1929:農化,5,816,921(1929);6,
248,257(1930).) これらの中で、特に次の4つの相違点を除けば形態学
的,培養的,生理生化学的試験において本菌株とよく類
似する細菌はクロストリジュウム・ディゾルベンスであ
る。 (1) 弱又は微弱ではあるが、リグニン可溶化能を有す
る。 (2) 生育適温65〜72℃,生育温度範囲40〜80
℃,40℃以下では生育しない。 (3) 繊維素を旺盛に発酵するが、ヘミセルロース,キシ
ラン,ペクチン等は発酵しない。 (4) リグニン可溶化能はサーマス属(genus Thermus)
のある種の細菌によって共生的に顕著に強調され、同時
に本菌の繊維素分解力,その他作用機能に対して好影響
が与えられる。 しかし現在、バージェーズマニアル(Bergsy’s Manual
of Systematic Bacteriology Vol.2(1986)p.1104,p.11
41.)において、好熱性分解菌として記載されているも
のはクロストリジュウム・サーモセルムのみである。ク
ロストリジュウム・ディゾルベンス以下全部当該菌の亜
種か変種として取り扱われ、又は研究不完全なものとさ
れている。そこで、本菌株の分類同定は、クロストリジ
ュウム・サーモセルムとその菌学的諸性質を比較検討す
ることとし、同時に他の好熱性繊維素分解菌についても
対比し、参考とした。Taxonomy position: SK522 which actively ferments fibrin at a growth optimum temperature of 65 to 72 ° C and a temperature range of 40 to 80 ° C.
The following thermophilic fibrinolytic bacteria are similar to the strains. Clostridium ther
mocellum Viljoen, Fred and Peterson, 1926 .: Jour. A
gr. Sci. (London), 16,7 (1926).) C. dissolvens
Bergey et al. 1925, aka Bacillus cellulose dessolve
ns Khouvine, 1923 .: Ann. Inst. Past.37,711 (1923); Be
rgey's Manual, 2nd.Ed. (1925) p.344.) C. thermocellulas
eum Enebo, 1951.: Bergey's Manual, 7th.Ed. (1957) p.68
9.) Bacillus thermocellolitex
ellulolyticus Coolhaas, 1928.: Cent Bakt.II, 75,101
(1928), 76,38 (1929).) Bacillus thermofibrinco
lus Itano and Arakawa, 1929: Farming, 5,816,921 (1929); 6,
248,257 (1930).) Among these, Clostridium disorbens is a bacterium that is very similar to this strain in morphological, culture, and physiological biochemical tests except for the following four differences. (1) It has a lignin-solubilizing ability although it is weak or weak. (2) Suitable growth temperature of 65 to 72 ° C, growth temperature range of 40 to 80
Does not grow at temperatures below 40 ° C. (3) Actively ferment fibrin, but not hemicellulose, xylan, pectin, etc. (4) Lignin solubilization ability is the genus Thermus
It is prominently symbiotically emphasized by certain types of bacteria, and at the same time has a positive effect on the fibrinolytic activity and other action functions of this bacterium. However, at present, Berges' Manual (Bergsy's Manual
of Systematic Bacteriology Vol.2 (1986) p.1104, p.11
In 41.), only Clostridium thermocellum is described as a thermophilic degrading bacterium. All of them are treated as subspecies or variants of Clostridium dissolvens, or incompletely studied. Therefore, for the classification and identification of this strain, it was decided to compare and examine Clostridium thermocellum and various mycological properties thereof, and at the same time, other thermophilic fibrinolytic bacteria were also compared for reference.
【0026】以上のようにして、菌学的諸性質を比較検
討した結果を総括すれば、本菌株と公知のクロストリジ
ュウム・サーモセルムをはじめ列挙した好熱性繊維素分
解菌との特徴的な性状の相違点として、前記の4項目が
あげられるが、その他各種炭水化物に対する作用,微量
栄養素の要求等,色々と数え上げることができるが、特
に強調したいことはリグニンに対する問題である。本菌
株がリグニン可溶化能をもち、その可溶化能がサーマス
・アクアティックスSK542(Thermus aquaticus bi
ovar SK542)との共生的混合培養によって顕著に
増強されることである。これに対して、クロストリジュ
ウム・サーモセルムをはじめその他の好熱性繊維素分解
菌のリグニンに関する記載(description)は全く見ら
れない。あったとしても、それは繊維素の分解に対する
阻害作用についてである。斯る理由によって、本菌株の
リグニン可溶化能の生理生化学的性質をひとつの根拠と
して新菌株(Strain)とし、クロストリジュウム・サー
モセルムSK522(Clostridium thermocellum biova
r SK522)と名称した。The results of comparative examination of various mycological properties as described above are summarized as follows: characteristic characteristics of this strain and known thermophilic fibrinolytic bacteria including Clostridium thermocellum. The differences are the above-mentioned four items, and they can be enumerated in various ways such as the action on various other carbohydrates, the requirement of micronutrients, etc., but what I would like to emphasize especially is the problem with lignin. This strain has a lignin solubilizing ability, and the solubilizing ability is thermus aquaticus SK542 (Thermus aquaticus bi
It is significantly enhanced by the symbiotic mixed culture with ovar SK542). In contrast, there is no description about lignin of other thermophilic fibrinolytic bacteria including Clostridium thermocellum. If so, it is about its inhibitory effect on fibrin degradation. For this reason, a new strain (Strain) was established based on the physiological and biochemical properties of the lignin-solubilizing ability of this strain as one basis, and Clostridium thermocellum SK522 (Clostridium thermocellum biova
r SK522).
【0027】微生物受託番号 本菌株の微生物受託番号は、微工研条寄第3459号
(FERMBP−3459)である。本菌株を標準的菌
株とし、クロストリジュウム・サーモセルム(Clostrid
ium thermocellum Viljoen, Fred and Peterson,192
6.)に属する菌種中、リグニン可溶化能を有し、サーマ
ス属(genus Thermus Brock and Freeze1969)に属する
ある種の細菌との共生的混合培養によって顕著にリグニ
ン可溶化能を増強することを特徴し、かつSK522菌
株及び自然並びに人工的変異株を抱括する生理生化学的
性状による新菌株である。Microorganism Accession Number The microorganism accession number of this strain is Microtechnology Research Institute No. 3459 (FERMBP-3459). This strain is the standard strain, and Clostridium thermocellum (Clostrid
ium thermocellum Viljoen, Fred and Peterson, 192
It has the ability to solubilize lignin among bacterial species belonging to 6.), and significantly enhances the ability to solubilize lignin by symbiotic mixed culture with certain bacteria belonging to the genus Thermus Brock and Freeze1969. It is a novel strain with physiological and biochemical properties that characterizes the SK522 strain and natural and artificial mutants.
【0028】SK522菌株のスクリーニング 土壌,海浜汚泥,堆・厩肥,人・家畜糞便を分離源とし
て55〜65℃で数回濃縮培養を繰り返した後、ビルジ
ョンら(Viljoen et al.)の提示する培地テトロール
(Tetrault)円形ろ紙寒天平板培養等によって定法通り
分離する。これによって、実質的に純粋に分離できる。
培地としてビルジョンら(Viljoen et al.)の提示する
塩類組成が適当であるが、必要に応じて微量の無機金属
塩類,ビタミン類,生長促進因子,例えば酵母エキス等
を添加するとよい。 ビルジョンらの培地 (Viljoen,Fred and Peterson 1926) ペプトン 5.0g 炭酸カルシウム 過剰 リン酸アンモニウムナトリウム 2.0g 酸性リン酸カリウム 1.0g 硫酸マグネシウム 0.3g 塩化カルシウム 0.1g 塩化第二鉄 痕跡 繊維素(ろ紙) 15.0g 井水 1000mlScreening of SK522 strain Soil, beach sludge, manure / manure, human / livestock faeces are used as separation sources, and concentrated culture is repeated several times at 55 to 65 ° C., then presented by Viljoen et al. Medium Tetrault circular filter paper Agar plates are used for isolation according to standard methods. This allows a substantially pure separation.
The salt composition suggested by Viljoen et al. Is suitable as the medium, but if necessary, a trace amount of inorganic metal salts, vitamins, growth promoting factors such as yeast extract, etc. may be added. Viljoen, Fred and Peterson 1926 Peptone 5.0 g Calcium carbonate Excess ammonium phosphate sodium 2.0 g Potassium acid phosphate 1.0 g Magnesium sulfate 0.3 g Calcium chloride 0.1 g Ferric chloride Trace fiber Element (filter paper) 15.0 g Imizu 1000 ml
【0029】SK522菌株の有用性 本SK522菌株は、もともと旺盛なる繊維素分解菌で
ある。この菌株が弱又は微弱ではあるが、リグニン可溶
化能を有することを本発明者たちが見出し、更にその可
溶化能が本菌株とサーマス属(genus Thermus)のある
種の細菌との共生的混合培養によって顕著に増強される
と共に、本菌株の繊維素分解力、その他作用機能にも好
影響を与えた。又、この混合培養の成功は、多種類の難
分解性有機資材の分解利用を可能にして、その生産性を
向上させ、野積堆肥化法を確実なものにし、好熱性高速
分解方式による堆肥の製造法を確立した。そして、SK
522菌株とSK542菌株を主要菌とする健全活性あ
る土壌微生物フロラーの形成は、環境要因の変化に対し
て有効菌の生育と作用機能を安定させて、土壌病害や連
作障害の消滅,克服した。又、土壌や植物の葉面に直接
撒布する新技術や屎尿の脱臭分解,硬タンパク質の分解
等、新しい次元の微生物利用法の展開が期待される。Utility of SK522 Strain The present SK522 strain is originally a vigorous fibrinolytic bacterium. The present inventors have found that this strain has a lignin solubilizing ability, although this strain is weak or weak, and the solubilizing ability of the strain is symbiotic mixing of this strain with a certain bacterium of the genus Thermus (genus Thermus). It was remarkably enhanced by culturing, and also had a favorable effect on the fibrinolytic activity and other action functions of this strain. In addition, the success of this mixed culturing enables the decomposition and utilization of many types of persistent organic materials, improves their productivity, and ensures the nodoko composting method. The manufacturing method was established. And SK
The formation of a healthy active soil microbial flora mainly consisting of the 522 strain and the SK542 strain stabilized the growth and action function of effective bacteria against changes in environmental factors, and eliminated or overcame soil diseases and continuous cropping disorders. In addition, it is expected that new technology of microbial utilization such as new technology of direct spraying on soil or leaf surface of plant and deodorization of human waste and decomposition of hard protein will be developed.
【0030】[III]SK522菌株とSK542菌
株の混合培養物 1.SK522菌株の多量培養物 前記ビルジョンら(Viljoen et al.,1926)の培地を用
い嫌気または半嫌気的条件下で、65〜70℃,48〜
60時間培養し、その培養液をもって菌体含有の培養物
とする。 2.SK542菌株の多量培養物 前記カステンホルツ(Castenholz,1969)の培地を用
い、通気または振盪等の好気的条件下で、70〜75
℃,24時間培養し、その培養液をもって培養物とす
る。[III] Mixed culture of SK522 strain and SK542 strain 1. Large-scale culture of SK522 strain Using the medium of Viljoen et al., 1926 described above, under anaerobic or semi-anaerobic conditions, 65-70 ° C, 48-
After culturing for 60 hours, the culture solution is used as a culture containing cells. 2. Large-scale culture of SK542 strain Using the culture medium of Castenholz (1969), 70-75 under aerobic conditions such as aeration or shaking
Cultivate at 24 ° C for 24 hours, and use the culture solution as a culture.
【0031】3.SK522菌株とSK542菌株の混
合培養物 下記の培地を用い、SK522菌株及びSK542菌株
の多量培養物の適量をそれぞれ接種して、65〜70
℃,24〜36時間培養して、両菌株の混合培養物とす
る。そして、これを本発明の提示する賦型剤を用いて粉
粒体となし、水分を5〜7%以下の製品にすると2ケ年
以上の保存に耐える。又、施用直前に両菌株の多量培養
物を等量にとってよく混和して、すぐに施用するか、或
いは賦型剤を用いて粉粒体に製造しても、その最終製品
の品質性能は全くかわらない。又、下記の共生混合培養
用E−培地を以下単にE−培地と称する。 下 記 共生混合培養用 E−培地 K2HPO4 5kg (NH4)2SO4 2kg 尿素 2kg ペプトン 5kg 酵母エキス 5kg ろ紙(繊維素) 15kg CaCO3 過剰 水(〜pH7.0) 1000l 4.両菌株の共生的混合培養のもたらす有用性 共に高温環境という条件下で、絶対好気性のサーマス属
(genus Thermus)のSK542菌株との共生的混合培
養は、嫌気性の好熱性繊維素分解菌SK522菌株単独
ではほとんど不可能な天然の難分解性有機資材の分解発
酵を顕著に高揚する。まず、SK542菌株の増殖はS
K522菌株の欠除するタンパク分解活性を充補する。
しかも、堆・厩肥をはじめ廃木材、都市下水汚泥等の難
分解性有機資材の分解発酵は、すべて固体発酵の状態
で、かなり通気のよい環境下におかれている。そして、
初期の段階におけるSK542菌株の増殖は高温嫌気的
環境へと変移せしめ、これによってSK522菌株の自
然界での生育を可能となる。3. Mixed culture of SK522 strain and SK542 strain Using the following medium, appropriate amounts of large-scale cultures of the SK522 strain and the SK542 strain were inoculated, respectively, to obtain 65-70.
Cultivate at 24 ° C for 36 to 36 hours to obtain a mixed culture of both strains. Then, this is made into a granular material by using the excipients of the present invention, and a product having a water content of 5 to 7% or less can be stored for 2 years or more. Further, a large amount cultures of both strains immediately before application and mixed well for equal amounts, either immediately applied, or be prepared in powder or granular material with a shaping agent, quality performance of the final product are quite Unchanged. Further, the following E-medium for mixed culture is simply referred to as E-medium. E-medium for co-cultivation mixed culture K2HPO4 5kg (NH4) 2SO4 2kg Urea 2kg Peptone 5kg Yeast extract 5kg Filter paper (fibrin) 15kg CaCO3 excess water (~ pH 7.0) 1000l 4. Utility of symbiotic mixed culture of both strains Under the condition of high temperature environment, symbiotic mixed culture with SK542 strain of absolutely aerobic Genus Thermus is anaerobic thermophilic fibrinolytic bacterium SK522. It remarkably enhances the decomposition and fermentation of natural refractory organic materials, which is almost impossible with the strain alone. First, the growth of the SK542 strain is S
It complements the proteolytic activity of the K522 strain.
Moreover, the decomposition and fermentation of persistent organic materials such as waste wood, manure, waste wood, and municipal sewage sludge are all solid-state fermentations, and are in a well-ventilated environment. And
The growth of the SK542 strain in the early stages is transformed into a high temperature anaerobic environment, which allows the SK522 strain to grow in nature.
【0032】(1) リグニンの可溶化 ここで、特に注目されることは、SK542菌株によっ
てSK522菌株のリグニン可溶化能が顕著に発揮され
ることです。難分解性有機資材は、実際には繊維素がリ
グニン、その他の有機成分と強く結合した状態で存在す
る。こうした天然のリグノセルロースは繊維素分解力の
旺盛なSK522菌株の単独施用ではほとんど分解が不
可能である。リグニンの化学構造は、未だ完全に明らか
にされていないが、ベンゼン環に炭素数3つの側鎖を持
つフエニルプロパンが基本単位になって、この単位体が
パーオキシダーゼによって触媒されるラジカル反応によ
りランダムに三次元的に重合した高分子化合物である。
微生物の分解に対する抵抗がきわめて強く、きわめて分
解困難であることは多くの研究者・技術者の認める一致
した見解である。ところが、本願発明で使用するSK5
22菌株は弱又は微弱ではあるが、リグニンを可溶化す
る。そして、このリグニン可溶化能が、SK542菌株
と共生的混合培養することによって、たとえば、イナワ
ラリグニンの50%以上が10日前後の短日時において
可溶化されるということが、今回、本願発明において、
はじめて明らかにされた。その実際的な施用が本願発明
の重要なる特徴的態様で、その作用効果は50〜80℃
以上という高温期段階で発揮され、分解発酵へその腐植
化が急速に進行する。(1) Solubilization of lignin Here, it is particularly noted that the SK542 strain exerts a remarkable lignin-solubilizing ability of the SK522 strain. The persistent organic material actually exists in a state in which the fibrin is strongly bound to lignin and other organic components. Such natural lignocellulose can hardly be decomposed by the single application of the SK522 strain having a strong fibrinolytic activity. Although the chemical structure of lignin has not been completely clarified yet, phenylpropane, which has a side chain with three carbon atoms in the benzene ring, serves as the basic unit, and this unit is formed by a radical reaction catalyzed by peroxidase. It is a polymer compound that is randomly and three-dimensionally polymerized.
It is a consensus view of many researchers and engineers that resistance to microbial decomposition is extremely strong and extremely difficult to decompose. However, SK5 used in the present invention
Twenty-two strains solubilize lignin, though weakly or weakly. And, this lignin solubilizing ability, by symbiotic mixed culture with the SK542 strain, for example, that 50% or more of Inawara lignin is solubilized in a short time around 10 days, this time, in the present invention,
It was revealed for the first time. Its practical application is an important characteristic aspect of the present invention, and its action and effect are 50 to 80 ° C.
It is exerted at the high temperature stage mentioned above, and the humification progresses rapidly to the decomposition fermentation.
【0033】(2) 黄色色素の産生 SK542菌株をはじめ、サーマス属(genus Thermu
s)の産生する黄色色素はカロチノイド系不溶性色素で
ある。菌体中のこの黄色色素が他の微生物等による分解
を受け水溶性の低分子量体のものとなり、植物体に吸収
され、必要部位に移行し、丁度都合の良い前駆物質とな
る。すなわち、このような黄色色素が、ほかの溶菌した
細胞内容物や分泌物、それに発酵生産物等とともに根茎
葉部位の増大繁茂を促すだけでなく、花芽の形成,着
果,果実の肥大等の生殖生長の代謝系に深く関与してい
ることが分子生物学的レベルで行なわれた研究によって
も明らかにされ、その応用利用へと発展し、すでに果実
の味,色沢,貯蔵性等品質向上に役立っている。(2) Production of yellow pigments genus Thermu (genus Thermu) including SK542 strain
The yellow pigment produced by s) is a carotenoid insoluble pigment. This yellow pigment in the bacterial cells is decomposed by other microorganisms to become a water-soluble low molecular weight substance, which is absorbed by the plant body and transferred to a necessary site, which is just a convenient precursor. That is, such a yellow pigment not only promotes the growth and proliferation of rhizomes along with other lysed cell contents and secretions, and fermentation products, but also causes the formation of flower buds, fruit set, fruit enlargement, etc. It has been clarified by research conducted at the molecular biological level that it is deeply involved in the metabolic system of reproductive growth, and it has been developed into its application and has already been improved in quality such as fruit taste, color and storage quality. Is useful to.
【0034】(3) 微生物生態系の混合複合化 また、この黄色色素を含むSK542菌株の菌体を施用
すると、それを基質として繁殖する一般の従属栄養微生
物,土壌有効菌等の増殖を促す。さらに重要なことは、
自然界の堆肥製造のような現場では、SK522菌株の
ような好熱性微生物が、すぐに増殖活性化するわけでは
ない。当初は、一般の常温性従属栄養微生物が増殖し、
これらの作用による発酵熱が蓄積されて品温の上昇に役
立つものであり、単一菌株の好熱性繊維素分解菌や少数
の微生物のみが、天然の難分解性有機資材の腐植化に関
与するわけではない。微生物フロラーの混合複合化多様
化が求められる。さまざまなタイプの有効菌が多く、さ
らに「エサ(基質)」も適度にあるという複合的内容が
望ましい。こうして、難分解性有機物の高温分解は、S
K522菌株を中心とする細菌フロラーがまず形成さ
れ、その腐植化過程が単純な構成成分の変化だけでな
く、きわめて複雑な微生物フロラーの相互作用やその変
遷等が、深い関わりをもって最も効率よく進行するので
ある。SK522菌株とSK542菌株の他の有効菌と
しては下記のものがある。(3) Mixed Complexation of Microbial Ecosystem Further, when the bacterial cells of the SK542 strain containing this yellow pigment are applied, the growth of general heterotrophic microorganisms, soil effective bacteria and the like that propagate using the same as substrates is promoted. More importantly,
In the field such as compost production in the natural world, thermophilic microorganisms such as the SK522 strain do not immediately proliferate and activate. Initially, general room temperature heterotrophic microorganisms grow,
Fermentation heat due to these actions is accumulated to help increase product temperature, and only a single strain of thermophilic fibrinolytic bacteria and a small number of microorganisms are involved in the humification of natural persistent organic materials. Do not mean. It is required to mix and diversify the microbial flora. It is desirable to have a complex content that there are many effective bacteria of various types and that "feed (substrate)" is also appropriate. Thus, the high-temperature decomposition of persistent organic matter is
The bacterial flora centering on the K522 strain is formed first, and the humification process not only involves simple changes in the constituent components, but also the extremely complex interactions and transitions of the microbial flora, which are most efficiently and closely related. Of. Other effective bacteria S K522 strain and SK542 strains are those below.
【0035】(1) ヘミセルロースの分解菌の培養物 ヘミセルロースは繊維素とともに植物体(細胞壁)を形
成し、これを構成する糖類によって、キシラン,アラバ
ン,デキストラン,マンナン,ガラクタン等と称せられ
る。ヘミセルロース分解菌の培養物は、イナワラキシラ
ン約1%の濃度に加えた岩田の培地(1936)を使用
し、30〜38℃,通性嫌気的に集殖する。通常、バク
テリウム・ブルガトゥス(Bacterium vulgatus),バク
テリウム・プロディギオサム(Bac. prodigiosum),バ
クテリウム・メセンテリクスルバー(Bac. mesentericu
s ruber),ミクロスピラ・アガーリクェフィセンス(M
icrospira ager-liquefaciens)等の1株又は2株以上
の混合培養物が獲得される。 (2) ペクチン物質分解菌の培養物 ペクチン物質を強力に分解する細菌は、好気性のもので
は枯草菌群細菌及びエタノール・アセトン菌に、又嫌気
性ではラク酸菌に属するものが多い。本発明では、モリ
シュの培地(Molisch1939)を用い、土壌,堆肥,
馬糞,バガスやチョ麻等の腐敗物を分離源として、27
〜35℃,培養日数3〜5日,厚層及び薄層で数回の集
殖培養で種菌を獲得する。本発明では、その1株以上の
数株の培養物を用いる。(1) Culture of Hemicellulose Degrading Bacteria Hemicellulose forms a plant (cell wall) together with fibrin, and is called xylan, araban, dextran, mannan, galactan, etc. depending on the sugars constituting the plant. As a culture of hemicellulose-decomposing bacteria, Iwata's medium (1936) added to a concentration of rice xylan about 1% was used, and the cells were facultatively and anaerobically collected at 30 to 38 ° C. Usually Bacterium vulgatus, Bac. Prodigiosum, Bacteria mesentericu
s ruber), Microspyra Agariquefisense (M
A mixed culture of one or more strains such as icrospira ager-liquefaciens) is obtained. (2) Cultures of pectin substance-degrading bacteria Many of the bacteria that strongly decompose pectin substances belong to the Bacillus subtilis group bacteria and ethanol / acetone bacteria when they are aerobic, and to the lactic acid bacteria when they are anaerobic. In the present invention, using a Morish medium (Molisch 1939), soil, compost,
Separation sources such as horse manure, bagasse and chomp are 27
The inoculum is obtained by collecting culture several times in a thick layer and a thin layer at ˜35 ° C. for 3 to 5 days of culturing. In the present invention, cultures of one or more strains are used.
【0036】(3) 土壌放線菌の培養物 放線菌(Actinomycetales Buchanan,1917)の土壌
中の働きについて一般的に言うことが難しい。しかし、
各種の有機性物質、特に難分解性の繊維素,リグニン等
を他の微生物とともに分解し、土壌肥沃のもとになる腐
植の生成に重要な働きをしており、又抗生物質の産生を
通してのミクロフローラ・コントロール面で重要な意義
をもつことは確かである。放線菌の培養は、ワックスマ
ンの培地(Waksman1919),分離源に肥沃な土壌,
又堆・厩肥を用い強力菌を集殖する。 (4) 土壌糸状菌及び酵母の培養物 土壌糸状菌の最も多く存在する場所は、細菌,放線菌と
同様土壌で、土壌中の糸状菌は当然植物根のある耕作土
に多く、特に根圏ではその働きも活発である。植物遺体
等の有機性物質の分解にあずかり、土壌の肥沃度に関係
する。糸状菌は主として分解の初期段階で活躍している
と考えられる。次に土壌酵母の働きについては不明の点
が多い。しかし、土壌中には相当数の酵母菌が存在し、
かつその含有する豊富なビタミン類や生育因子をめぐっ
て他微生物との共存共棲や土壌活性等に影響のあること
は確かである。土壌糸状菌や酵母の培養物は、ツアペッ
ク・ドックスの培地(Czapek & Dox,1910)を用
い、土壌或いは堆・厩肥より分離,培養する。(3) Soil actinomycete culture It is generally difficult to say about the action of actinomycetes (Actinomycetales Buchanan, 1917) in soil. But,
It decomposes various organic substances, especially incombustible fibrin and lignin, together with other microorganisms, and plays an important role in the formation of humus, which is the source of soil fertility. Certainly, it has important significance in terms of microflora control. The culture of actinomycetes is Waxman's medium (Waksman 1919), fertilized soil as a separation source,
In addition, strong bacteria are collected by using the manure and manure. (4) Cultures of soil filamentous fungi and yeasts The most abundant locations of soil filamentous fungi are soils like bacteria and actinomycetes, and filamentous fungi in soils are naturally abundant in cultivated soil with plant roots, especially rhizosphere. Then, the work is also active. It is involved in the decomposition of organic substances such as plant remains and is related to soil fertility. It is considered that filamentous fungi are mainly active in the initial stage of decomposition. Next, there are many unclear points about the function of soil yeast. However, there are quite a few yeasts in the soil,
In addition, it is certain that the abundant vitamins and growth factors contained therein will affect coexistence with other microorganisms and soil activity. Soil filamentous fungi and yeast cultures are separated and cultured from soil or manure / manure using a Tuapeck Dox medium (Czapek & Dox, 1910).
【0037】(5) 好熱性バチルス属の培養物 一般に好気性,運動性,内生胞子を有する桿菌で、土
壌,葉面,枯草等自然界に広く分布する一群の細菌で、
ほとんどの菌株が強い熱抵抗性をもち、55℃以上でも
生育できるものが多いことから堆肥製造中の主要な微生
物であるという報告もある。又、バシトラシンやバシリ
シン等の抗菌物質を分泌することが、近年土壌植物病理
学の分野で注目されている。このようなことから、本菌
群が作物生産に利用しようとする試みが可能で、肥沃な
土壌や堆・厩肥等の懸濁液を80℃,10分間,加熱処
理して分離源とする。ワックスマンの培地(Waksman,19
22)を用い、50〜60℃,好気的に本菌群を集殖す
る。本発明では、これらの菌の単独又は混合培養を用い
る。 (6) 黄色色素産生菌の培養物 細菌の産生する黄色色素はカロチノイド系の色素であ
る。特に完熟堆肥中に数多く存在し、種類も多い。また
植物葉面にもよく存在する。これらを分離源とし、通常
の肉エキス培地又はペプトン・酵母エキス培地を用い、
25〜35℃,好気的条件下で培養,分離する。黄緑
色,黄色,黄褐色,紅色等の呈色によって容易に識別さ
れ、一般にフラボバクテリウム(Flavobacterium),ク
ロモバクテリウム(Chromobacterium),シュドモナス
(Pseudomonas),セラテラ(Serratia),光合成細菌
(Phototrophic bacteria)等に属する1株又は2株以
上の培養物が獲得される。(5) Culture of thermophilic Bacillus genus Bacillus which is generally aerobic, motile, and has endospores, and is a group of bacteria widely distributed in nature such as soil, leaf surface, and hay.
It is also reported that most of the bacterial strains have strong heat resistance and can grow even at 55 ° C. or higher, and thus are major microorganisms during compost production. In addition, secreting antibacterial substances such as bacitracin and basilicin has attracted attention in the field of soil plant pathology in recent years. From these reasons, it is possible to try to utilize this bacterial group for crop production, and fertilized soil or a suspension of manure / manure is heat treated at 80 ° C. for 10 minutes to be used as a separation source. Waxman's medium (Waksman, 19
22), and aerobically collect the bacterial group at 50 to 60 ° C. In the present invention, single or mixed culture of these bacteria is used. (6) Culture of yellow pigment-producing bacteria Yellow pigment produced by bacteria is a carotenoid pigment. There are many types of ripe compost, and many types. It is also often found on the leaves of plants. Using these as a separation source, a normal meat extract medium or peptone-yeast extract medium,
Incubate and separate at 25-35 ° C under aerobic conditions. It is easily identified by colors such as yellow-green, yellow, yellow-brown, and red, and is generally flavobacterium (Flavobacterium), chromobacterium (Chromobacterium), pseudomonas (Pseudomonas), serratia (Serratia), phototrophic bacteria (Phototrophic bacteria) A culture of one or more strains belonging to, etc. is obtained.
【0038】[IV]SK522菌株とSK542菌株
の共生的混合培養物の応用例 応用例1.リグニン可溶化法の応用例 1.種菌:SK522菌株とSK542菌株共生的多量
混合培養物 SK522菌株の多量培養物 SK542菌株の多量培養物 各培養物いずれも本明細書前記の通りである。 2.試料:試験に使用した原材料はイナワラ(粳)で、
その分析値は表1の通りである。[IV] Application Example of Symbiotic Mixed Culture of SK522 Strain and SK542 Strain Application Example 1. Application example of lignin solubilization method 1. Inoculum: SK522 strain and SK542 strain symbiotic high-volume mixed culture Large-scale culture of SK522 strain Large-scale culture of SK542 strain Each culture is as described above in the present specification. 2. Specimen: The raw material used for the test is rice straw (rice cake),
The analysis values are shown in Table 1.
【表1】 3.試験方法 1000ml三角フラスコに風乾試料10.0g,E−培
地700ml(ろ紙を除いたもの)注加、常法通り加圧殺
菌後、適量の種菌を接種,65℃,20日間培養。蒸発
欠減を補い、分析に供する。4.結果の表示[Table 1] 3. Test method 10.0 g Erlenmeyer flask was injected with 10.0 g of air-dried sample, 700 ml of E-medium (without filter paper), sterilized under pressure as usual, and inoculated with an appropriate amount of inoculum, and cultured at 65 ° C for 20 days. Evaporation depletion is compensated and used for analysis. 4. Display results
【数1】 [Equation 1]
【数2】 [Equation 2]
【数3】 5.試験結果[Equation 3] 5. Test results
【表2】 表2を見ても明らかなように、この522菌株と542
菌株の二つの新株の好熱的な共生的混合培養は、それぞ
れの機能の相加的作用だけでなく、相互作用による相乗
的効果によって互いにその機能を高め、イナワラのよう
な天然の難分解性繊維物質を10〜12日という短日時
で、リグニンの50%以上を可溶化し、繊維素は90%
以上、タンバク質は70%以上を強力かつ安定して発酵
分解している。522菌株,542菌株それぞれ単独培
養とは、顕著な差異がある。[Table 2] As is clear from Table 2, this 522 strain and 542 strain
Thermophilic symbiotic mixed culture of two new strains enhances each other's functions not only by the additive effects of their respective functions but also by the synergistic effect of the interaction, which makes them naturally persistent such as rice straw. In a short time of 10 to 12 days, fibrous material solubilizes 50% or more of lignin and 90% fibrin.
As described above, 70% or more of the protein is fermented and decomposed strongly and stably. There are remarkable differences from the individual cultures of the 522 strain and the 542 strain, respectively.
【0039】応用例2.有機資材の発酵分解剤によるノ
コクズの発酵 1.発酵分解剤:SK522菌株とSK542菌株との
共生的混合培養物に酵母エキス10%(wt/vo
l),ペプトン10%(wt/vol)を添加して発酵
分解剤とする。 2.試料:ノコクズ。樹種杉,樹齢17〜20年,木質
部を縦引にし、粒子の大きさ3.0〜1.0mm。Application Example 2. Fermentation of sawdust by fermentation decomposer of organic materials 1. Fermentative decomposition agent: yeast extract 10% (wt / vo) in a symbiotic mixed culture of SK522 strain and SK542 strain.
l) and peptone 10% (wt / vol) are added to make a fermentation decomposer. 2. Sample: sawdust. Cedar tree, 17 to 20 years old, the wood part is pulled vertically, and the particle size is 3.0 to 1.0 mm.
【表3】 3.試験方法及び結果の表示 1000ml三角フラスコに風乾試料10g,E−培地7
00ml(除ろ紙,本明細書第19頁参照)注加,常法通
り加圧殺菌後、適量の発酵分解剤を接種,65℃,20
日間培養。蒸発欠減を補い、分析に供する。その結果に
より、応用例1.と同様にしてリグニン可溶化率,繊維
素発酵率,タンパク質分解率を算出する(本明細書第2
4頁参照)。 4.試験結果 試験結果は表4に示される通りで、[Table 3] 3. Display of test method and results 10 g of air-dried sample, 1000 ml Erlenmeyer flask, E-medium 7
Add 00 ml (filter paper, see page 19 of this specification), sterilize under pressure according to the usual method, and inoculate with an appropriate amount of fermentation decomposition agent, 65 ° C, 20
Day culture. Evaporation depletion is compensated and used for analysis. According to the result, the application example 1. The lignin solubilization rate, the fibrin fermentation rate, and the protein decomposition rate are calculated in the same manner as in (2nd in this specification).
(See page 4). 4. Test results The test results are shown in Table 4,
【表4】 二つの新菌株SK522菌株とSK542菌株の共生的
混合培養を主成分とする有機資材の発酵分解剤が、ノコ
クズのような天然の難分解性繊維物質を10〜12日前
後で、リグニンの50%以上を可溶化し、繊維素及びタ
ンパク質もSK522菌株単独の場合と同様か、それ以
上に強力かつ安定して発酵分解している。[Table 4] Fermentative decomposing agent for organic materials whose main component is symbiotic mixed culture of two new strains, SK522 strain and SK542 strain, uses natural refractory fiber material such as sawdust for about 10 to 12 days and 50% of lignin. The above was solubilized, and the fibrin and protein were fermented and decomposed similarly to the case of the SK522 strain alone, or more strongly and stably.
【0040】応用例3.粉粒状の発酵分解剤と野積堆肥
化法 1.発酵分解剤の製造 そして、本発明の粉粒状の発酵分解剤の製造は、SK5
22菌株とSK542菌株の多量培養物とともに、微量
栄養素と微量ミネラルのある種のものを選択して、石灰
岩岩粉等の賦型剤に添加し、よく攪拌混和して製造基準
に制定された形状の発酵分解剤とする。原材料の配合の
一例は下記の通りで、粉状の製品とする。もし、必要が
あれば、つなぎ剤等を加え顆粒状とすることもできる。 原材料の配合割合 (賦型剤:石灰岩岩粉1000kgに対して) SK522菌株の多量培養物 2kg SK542菌株の多量培養物 2kg ビオチン 1g ビタミンB12 1g シスチン 5g メチオニン 10g マンガン 10g コバルト 1g イーストエキス 100g けいそう土 50kg 2.堆肥の製造 スギ材のバークを主原料として、粉状の本発酵分解剤を
使用し、従来の野積堆肥化法によって堆肥を製造し、そ
の施用効果をみた。 (1) バークの成分組成Application Example 3. Fermented decomposing agent in the form of powder and Noduki composting method Manufacture of Fermentation Decomposition Agent And, the manufacture of the powdery fermentation decomposition agent of the present invention is SK5
Along with the large-scale cultures of 22 strains and SK542 strains, some kinds of micronutrients and trace minerals were selected, added to the excipient such as limestone rock powder, and well mixed by stirring to obtain the shape established in the manufacturing standard. It is used as a fermentative decomposition agent. An example of the blending of raw materials is as follows, and it is a powdery product. If necessary, a binder or the like may be added to form a granule. Mixing ratio of raw materials (vehicle: based on 1000 kg of limestone rock powder) Large-scale culture of SK522 strain 2 kg Large-scale culture of SK542 strain 2 kg Biotin 1 g Vitamin B 12 1 g Cystine 5 g Methionine 10 g Manganese 10 g Cobalt 1 g Yeast extract 100 g Soil 50kg 2. Manufacture of compost Using bark made of cedar wood as the main raw material and a powdery main fermentation decomposing agent, compost was manufactured by the conventional Nozomi composting method, and its application effect was observed. (1) Composition of bark
【表5】 (2) 原材料の配合 バーク(選別篩の目20mm) 1000kg 米ヌカ 250kg 硫安 2kg 過リン酸石炭(第2回切り返し時に添加) 20kg 本発酵分解剤 60kg 水分 約60% (3) 堆肥の積込み バーク粉砕物1000kgを使用し、本発酵分解剤,そ
の他をよく混合しながら水分が約60%になるように給
水し、180×180cmの木枠の中に積込み、上部を
ビニールシートで覆った。 (4) 製造経過 積込み作業は5月10日に実施し、切り返し3回、2回
目の切り返し時に過リン酸石灰20kgを添加し、7月
10日終了する。品温の上昇は速かで、積込み後5日目
で61℃に達し、爾後60℃以上を継続する。2回目の
切り返し後最高温72℃に達し、暫時次第に品温が低下
し、後熟期にはいった。7月10日までの積算温度の算
出をすると4,383℃・日となり、単純に平均する
と、従来の場合と比較して、1日当り2.7〜3.2℃
の温度上昇を示している。また、切り返し時における色
の変化をマンセルの色彩図表でみると、表6の通りであ
った。[Table 5] (2) Blending of raw materials Burk (20 mm mesh size) 1000 kg Rice bran 250 kg Ammonium sulphate 2 kg Coal superphosphate (added during the second turning) 20 kg Main fermentation decomposer 60 kg Moisture approx. 60% (3) Compost loading Burk crushed Using 1000 kg of the product, water was supplied so that the water content was about 60% while thoroughly mixing the main fermentation decomposing agent and the like, the product was loaded into a wooden frame of 180 × 180 cm, and the upper part was covered with a vinyl sheet. (4) Manufacturing progress The loading operation will be carried out on May 10, and 20kg of superphosphate will be added at the time of turning back three times and the time of turning the second time. The product temperature rises quickly, reaching 61 ° C on the 5th day after loading, and continuing to be 60 ° C or higher after that. After the second turning, the maximum temperature reached 72 ° C, the product temperature gradually decreased, and it entered the post-ripening period. When the integrated temperature up to July 10 is calculated, it will be 4,383 ° C / day. Simply averaging, it will be 2.7 to 3.2 ° C per day compared to the conventional case.
Shows an increase in temperature. In addition, the change in color at the time of switching back is as shown in Table 6 when viewed in the Munsell color diagram.
【表6】 (5) 結果 結果は表7に示される通りである。従来3〜6ケ月以上
かかって製造されていたバーク堆肥が60日で可能とな
った。また、60日間の積算温度4,383℃・日、従
来の場合と比較して、1日当り2.7〜3.2℃の品温
上昇を示すことになり、熟成に伴う色彩の変化も好結果
を示している。以上のようにして、バーク堆肥化に本発
酵分解剤の腐熟効果が充分にあったと考えられる。この
ことは最終的に製品の分析結果によってもよくわかる。
いずれの分析値も日本バーク堆肥協会及び全国バーク堆
肥工業会の統一基準値を上まわる優れた結果を示してい
る。[Table 6] (5) Results The results are shown in Table 7. Bark compost, which used to take 3 to 6 months or more to manufacture, can now be done in 60 days. In addition, the accumulated temperature for 60 days is 4,383 ° C / day, which means that the product temperature rises by 2.7 to 3.2 ° C per day compared to the conventional case, and the change in color due to aging is also favorable. The results are shown. As described above, it is considered that the fermentation effect of the present fermentation decomposing agent was sufficient for bark composting. This is finally well understood by the analysis results of the product.
All of the analysis values show excellent results exceeding the unified standard values of the Japan Burk Compost Association and the National Burk Compost Industry Association.
【表7】 [Table 7]
【0041】尚、本明細書中の賦型剤とは、石灰岩岩
粉,ドロマイト岩粉,貝化石粉末,カニ・シャコ,貝殻
等の甲殻・貝殻粉末,炭酸カルシウム,消石灰,パーラ
イト,バーミキュライト,ゼオライト,けいそう土,塩
基性岩岩粉,粉砕ピートモス,木炭・くん炭末等の粉粒
体があり、これらの一種又は複数種の混合物を示す。本
明細書中の微量栄養素及び微量ミネラルとは、ビオチ
ン,ニコチン酸アミド,チアミン,ピリドキサミン,ビ
タミンB12,バラアミノ安息香酸等のビタミン類、アル
ギニン,シスチン,グルタミン酸,イソロイシン,ロイ
シン,リジン,メチオニン,フェニルアラニン,スレオ
ニン,トリプトファン,チロシン,バリン等のアミノ酸
類、又、鉄,マンガン,コバルト,カルシウム等の微量
ミネラル等がある。以上、これらの賦型剤・微量栄養素
及び微量ミネラルは、有効菌・使用目的・土壌環境・風
土・気候に応じて適宜選択される。The excipients used in the present specification include limestone rock powder, dolomite rock powder, shell fossil powder, crab shell, shellfish powder such as shellfish, calcium carbonate, slaked lime, perlite, vermiculite and zeolite. , Diatomaceous earth, basic rock rock powder, crushed peat moss, charcoal, charcoal powder, etc. are present, and a mixture of one or more of these is shown. Micronutrients and trace minerals in the present specification include vitamins such as biotin, nicotinic acid amide, thiamine, pyridoxamine, vitamin B12, balaaminobenzoic acid, arginine, cystine, glutamic acid, isoleucine, leucine, lysine, methionine, phenylalanine, There are amino acids such as threonine, tryptophan, tyrosine and valine, and trace minerals such as iron, manganese, cobalt and calcium. As described above, these excipients, micronutrients and trace minerals are appropriately selected according to effective bacteria, purpose of use, soil environment, climate and climate.
【0042】[0042]
【発明の効果】以上のように、本発明によれば有用な新
菌SK542菌株を提供する。又、新菌SK542とS
K522の両菌株の共生的混合培養の成功は、自然生態
系や農耕生態系、又工場生産システムにおける微生物フ
ロラーの作用システムの次のような利用を可能にし、そ
の実用化を確実なものとした。 (1) 多種類の天然の難分解性有機質の分解利用を可能に
し、ただ分解消化するだけでなく、いくつかの有用な物
質の生産ができた。 (2) 分解が迅速,安定して、その生産性が向上し、野積
堆肥化法を確実なものにした。 (3) そしてまた、難分解性有機質の複雑な分解工程をで
きるだけ短時間に一段階で行う好熱性高速分解方式を確
立した。 (4) SK522菌株とSK542菌株を主要菌とする健
全活性ある強力な土壌微生物フロラーの形成は、環境要
因の変化に対して有効菌の生育と作用機能を安定させ
た。 (5) また同時に雑菌や有害菌等の汚染に抵抗性を増強し
て、土壌病害や連作障害を消滅,克服することができ
た。 (6) 前項のようにして製造された良質の堆肥,コンポス
トの施用、又は土壌や植物の葉面に直接施用する撒布技
術の開発等は、土壌の物理的性状を改善し、特に緑農地
の植物を活性化し、生産性を維持するための地力を増大
させることができた。 (7) 屎尿の脱臭分解、又硬タンパク質,余剰汚泥の分解
にも有効で、これらを窒素源として難分解性繊維物質を
効果的に分解する。INDUSTRIAL APPLICABILITY As described above, according to the present invention, a useful new strain SK542 is provided. In addition, new bacteria SK542 and S
The success of the symbiotic mixed culture of both strains of K522 enabled the following applications of the action system of microbial flora in natural ecosystems, agricultural ecosystems, and factory production systems, and ensured its practical application. . (1) It enabled the decomposition and utilization of various kinds of natural persistent organic substances, and not only decomposed and digested it, but also produced some useful substances. (2) The decomposition was quick and stable, the productivity was improved, and the field composting method was secured. (3) Furthermore, a thermophilic rapid decomposition method was established in which a complicated decomposition process of persistent organic substances was carried out in one step in the shortest possible time. (4) The formation of a strong and active soil microbial flora mainly consisting of the SK522 strain and the SK542 strain stabilized the growth and action function of effective bacteria against changes in environmental factors. (5) At the same time, it was possible to enhance the resistance to contamination by various bacteria and harmful bacteria, and eliminate and overcome soil diseases and continuous crop damage. (6) The application of good quality compost produced as described in the preceding paragraph, composting, or the development of a sprinkling technique to be applied directly to the foliage of soil or plants improves the physical properties of the soil, especially in the case of green farmland. It was possible to activate plants and increase the fertility for maintaining productivity. (7) It is also effective for deodorizing human waste and decomposing hard proteins and excess sludge, and effectively decomposes hardly decomposable fiber substances by using these as nitrogen sources.
フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 FI 技術表示箇所 C12N 9/52 9152−4B C12P 5/00 8114−4B 39/00 9452−4B (C12N 1/20 C12R 1:01) (C12P 39/00 C12R 1:145 1:01) Continuation of front page (51) Int.Cl. 6 Identification number Internal reference number FI Technical indication C12N 9/52 9152-4B C12P 5/00 8114-4B 39/00 9452-4B (C12N 1/20 C12R 1: 01) (C12P 39/00 C12R 1: 145 1:01)
Claims (1)
で、40〜82℃の温度範囲で通常濃度の培地に生育
し、作用適温75〜85℃、作用水素イオン濃度pH=
4.0〜11.3の高温性広域作用水素イオン濃度活性
のタンパク質分解酵素とカロチノイド系黄色色素を産生
するサーマス・アクアティクス菌(Thermus aquaticus
biovar SK542,微工研条寄第3382号)。1. Absolutely aerobic, optimum temperature for growth is 72 to 76 ° C.
In the temperature range of 40 to 82 ℃, it grows in a medium of normal concentration, the optimum temperature of action is 75 to 85 ℃, and the action hydrogen ion concentration is pH =
Thermus aquaticus producing thermolytic broad-range action hydrogen ion concentration active proteolytic enzyme and carotenoid yellow pigment of 4.0 to 11.3 (Thermus aquaticus)
biovar SK542, Microtech Research Institute No. 3382).
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP31583692A JPH0757185B2 (en) | 1992-10-30 | 1992-10-30 | Thermus aquaticus |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP31583692A JPH0757185B2 (en) | 1992-10-30 | 1992-10-30 | Thermus aquaticus |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP3299897A Division JPH0734739B2 (en) | 1991-10-19 | 1991-10-19 | Thermophilic fibrinolytic bacteria |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH06105679A JPH06105679A (en) | 1994-04-19 |
JPH0757185B2 true JPH0757185B2 (en) | 1995-06-21 |
Family
ID=18070162
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP31583692A Expired - Lifetime JPH0757185B2 (en) | 1992-10-30 | 1992-10-30 | Thermus aquaticus |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH0757185B2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2014508536A (en) * | 2011-03-24 | 2014-04-10 | アニッキ ゲーエムベーハー | Method for producing lignin derivative |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH08140668A (en) * | 1994-11-15 | 1996-06-04 | Hide Kume | Agent for fermentation treatment of raw garbage |
TW200301303A (en) | 2001-12-25 | 2003-07-01 | Sanyu Co Ltd | Novel microorganism |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05299897A (en) * | 1992-04-20 | 1993-11-12 | Takara Seisakusho:Kk | Base material conveyor |
-
1992
- 1992-10-30 JP JP31583692A patent/JPH0757185B2/en not_active Expired - Lifetime
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05299897A (en) * | 1992-04-20 | 1993-11-12 | Takara Seisakusho:Kk | Base material conveyor |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2014508536A (en) * | 2011-03-24 | 2014-04-10 | アニッキ ゲーエムベーハー | Method for producing lignin derivative |
Also Published As
Publication number | Publication date |
---|---|
JPH06105679A (en) | 1994-04-19 |
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