JP2005117973A - Highly functional denitrifying and oil degrading bacterium, and method for purifying wastewater using the same - Google Patents
Highly functional denitrifying and oil degrading bacterium, and method for purifying wastewater using the same Download PDFInfo
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Abstract
Description
本発明は、高機能脱窒油脂分解菌等に関するものである。特には、比較的低温で増殖が可能で、かつ動植物脂質分解能及び脱窒能を有する高機能脱窒油脂分解菌等に関する。 The present invention relates to a highly functional denitrifying oil-degrading bacterium and the like. In particular, the present invention relates to a highly functional denitrifying oil-degrading bacterium capable of growing at a relatively low temperature and having the ability to degrade animal and plant lipids and denitrification.
病院等の厨房施設、レストラン等の業務用の厨房施設からは大量の廃水が発生しているが、この廃水中には、調理に用いられた油脂に由来する多量の油脂が含まれている。現在、これらの廃水処理施設には、油脂分を処理するためのグリーストラップという設備の設置が義務づけられている。グリーストップは、上述したような厨房施設由来の廃水中に含まれる油脂分を分離し、油脂分が廃水管中に流入して管を詰まらせることを防ぐ役割を果たすものである。しかし、現実には、グリーストラップにおける油脂分の滞留時間は通常のシステムを適用しても十分ではなく、油脂分を処理しきれていないことは明らかである。また、グリーストラップからの悪臭の発生、真菌類の増殖、油脂分が分解しないことによる油脂分の滞留等の問題が常に起こっている。 A large amount of waste water is generated from kitchen facilities such as hospitals and commercial kitchen facilities such as restaurants. The waste water contains a large amount of fats and oils derived from the fats and oils used for cooking. Currently, these wastewater treatment facilities are obligated to install equipment called grease traps for treating fats and oils. The grease top serves to separate the oil and fat contained in the wastewater derived from the kitchen facility as described above and prevent the oil and fat from flowing into the wastewater pipe and clogging the pipe. However, in reality, the residence time of the oil and fat in the grease trap is not sufficient even if a normal system is applied, and it is clear that the oil and fat cannot be completely processed. In addition, problems such as generation of malodor from the grease trap, growth of fungi, and retention of fats and oils due to the fats and oils not being decomposed are constantly occurring.
近年、ある種の細菌や真菌類の微生物の菌体、又は混合液をグリーストラップ内に投入して、微生物によって油脂を分解させようとする方法が提案されている。しかし、投入する微生物株の脂質分解活性が十分に強力でない点や、グリーストラップ内の多様な環境に微生物自身が適応しきれない、また、グリーストラップにおける滞留時間が短すぎる等の問題があった。 In recent years, a method has been proposed in which a cell or mixture of microorganisms of a certain kind of bacteria or fungi is introduced into a grease trap and oils and fats are decomposed by the microorganisms. However, there are problems such as that the lipolytic activity of the microorganism strain to be introduced is not sufficiently strong, the microorganism itself cannot adapt to various environments in the grease trap, and the residence time in the grease trap is too short. .
また、上記廃水はアンモニア性窒素を含有していることが多い。このようなアンモニア性窒素は、亜硝酸や硝酸に変化した後、脱窒工程により窒素ガスに変換される。この脱窒工程が不十分であると、亜硝酸や硝酸が十分に脱窒されずに、処理水中に残留して放流されてしまうので、湖沼、河川等が、富栄養化を起こし環境汚染の原因となっている。 In addition, the wastewater often contains ammoniacal nitrogen. Such ammoniacal nitrogen is converted into nitrous acid or nitric acid, and then converted into nitrogen gas by a denitrification step. If this denitrification process is inadequate, nitrous acid and nitric acid will not be sufficiently denitrified and will remain in the treated water and be released, so lakes, rivers, etc. will become eutrophication and cause environmental pollution. It is the cause.
廃水を実際に処理する場合の温度条件は、通常の微生物が生育し、有効に酵素活性を発揮する温度よりも低い温度である。従って、１０〜２０℃程度の比較的低温の温度域で活性を発揮することが望まれる。従って、比較的低温で増殖が可能で、かつ動植物脂質分解能及び脱窒能を有する微生物が望まれている。
従って、本発明の目的は、比較的低温で増殖が可能で、かつ動植物脂質分解能及び脱窒能を有する微生物を提供することにある。
The temperature condition for actually treating the wastewater is a temperature lower than the temperature at which normal microorganisms grow and effectively exhibit enzyme activity. Therefore, it is desirable to exhibit activity in a relatively low temperature range of about 10 to 20 ° C. Therefore, there is a demand for a microorganism that can grow at a relatively low temperature and has the ability to resolve animal and plant lipids and denitrification.
Accordingly, an object of the present invention is to provide a microorganism that can be grown at a relatively low temperature and that has the ability to resolve animal and plant lipids and denitrification.
本発明者は、上記目的を達成すべく鋭意検討した結果、生ゴミ発酵試料中に、高機能脱窒油脂分解活性が観察されることを見出した。今回、本発明者らは、この生ゴミ発酵試料中から、高機能脱窒油脂分解菌単離することに成功した。単離した高機能脱窒油脂分解菌（Ｆ−１株と命名）の特徴を分析し、この菌株がシュードモナス デニトリフィカンス(Pseudomonas denitrificans)株であるという知見を得、この知見に基づいて本発明を完成させた。 As a result of intensive studies to achieve the above object, the present inventor has found that a high-performance denitrifying oil / fat decomposition activity is observed in the raw garbage fermentation sample. This time, the present inventors succeeded in isolating a highly functional denitrified oil-degrading bacterium from this raw garbage fermentation sample. The characteristics of the isolated highly functional denitrifying oil-degrading bacterium (named F-1 strain) were analyzed, and the knowledge that this strain was Pseudomonas denitrificans strain was obtained. Completed the invention.
すなわち、本発明は、４〜４０℃の範囲の温度で生育することができるシュードモナス デニトリフィカンス(Pseudomonas denitrificans)株であって、油脂分解能力、及び脱窒能力を有することを特徴とする、高機能脱窒油脂分解菌を提供するものである。
本発明の高機能脱窒油脂分解菌の好ましい具体例としては、受託番号ＦＥＲＭ Ｐ−１９４７６を有する菌株である。
That is, the present invention is a Pseudomonas denitrificans strain capable of growing at a temperature in the range of 4 to 40 ° C., characterized in that it has an oil and fat decomposition ability and a denitrification ability. A highly functional denitrifying oil-degrading bacterium is provided.
A preferable specific example of the highly functional denitrifying oil-degrading bacterium of the present invention is a strain having the accession number FERM P-19476.
また、本発明は、上記高機能脱窒油脂分解菌を含有する、脱窒油脂分解用組成物を提供するものである。
また、本発明は、上記高機能脱窒油脂分解菌を固相担体に固定した、脱窒油脂分解用組成物を提供するものである。
また、本発明は、上記高機能脱窒油脂分解菌、又は脱窒油脂分解用組成物を油脂及び窒素分を含有する廃水と接触することを特徴とする、油脂及び窒素分を分解する方法を提供するものである。
また、本発明は上記高機能脱窒油脂分解菌、又は脱窒油脂分解用組成物を、油脂及び窒素分を含有する廃水と接触することを特徴とする、廃水浄化方法を提供するものである。
The present invention also provides a composition for denitrifying oil and fat decomposition, which contains the above-described highly functional denitrifying oil and fat degrading bacteria.
The present invention also provides a composition for denitrifying oil and fat decomposition, wherein the high-performance denitrifying oil and fat degrading bacterium is fixed to a solid phase carrier.
The present invention also provides a method for decomposing oil and nitrogen, characterized in that the high-performance denitrifying oil-degrading bacterium or the denitrifying oil-decomposing composition is contacted with waste water containing oil and nitrogen. It is to provide.
The present invention also provides a method for purifying waste water, characterized in that the high-performance denitrifying oil-degrading bacterium or the composition for denitrifying oil-degrading is brought into contact with waste water containing fat and nitrogen. .
本発明により、新規な高機能脱窒油脂分解菌、及びそれを用いた油脂及び窒素分を分解する方法、廃水浄化方法が提供される。本発明の高機能脱窒油脂分解菌は、固体培地のみならず、液体培地においても脂質分解活性を示す。また、その培養上清は培地中の基質に油脂が含まれない場合においても脂質分解活性を示し、界面活性剤を添加した時には、僅か培養1日で培養上清に著量の脂質分解を示した。また、本発明の高機能脱窒油脂分解菌は比較的低温で増殖できるので、比較的低温での廃水浄化が可能である。したがって、業務用厨房や食品工場から排出される油脂含有廃水や油脂に汚染された土壌の浄化および生ゴミの処理にも応用できる微生物である。 INDUSTRIAL APPLICABILITY According to the present invention, there are provided a novel highly functional denitrifying oil-degrading bacterium, a method for decomposing oil and nitrogen using the same, and a method for purifying waste water. The highly functional denitrifying oil-degrading bacterium of the present invention exhibits lipolytic activity not only in a solid medium but also in a liquid medium. In addition, the culture supernatant shows lipolytic activity even when the substrate in the medium does not contain fats and oils. When a surfactant is added, the culture supernatant shows a significant amount of lipolysis in just one day of culture. It was. Moreover, since the highly functional denitrifying oil-degrading bacterium of the present invention can grow at a relatively low temperature, it is possible to purify wastewater at a relatively low temperature. Therefore, it is a microorganism that can be applied to the purification of soil contaminated with fat and oil-containing wastewater discharged from commercial kitchens and food factories and the treatment of raw garbage.
以下、本発明について詳細に説明する。
先ず、本発明の高機能脱窒油脂分解菌について説明する。
本明細書において、「油脂」とは、動植物を起源とするあらゆる油脂を含むものとし、例えば、動植性食用油、不飽和脂肪酸、魚油等を含む油脂が挙げられる。
Hereinafter, the present invention will be described in detail.
First, the highly functional denitrifying oil-degrading bacterium of the present invention will be described.
In this specification, “oil and fat” includes all fats and oils originating from animals and plants, and examples thereof include fats and oils including animal and plant edible oils, unsaturated fatty acids, fish oils and the like.
本発明の高機能脱窒油脂分解菌は、４〜４０℃の範囲の温度で生育することができるシュードモナス デニトリフィカンス(Pseudomonas denitrificans)株であり、例えば、シュードモナス デニトリフィカンス Ｆ−１株が挙げられる。これらの菌株は、変種又は変異株であってもよい。 The highly functional denitrifying oil-degrading bacterium of the present invention is a Pseudomonas denitrificans strain that can grow at a temperature in the range of 4 to 40 ° C., for example, Pseudomonas denitrificans F-1 strain Is mentioned. These strains may be variants or mutants.
本発明者らが単離した一例の菌株（Ｆ−１株）は、受託番号ＦＥＲＭ Ｐ−１９４７６として寄託されている。上記菌株は、牛の糞尿に有機物（生ゴミ）を加えて分解させたものから単離されたものである。Ｆ−１株の分離のために、オリーブオイル入りの寒天培地を使用し、ハロー形成能を指標として選択した。
得られたＦ−１株の細菌分類学的性質は以下の通りである。
An example strain (F-1 strain) isolated by the present inventors has been deposited under the accession number FERM P-19476. The above strain is isolated from a product obtained by decomposing cattle manure by adding organic matter (garbage). For isolation of the F-1 strain, an agar medium containing olive oil was used, and halo-forming ability was selected as an index.
The bacterial taxonomic properties of the obtained F-1 strain are as follows.
〔１〕形態学的性質
（１）グラム染色：陰性
（２）芽胞：無し
（３）運動性：有り
（３）菌形：桿菌
[1] Morphological properties (1) Gram staining: Negative (2) Spore: None (3) Motility: Existence (3) Fungal form: Neisseria gonorrhoeae
〔２〕培地における生育特性
色調は白色であり、コロニーの形状は円形、であり、コロニーの表面は平滑である。
〔３〕化学分類学的特性
ＤＮＡのＧ＋Ｃモル含有量は６２．８％である。
[2] Growth characteristics in medium The color tone is white, the shape of the colony is circular, and the surface of the colony is smooth.
[3] Chemical taxonomic characteristics The G + C molar content of DNA is 62.8%.
〔４〕生理・生化学的特性
オキシダーゼ試験：陽性
カタラーゼ試験：陽性
尿素分解：陰性
ONPG試験：陰性
硫化水素の産生：陽性
メチルレッド試験：陰性
VP試験：陰性
インドール産生：陰性
硝酸塩の還元：陽性
オリーブ油分解：陽性
ラード分解：陽性
デンプン分解：陰性
ゼラチン分解：陰性
カゼイン分解：陰性
DNA分解：陰性
アルギン酸分解：陰性
Tween 20の分解：陽性
Tween 40の分解：陽性
Tween 60の分解：陽性
Tween 80の分解：陽性
[4] Physiological and biochemical characteristics Oxidase test: positive Catalase test: positive Urea degradation: negative
ONPG test: negative Hydrogen sulfide production: positive Methyl red test: negative
VP test: negative Indole production: negative nitrate reduction: positive olive oil degradation: positive lard degradation: positive starch degradation: negative gelatin degradation: negative casein degradation: negative
DNA degradation: negative Alginic acid degradation: negative
Tween 20 degradation: positive
Tween 40 degradation: positive
Tween 60 degradation: positive
Tween 80 degradation: positive
各温度における生育
-1℃：−
4℃：＋
10℃：＋
25℃: ＋
40℃: ＋
45℃: −
50℃: −
Growth at each temperature
-1 ° C:-
4 ° C: +
10 ° C: +
25 ℃: +
40 ℃: +
45 ° C: −
50 ° C: −
本発明の高機能脱窒油脂分解菌は、Ｌ−アラビノース、Ｄ−フルクトース、ラクトース、Ｄ−マルトース、Ｄ−マンノース、メリビオース、シュークロース、Ｄ−キシロース、ラフィノース、イノシトール、マンニトール、ソルビトール、ガラクトース、Ｌ−ラムノース、及びトレハロースから酸を産生しない。 The highly functional denitrifying oil-degrading bacterium of the present invention is L-arabinose, D-fructose, lactose, D-maltose, D-mannose, melibiose, sucrose, D-xylose, raffinose, inositol, mannitol, sorbitol, galactose, L -No acid is produced from rhamnose and trehalose.
本発明の高機能脱窒油脂分解菌は、Ｄ−マルトース、Ｌ−アラニン、Ｌ−グルタミン酸、Ｌ−ロイシン、及びＬ−プロリンを唯一の炭素源として資化する。
本発明の高機能脱窒油脂分解菌は、Ｌ−アラビノース、Ｄ−フルクトース、Ｄ−グルコース、グリセロール、ラクトース、Ｄ−マンノース、メリビオース、シュークロース、Ｄ−キシロース、ソルビトール、Ｌ−ラムノース、Ｄ−セロビオース、イヌリン、Ｌ−トリプトファン、Ｌ−スレオニン、Ｌ−ヒスチジン、Ｌ−リジン、Ｌ−アスパラギン、及びＬ−グリシンを唯一の炭素源として資化しない。
The highly functional denitrifying oil-degrading bacterium of the present invention assimilate using D-maltose, L-alanine, L-glutamic acid, L-leucine, and L-proline as the sole carbon source.
The highly functional denitrifying oil-degrading bacterium of the present invention includes L-arabinose, D-fructose, D-glucose, glycerol, lactose, D-mannose, melibiose, sucrose, D-xylose, sorbitol, L-rhamnose, D-cellobiose. Inulin, L-tryptophan, L-threonine, L-histidine, L-lysine, L-asparagine, and L-glycine are not assimilated as the sole carbon source.
Ｆ−１株のＤＮＡを抽出し、ＰＣＲ法により１６Ｓ ｒＲＮＡ遺伝子を増幅し、増幅して得られた１６Ｓ ｒＲＮＡ遺伝子の塩基配列をオートシークエンサーで解析し、ブラストサーチにより類似した塩基配列を調べた。その結果に基づき、類似した塩基配列と多重アラインメントを取り、得られた結果に基づき近隣結合法（Neighbor-Joining法）により系統樹を作成したところ、Ｆ−１株はシュードモナス（Pseudomonas）属細菌に分類されることが判明した。作成した系統樹を図1に示す。図１は、Pseudomonasdenitrificans F-1株の16S rRNA遺伝子に基づく系統的位置を示す図であり、系統樹内の数字はブートストラップ値が５００以上を示す。バーは、０．１K uncユニットを示す。
同属細菌の中でもシュードモナス デニトリフィカンス（Pseudomonas denitrificans） と高い相同性を示す（９９．４％）ことが示された。
The DNA of F-1 strain was extracted, the 16S rRNA gene was amplified by the PCR method, the base sequence of the 16S rRNA gene obtained by the amplification was analyzed with an autosequencer, and similar base sequences were examined by blast search. Based on the results, multiple alignments with similar base sequences were taken, and a phylogenetic tree was created based on the results obtained by the Neighbor-Joining method. As a result, the F-1 strain was identified as a Pseudomonas genus bacterium. It turned out to be classified. The created phylogenetic tree is shown in FIG. FIG. 1 is a diagram showing a systematic position based on the 16S rRNA gene of Pseudomonas denitrificans F-1 strain, and the numbers in the phylogenetic tree indicate a bootstrap value of 500 or more. Bars indicate 0.1 K unc units.
Among homologous bacteria, it was shown to show high homology (99.4%) with Pseudomonas denitrificans ( Pseudomonas denitrificans ).
上記１６Ｓ ｒＲＮＡ遺伝子の塩基配列における相同性において最も相同性の高かったシュードモナス デニトリフィカンス（Pseudomonas denitrificans）、 及びその次に相同性が高かったPseudomonaspertucinogena（３８．９％）について、菌株を保存機関より入手し、Ｆ−１株および入手した２菌株からＤＮＡを抽出し、Ｆ−１株のＤＮＡをフォトビオチンでラベルしプローブを作成し、ブラックマイクロプレートに被検ＤＮＡを固定し、相同性を蛍光測定により算出した。結果は、Ｆ−１株がP.denitrificansおよびP. pertucinogenaとそれぞれ９６％及び３９％の相同性を示した。ＤＮＡ−ＤＮＡ相同性試験において７０％以上であると同一種と考えられることから、Ｆ−１株はシュードモナス デニトリフィカンスと同一種であることが示された。 The 16S rRNA gene most homologous higher was Pseudomonas de nitrilase in homology in nucleotide sequence of denitrificans (Pseudomonas denitrificans), and the Pseudomonaspertucinogena (38.9%) of the next homology tinged high, save engine strain DNA is extracted from F-1 strain and the two obtained strains, the DNA of F-1 strain is labeled with photobiotin, a probe is prepared, and the test DNA is immobilized on a black microplate. Calculated by fluorescence measurement. The results showed that the F-1 strain had 96% and 39% homology with P. denitrificans and P. pertucinogena , respectively. In the DNA-DNA homology test, it was considered to be the same species as 70% or more, indicating that the F-1 strain was the same species as Pseudomonas denitrificans.
本発明の高機能脱窒油脂分解菌は、動植物性油脂を分解すると同時に不要な窒素分を脱窒により除去するのに用いることができる。特に、廃水中および生ゴミ中の動植物油脂分および窒素分を分解するのに用いることができる。細菌そのものを油脂および不要窒素分を含む廃水に添加しても良いし、固相担体に菌体を固定してから用いても良い。この際、廃水の温度は４〜４０℃程度が望ましく、pHは６．５〜９．０が望ましい。本発明の高機能脱窒油脂分解菌は、４〜４０℃の範囲の温度で増殖することができるので、上記廃水の温度範囲で好ましく用いられる。 The highly functional denitrifying oil-degrading bacterium of the present invention can be used for decomposing animal and vegetable oils and removing unnecessary nitrogen components by denitrification. In particular, it can be used for decomposing animal and vegetable fats and oils and nitrogen in wastewater and garbage. Bacteria themselves may be added to waste water containing fats and oils and unnecessary nitrogen, or may be used after the cells are fixed to a solid phase carrier. At this time, the temperature of the waste water is desirably about 4 to 40 ° C., and the pH is desirably 6.5 to 9.0. Since the highly functional denitrifying oil-degrading bacterium of the present invention can grow at a temperature in the range of 4 to 40 ° C., it is preferably used in the temperature range of the waste water.
次に、本発明の脱窒油脂分解用組成物について説明する。本発明の高機能脱窒素油脂分解菌は、そのままで廃水の浄化に用いることもでき、またそのままで生ゴミ中の動植物油脂分及び窒素分を分解するのに用いることができる。しかし、本発明の高機能脱窒油脂分解菌を含有する脱窒油脂分解用組成物として用いてもよい。
このような脱窒油脂分解用組成物としては、例えば液状製剤や粉末製剤が挙げられる。
Next, the composition for denitrifying fat and oil decomposition of the present invention will be described. The highly functional denitrifying oil-degrading bacterium of the present invention can be used as it is for purification of waste water, or can be used as it is for decomposing animal and vegetable oils and nitrogen in raw garbage. However, you may use as a composition for denitrification fat-and-oil decomposition | disassembly containing the highly functional denitrification fat-and-oil degradation microbe of this invention.
Examples of such a composition for denitrifying oil and fat decomposition include liquid preparations and powder preparations.
液状製剤とするには、例えば下記方法によって実施可能である。
高機能脱窒油脂分解菌を培養し、この培養物を遠心分離し、菌体を回収し、生理食塩水を加えて適当な濃度となるように懸濁し、脱窒油脂分解用組成物とする。また、この脱窒油脂分解用組成物に凍結乾燥などにより適度に濃縮してもよい。また、脱窒油脂分解用組成物には、必要に応じてｐＨ調整剤等を加えてもよい。
In order to obtain a liquid preparation, for example, the following method can be used.
High-performance denitrifying oil-degrading bacteria are cultured, the culture is centrifuged, the cells are collected, suspended in an appropriate concentration by adding physiological saline, and used as a denitrifying oil-decomposing composition. . Further, the composition for denitrifying oil / fat decomposition may be appropriately concentrated by freeze-drying or the like. Moreover, you may add a pH adjuster etc. to the composition for denitrification oil-fat decomposition | disassembly as needed.
粉末製剤とするには、例えば下記方法によって実施可能である。
高機能脱窒油脂分解菌を培養し、この培養液を凍結乾燥等によって乾燥し、粉末製剤とする。また、培養液から菌体を遠心分離によって回収し、生理食塩水や新鮮な培地と懸濁した後、凍結乾燥等によって乾燥してもよい。また、凍結乾燥前にｐＨ調整剤等を加えてもよい。
In order to obtain a powder formulation, for example, the following method can be used.
High-performance denitrifying oil-degrading bacteria are cultured, and this culture solution is dried by freeze drying or the like to obtain a powder preparation. Alternatively, the cells may be collected from the culture solution by centrifugation, suspended in physiological saline or a fresh medium, and then dried by freeze drying or the like. Further, a pH adjusting agent or the like may be added before lyophilization.
また、本発明の脱窒油脂分解用組成物は、上記高機能脱窒油脂分解菌を固相担体に固定して製造することができる。高機能脱窒油脂分解菌を固定するための固相担体としては、微生物の固定に使用することのできるものであればいずれでも良く、特に限定されるものではない。例えば、アルギン酸、活性炭、珪藻土セラミック多孔体等があげられる。このような固相担体の形状としては、球状又は円柱状が好適であり、球状の場合、粒径は好ましくは０．３〜２．８ｍｍ程度であり、更に好ましくは０．３〜１．２ｍｍ程度である。 Moreover, the composition for denitrifying fat and oil decomposition of the present invention can be produced by fixing the high-performance denitrifying fat and oil-degrading bacterium to a solid phase carrier. The solid phase carrier for immobilizing the highly functional denitrifying oil-degrading bacterium is not particularly limited as long as it can be used for immobilizing microorganisms. For example, alginic acid, activated carbon, diatomaceous earth ceramic porous body and the like can be mentioned. As the shape of such a solid phase carrier, a spherical shape or a cylindrical shape is suitable. In the case of a spherical shape, the particle size is preferably about 0.3 to 2.8 mm, more preferably 0.3 to 1.2 mm. Degree.
次に、本発明の油脂及び窒素分を分解する方法について説明する。本発明の油脂及び窒素分を分解する方法は、本発明の高機能脱窒油脂分解菌、又は脱窒油脂分解用組成物を、油脂及び窒素分を含有する廃水と接触することを特徴とする。
本発明の油脂及び窒素分を分解する方法は、油脂及び窒素分を含有する廃水（生ゴミも含む）に、本発明の高機能脱窒素油脂分解菌、又は脱窒油脂分解用組成物を投与するこおによって実施することができる。高機能脱窒素油脂分解菌、又は脱窒油脂分解用組成物の投与量に特に制限はなく、廃水中に含まれる油脂及び窒素分を分解することのできる量でよい。なお、廃水の温度は４〜４０℃の範囲が好ましく、ｐＨは６．５〜９．０の範囲が好ましい。本発明の油脂及び窒素分を分解する方法によれば、廃水を浄化することが可能となる。
Next, the method for decomposing oil and fat and nitrogen content of the present invention will be described. The method for decomposing oil and nitrogen of the present invention is characterized by contacting the highly functional denitrifying oil-degrading bacterium of the present invention or the denitrifying oil-decomposing composition with waste water containing oil and nitrogen. .
The method for decomposing oil and nitrogen of the present invention comprises administering the highly functional denitrifying oil-degrading bacterium of the present invention or the composition for denitrifying oil and fat decomposition to waste water (including garbage) containing oil and nitrogen. It can be implemented by doing things. There is no restriction | limiting in particular in the dosage amount of a highly functional denitrifying oil-degrading microbe or the composition for denitrifying fat-and-oil decomposition | disassembly, The quantity which can decompose | disassemble the fats and oils and nitrogen content which are contained in wastewater is sufficient. In addition, the temperature of wastewater has the preferable range of 4-40 degreeC, and pH has the preferable range of 6.5-9.0. According to the method for decomposing oil and fat and nitrogen of the present invention, it becomes possible to purify waste water.
以下、実施例により本発明を具体的に説明する。但し、本発明はこれらの実施例にその技術範囲が限定されるものではない。なお、以下の実施例において、％は、特に断りのない限り質量％を表す。
実施例１
牛の糞尿に生ゴミを加えて分解させた分解物を用いて高機能脱窒油脂分解菌を単離するための材料として用いた。
基礎培地（ペプトン、５ｇ; 酵母エキス、３ｇ;ＮａＣｌ、５ｇ; 寒天、１５ｇ; 蒸留水ｍ９００ｍｌ,ｐＨ７．８）、およびオリーブオイル入り寒天溶液（０．５％ 寒天溶液、４０ｍｌ; オリーブオイル、１０ｍｌ；Ｔｗｅｅｎ８０、０．５ｍｌ）をそれぞれ別滅菌する。滅菌後オリーブ油入り寒天溶液を超音波で乳化し、基礎培地と混合し、シャーレーに分注し固化して高機能脱窒油脂分解菌単離用培地とした。
Hereinafter, the present invention will be described specifically by way of examples. However, the technical scope of the present invention is not limited to these examples. In the following examples, “%” represents “% by mass” unless otherwise specified.
Example 1
It was used as a material for isolating high-performance denitrifying oil-degrading bacteria using a decomposition product obtained by adding raw garbage to cattle manure and decomposing it.
Basal medium (peptone, 5 g; yeast extract, 3 g; NaCl, 5 g; agar, 15 g; distilled water m900 ml, pH 7.8), and agar solution with olive oil (0.5% agar solution, 40 ml; olive oil, 10 ml; Tween 80, 0.5 ml) is sterilized separately. After sterilization, the agar solution containing olive oil was ultrasonically emulsified, mixed with a basal medium, dispensed into a Petri dish and solidified to obtain a medium for isolating highly functional denitrifying oil-degrading bacteria.
上記高機能脱窒油脂分解菌を単離するための材料を上記高機能脱窒油脂分解菌単離用培地に接種し、２７℃の温度で培養した。３日培養した後、油脂を分解したことを示す透明帯がコロニー周辺部に観察された。そのコロニーを、新鮮な高機能脱窒油脂分解菌単離用培地に接種し、同様に培養を行った。この操作を５回繰り返し、高機能脱窒油脂分解菌を単離し、これをＦ−１と命名した。このＦ−１は独立行政法人産業技術総合研究所特許生物センターに受託番号ＦＥＲＭ Ｐ−１９４７６として寄託されている。 The material for isolating the highly functional denitrifying oil-degrading bacteria was inoculated into the medium for isolating the highly functional denitrifying oil-degrading bacteria and cultured at a temperature of 27 ° C. After culturing for 3 days, a zona pellucida indicating that the fats and oils were decomposed was observed around the colony. The colony was inoculated into a fresh high-performance denitrifying oil-degrading bacterium isolation medium and cultured in the same manner. This operation was repeated 5 times to isolate a highly functional denitrifying oil-degrading bacterium, which was designated F-1. This F-1 is deposited under the accession number FERM P-19476 at the National Institute of Advanced Industrial Science and Technology.
実施例２
実施例１で用いた培地にＦ−１株を接種し、２７℃の温度で培養した。３日間培養した後、油脂を分解したことを示す透明帯がコロニー周辺部に観察された。その結果を図２に示す。図２に示すように、Ｆ−１株のコロニー周辺部には透明帯が形成されており、Ｆ−１株がプレート中の脂質を分解する能力を有していることがわかる。なお、シャーレの周縁部の白く見える部分は、この近くに菌体が存在しないため、脂質分が分解されずに残っていることを示す。
Example 2
The medium used in Example 1 was inoculated with the F-1 strain and cultured at a temperature of 27 ° C. After culturing for 3 days, a zona pellucida indicating that the fats and oils were decomposed was observed around the colony. The result is shown in FIG. As shown in FIG. 2, a zona pellucida is formed around the colony of the F-1 strain, and it can be seen that the F-1 strain has the ability to degrade lipids in the plate. In addition, since the microbial cell does not exist in the vicinity of the peripheral portion of the petri dish, the lipid component remains without being decomposed.
実施例３
５００ｍｌのＬＢ培地（トリプトン,１０ｇ; 酵母エキス,５ｇ;ＮａＣｌ,１０ｇ; 蒸留水, １０００ｍｌ）に５ｍｌ（１％）のオリーブオイルを加え、培地量の１／１０００容のＦ−１株の前培養液を接種し、２７℃で１２０ｒｍｐの回転速度で、１日、３日及び７日培養した後、培養液を１ｍｌをとり、この培養液から脂質を定法により抽出した。この脂質の一部の一定量をシリカゲルプレートを用いた薄層クロマトグラフィーに供した。展開溶媒としてはヘキサン／ジエチルエーテル／酢酸 （８０／３０／１ v/v/v）を用いた。
Example 3
5 ml (1%) of olive oil is added to 500 ml of LB medium (tryptone, 10 g; yeast extract, 5 g; NaCl, 10 g; distilled water, 1000 ml), and preculture of 1/1000 volume of F-1 strain of the medium amount After inoculating the solution and culturing at 27 ° C. at a rotation speed of 120 rpm for 1 day, 3 days and 7 days, 1 ml of the culture solution was taken, and lipid was extracted from this culture solution by a conventional method. A certain amount of this lipid was subjected to thin layer chromatography using a silica gel plate. As a developing solvent, hexane / diethyl ether / acetic acid (80/30/1 v / v / v) was used.
結果を図３に示す。図３は、Ｆ−１株を、オリーブオイル存在下で培養した後の抽出脂質の薄層クロマトグラムを示した図である。図３において、ＴＧはトリアシルグリセロールを示し、ＦＦＡは遊離脂肪酸を示す。図３は、左側からＦ−１株を培養していない培地、１日培養したもの、３日培養したもの、７日培養したものである。図３に示すように、Ｆ−１株を培養した培地においては、培地中に含まれるオリーブオイルの主成分であるトリアシルグリセロールの量が減少していることがわかる。 The results are shown in FIG. FIG. 3 is a diagram showing a thin-layer chromatogram of extracted lipids after culturing F-1 strain in the presence of olive oil. In FIG. 3, TG represents triacylglycerol and FFA represents free fatty acid. FIG. 3 is a culture medium in which the F-1 strain is not cultured from the left side, one day culture, three days culture, and seven days culture. As shown in FIG. 3, it can be seen that in the medium in which the F-1 strain was cultured, the amount of triacylglycerol which is the main component of olive oil contained in the medium is decreased.
実施例４
５００ｍｌ のLB培地（Ｔｗｅｅｎ８０無添加）、及びＴｗｅｅｎ ８０を１％添加したＬＢ培地を培地として用い、培地量の１／１０００容のＦ−１株の前培養液を接種し、２７℃の温度で１２０ｒｐｍの回転速度で１〜３日間培養した後、培養液を遠心分離により菌体を取り除き、上清を粗酵素液とした。得られた粗酵素液の脂質分解活性（リパーゼ活性）を、はｐ-ニトロフェニル パルミテート（ｐ−ＮＰＰ）を基質として用い、反応量をp-ニトロフェノールの遊離と見なし分光学的な測定により測定した。
Example 4
500 ml of LB medium (without Tween 80 added) and LB medium supplemented with 1% Tween 80 were used as the medium, inoculated with a preculture of 1/1000 volume of F-1 strain of the medium volume at a temperature of 27 ° C. After culturing for 1 to 3 days at a rotational speed of 120 rpm, the cells were removed from the culture by centrifugation, and the supernatant was used as a crude enzyme solution. The obtained crude enzyme solution of lipolytic activity (lipase activity), the p - using nitrophenyl palmitate the (p -NPP) as substrate, determined by free and regarded spectroscopic measurement of the reaction amount p- nitrophenol did.
測定結果を図4に示す。図４は、Ｆ−１の培養上清を粗酵素液として脂質分解活性を試験した結果を示したものである。
図４において、横軸は反応時間であり、縦軸は４５０ｎｍにおける吸光度である。また、黒丸はＴｗｅｅｎ８０を添加した培地で１日培養したもの、黒三角はＴｗｅｅｎ８０を添加した培地で３日培養したもの、四角はＴｗｅｅｎ８０を添加しない培地で１日培養したもの、三角はＴｗｅｅｎ８０を添加しない培地で３日培養したものの結果である。図４に示すように、オリーブオイルやＴｗｅｅｎ８０を培地に添加しなくても時間が多少かかるものの酵素の誘導が起こることが示された。一方、Ｔｗｅｅｎ８０を添加することにより、僅か培養1日で酵素の誘導が起こることが示された。
The measurement results are shown in FIG. FIG. 4 shows the results of testing the lipolytic activity using the culture supernatant of F-1 as a crude enzyme solution.
In FIG. 4, the horizontal axis is the reaction time, and the vertical axis is the absorbance at 450 nm. Also, black circles are cultured for 1 day in medium supplemented with Tween 80, black triangles are cultured for 3 days in medium supplemented with Tween 80, squares are cultured for 1 day in medium without Tween 80, triangles are supplemented with Tween 80 It is the result of what culture | cultivated for 3 days with the culture medium which does not. As shown in FIG. 4, it was shown that the induction of the enzyme occurred although it took some time without adding olive oil or Tween 80 to the medium. On the other hand, addition of Tween 80 was shown to induce enzyme in as little as one day of culture.
実施例５
肉汁ブイヨン（肉エキス,１０ｇ; ペプトン, １０ｇ;ＮａＣｌ,５ｇ; 蒸留水, １０００ｍｌ）を２本の試験管に用意し、一方に１％濃度となるように硝酸ナトリウムを添加し、もう一方には硝酸ナトリウムを添加せずコントロールとした。Ｆ−１株培養菌体をそれぞれの培地が入った試験管にそれぞれ接種し、流動パラフィン：白色ワセリンを１：１（ｖ・ｖ）で混合したものを重層し、簡易的な嫌気条件で４℃、１０℃、及び２７℃の温度で培養した。その結果、それぞれの温度で脱窒反応が確認された。
Example 5
Prepare broth broth (meat extract, 10 g; peptone, 10 g; NaCl, 5 g; distilled water, 1000 ml) in two test tubes, add sodium nitrate so that the concentration is 1%, and add the other to the other. Sodium nitrate was not added as a control. F-1 strain cultured cells are inoculated into each test tube containing each medium, and liquid paraffin: white petrolatum mixed at 1: 1 (v · v) is layered, and 4 under simple anaerobic conditions. Culturing was performed at temperatures of 10 ° C, 10 ° C, and 27 ° C. As a result, denitrification reaction was confirmed at each temperature.
Claims (8)
A wastewater purification method comprising contacting the composition for denitrifying fat and oil decomposition according to claim 3 or 4 with wastewater containing fat and nitrogen.
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| JP2010214310A (en) * | 2009-03-17 | 2010-09-30 | Prima Meat Packers Ltd | Microorganism having oil-and-fat decomposition capability and method for treating oil-and-fat containing wastewater using the same |
| JP2011125825A (en) * | 2009-12-21 | 2011-06-30 | Sumitomo Heavy Ind Ltd | Lypolytic bacteria and lipolytic agent |
| JP2011182782A (en) * | 2010-02-09 | 2011-09-22 | Sekisui Aqua System Kk | Oil-and-fat splitting microorganism, microorganism-immobilization carrier, method for treating waste water and system for treating waste water |
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| JP2011125825A (en) * | 2009-12-21 | 2011-06-30 | Sumitomo Heavy Ind Ltd | Lypolytic bacteria and lipolytic agent |
| JP2011182782A (en) * | 2010-02-09 | 2011-09-22 | Sekisui Aqua System Kk | Oil-and-fat splitting microorganism, microorganism-immobilization carrier, method for treating waste water and system for treating waste water |
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