JP2018126085A - Sludge volume reduction microbial material and excess sludge volume reduction method - Google Patents
Sludge volume reduction microbial material and excess sludge volume reduction method Download PDFInfo
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Abstract
【課題】活性汚泥法による水処理過程で発生する余剰汚泥を高効率に減容化することができ且つ低コストに実現可能な余剰汚泥の減容化方法を提供する。
【解決手段】活性汚泥にエキシグオバクテリウム・エスピー（Exiguobacterium sp.)Z153-2株（受託番号NITE P-02409）若しくは、エキシグオバクテリウム・アセチリカム（Exiguobacterium acetylicum)Z153-3株（受託番号NITE P-02410）、又はその両方を主成分として含有する汚泥減容微生物資材を添加することにより余剰汚泥を減容化する。
【選択図】図５A surplus sludge volume reduction method capable of reducing the volume of excess sludge generated in a water treatment process by an activated sludge method with high efficiency and at a low cost.
SOLUTION: Activated sludge is treated with Exiguobacterium sp. Z153-2 strain (Accession No. NITE P-02409) or Exiguobacterium acetylicum Z153-3 strain (Accession No. NITE). The volume of excess sludge is reduced by adding sludge volume-reducing microbial materials containing P-02410) or both as main components.
[Selection] Figure 5
Description
本発明は、活性汚泥法を利用した水処理設備において発生する余剰汚泥を減容化するための汚泥減容微生物資材、及び該微生物資材を用いた余剰汚泥の減容化方法に関する。 The present invention relates to a sludge-reducing microbial material for reducing the volume of excess sludge generated in water treatment facilities using an activated sludge method, and a method for reducing excess sludge using the microbial material.
活性汚泥法は、微生物の集合体から成る汚泥（活性汚泥）によって排水を処理する方法であり、処理能力が高く、ランニングコストが比較的低いことから有機性排水の処理方法として広く用いられている。活性汚泥法による水処理設備では、曝気槽とよばれる水槽内に活性汚泥を入れ、該曝気槽に空気を送り込むことにより微生物に酸素を供給する。そして該曝気槽に処理しようとする排水（汚水）を流入させて、該汚水中の有機性汚濁物質を微生物に分解・除去させる。曝気槽で処理された水は汚泥と共に後段の沈殿槽に流入して該沈殿槽にて汚泥と分離され、必要に応じて殺菌処理等を施された上で公共水域に放流される。 The activated sludge method is a method for treating wastewater with sludge (active sludge) composed of a collection of microorganisms, and is widely used as a method for treating organic wastewater because of its high treatment capacity and relatively low running costs. . In a water treatment facility using an activated sludge method, activated sludge is placed in a water tank called an aeration tank, and oxygen is supplied to microorganisms by sending air into the aeration tank. Then, wastewater (sewage) to be treated is introduced into the aeration tank, and organic pollutants in the wastewater are decomposed and removed by microorganisms. The water treated in the aeration tank flows into the subsequent sedimentation tank together with the sludge, is separated from the sludge in the sedimentation tank, and is discharged to the public water area after being sterilized as necessary.
このように、活性汚泥法は有機性汚濁物質を微生物に分解させるものであるため、必然的に微生物が増殖して系内の汚泥量が増大する。上記沈殿槽で水と分離された汚泥は該沈殿槽から引き抜かれ、その一部が曝気槽に返送され再利用されるが、残りの余剰な汚泥は系外に取り出されて凝集・脱水等の処理を施される。その後、該汚泥は主に建設資材（セメント原料等）、肥料等としてリサイクルされているが、約30％は埋め立て処分される。しかし、近年では埋め立て地の確保が難しくなっており、これに伴い汚泥の処分費用が高騰しているため余剰汚泥の減容化が求められている。 As described above, since the activated sludge method is to decompose organic pollutants into microorganisms, the microorganisms inevitably grow and the amount of sludge in the system increases. The sludge separated from the water in the settling tank is withdrawn from the settling tank, and a part of the sludge is returned to the aeration tank and reused, but the remaining excess sludge is taken out of the system for aggregation / dehydration, etc. Processed. Thereafter, the sludge is mainly recycled as construction materials (cement raw materials, etc.), fertilizers, etc., but about 30% is disposed of in landfills. However, in recent years, it has become difficult to secure landfills, and along with this, the disposal cost of sludge has soared, so there is a demand for volume reduction of excess sludge.
こうした汚泥の減容化を実現する技術としてこれまでに種々のものが提案されている。例えば非特許文献１には、汚泥減容化技術として、化学的処理方法、物理的処理方法、及び生物的処理方法等が挙げられている。化学的処理方法は、オゾン、アルカリ又は次亜塩素酸等を余剰汚泥に添加し、微生物の細胞壁を可溶化するなどして汚泥の減量化を図る手法である。また、物理的処理方法は、ビーズミル、高速回転ディスク又は超音波などによって微生物を破砕したり、熱処理によって微生物を構成するタンパク質を変性させたりすることで汚泥を減容化するものである。しかしながら、こうした化学的処理方法や物理的処理方法では、余剰汚泥の処理のための設備を新たに設ける必要があり、その設置スペースやコストが問題となる。
Various techniques have been proposed so far for reducing the volume of sludge. For example, Non-Patent
一方、生物的処理方法は、微生物や酵素を排水処理設備の曝気槽などに添加して、該微生物や酵素の作用により汚泥を減容化するものであり、上記の化学的処理方法や物理的処理方法に比べて低コストに実施できる場合が多いことから近年注目されている。例えば特許文献１には、汚水処理施設で分離された汚泥をバチルス属細菌に分解資化させて減容化する方法が記載されており、特許文献２には、汚泥とストレプトマイセス属に属する放線菌を含む微生物群を含有する汚泥分解剤とを接触させることで汚泥を分解する方法が記載されている。しかしながら、こうした生物的処理方法には処理効率の点でいまだ改善の余地がある。
On the other hand, the biological treatment method involves adding microorganisms or enzymes to an aeration tank of a wastewater treatment facility and reducing the volume of sludge by the action of the microorganisms and enzymes. In recent years, it has attracted attention because it can often be implemented at a lower cost than the processing method. For example,
本発明は上記の点に鑑みてなされたものであり、その目的とするところは、活性汚泥法による水処理過程で発生する余剰汚泥を高効率に減容化することができ且つ低コストに実現可能な汚泥減容微生物資材及び余剰汚泥の減容化方法を提供することにある。 The present invention has been made in view of the above points, and the object of the present invention is to reduce the excess sludge generated in the water treatment process by the activated sludge method with high efficiency and at a low cost. An object is to provide a microbial material capable of reducing sludge volume and a method for reducing excess sludge.
本発明者は上記課題を解決するために鋭意検討を重ね、その結果、余剰汚泥を効率よく分解可能な微生物としてエキシグオバクテリウム・エスピー（Exiguobacterium sp.)Z153-2株及びエキシグオバクテリウム・アセチリカム（Exiguobacterium acetylicum)Z153-3株を単離した。 The present inventor has made extensive studies to solve the above-mentioned problems, and as a result, Exiguobacterium sp. Z153-2 strain and Exigobacterium sp. The acetylicum ( Exiguobacterium acetylicum ) Z153-3 strain was isolated.
すなわち上記課題を解決するために成された本発明に係る汚泥減容微生物資材は、
エキシグオバクテリウム・エスピー（Exiguobacterium sp.)Z153-2株（受託番号NITE P-02409）若しくは、エキシグオバクテリウム・アセチリカム（Exiguobacterium acetylicum)Z153-3株（受託番号NITE P-02410）、又はその両方を主成分として含有することを特徴としている。
That is, the sludge volume-reducing microbial material according to the present invention made to solve the above problems is
Exiguobacterium sp. Z153-2 strain (Accession number NITE P-02409) or Exiguobacterium acetylicum Z153-3 strain (Accession number NITE P-02410), or its It is characterized by containing both as main components.
また、上記課題を解決するために成された本発明に係る余剰汚泥の減容化方法は、
活性汚泥にエキシグオバクテリウム・エスピー（Exiguobacterium sp.)Z153-2株（受託番号NITE P-02409）若しくは、エキシグオバクテリウム・アセチリカム（Exiguobacterium acetylicum)Z153-3株（受託番号NITE P-02410）、又はその両方を主成分として含有する汚泥減容微生物資材を添加することにより余剰汚泥を減容化することを特徴としている。
Moreover, the volume reduction method of the excess sludge which concerns on this invention made in order to solve the said subject,
Exiguobacterium sp. Z153-2 strain (Accession number NITE P-02409) or Exiguobacterium acetylicum Z153-3 strain (Accession number NITE P-02410) Further, the present invention is characterized in that excess sludge is reduced in volume by adding a sludge-reducing microbial material containing both of them as main components.
なお、余剰汚泥とは活性汚泥法を利用した排水処理系において、処理槽（例えば曝気槽）内の活性汚泥量を適量に維持するために系外に引き抜かれる汚泥のことであり、本発明における「余剰汚泥の減容化」は、この余剰汚泥の量を減らすこと又は余剰汚泥の発生をなくすことを意味している。 In addition, surplus sludge is sludge drawn out of the system in order to maintain an appropriate amount of activated sludge in a treatment tank (for example, an aeration tank) in a wastewater treatment system using the activated sludge method. “Reducing excess sludge” means reducing the amount of excess sludge or eliminating the generation of excess sludge.
上記本発明に係る汚泥減容微生物資材及び余剰汚泥の減容化方法によれば、前記資材を活性汚泥に添加するだけで余剰汚泥を効率よく且つ低コストに減容化することが可能となる。 According to the sludge volume reducing microbial material and the excess sludge volume reducing method according to the present invention, it is possible to efficiently reduce the excess sludge at low cost simply by adding the material to the activated sludge. .
本発明に係る２種類の微生物は、いずれも土壌から分離された菌株であり、それぞれZ153-2株、Z153-3株と命名した。これらの菌株の分析結果を以下に示す。 The two types of microorganisms according to the present invention are both strains isolated from soil, and are designated as Z153-2 and Z153-3 strains, respectively. The analysis results of these strains are shown below.
1. Ｚ１５３−２株の分析
1-A. 16S rDNA塩基配列解析
DNA抽出はアクロモペプチダーゼ（和光純薬製）により、PCR増幅はPrime STAR HS DNA Polymerase（タカラバイオ製）により、サイクルシークエンスはBigDye Terminator v3.1 Cycle Sequencing Kit（Applied Biosystems製、CA、米国）により、それぞれ実施した。使用プライマーはPCR増幅が9F, 1510R、シークエンスが9F, 785F, 802R, 1510Rである。シークエンスはABI PRISM 3130 xl Genetic Analyzer System（Applied Biosystems製、CA、米国）により行い、塩基配列決定にはChromasPro 1.7（Technelysium Pty Ltd., Tewantin, AUS）を使用した。BLAST相同性検索及び簡易分子系統解析にはソフトウェアとしてアポロン2.0（テクノスルガ・ラボ製）を使用し、データベースとしてアポロンDB-BA9.0（テクノスルガ・ラボ製）及び国際塩基配列データベース（Genbank/DDBJ/EMBL）を使用した。
1. Analysis of Z153-2 strain
1-A. 16S rDNA nucleotide sequence analysis
DNA extraction using achromopeptidase (Wako Pure Chemical Industries), PCR amplification using Prime STAR HS DNA Polymerase (Takara Bio), cycle sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) , Respectively. The primers used are PCR amplification 9F, 1510R, and sequences 9F, 785F, 802R, 1510R. Sequencing was performed using ABI PRISM 3130 xl Genetic Analyzer System (Applied Biosystems, CA, USA), and ChromasPro 1.7 (Technelysium Pty Ltd., Tewantin, AUS) was used for nucleotide sequencing. For BLAST homology search and simple molecular phylogenetic analysis, Apollon 2.0 (manufactured by Technosuruga Lab) is used as software, Apollon DB-BA9.0 (manufactured by Technosuruga Lab) and international base sequence database (Genbank / DDBJ) / EMBL).
上記シークエンスの結果、Z153-2株の16S rDNA（16S rRNA遺伝子）の塩基配列は、配列表の配列番号１に示す通りであった。 As a result of the above sequence, the base sequence of 16S rDNA (16S rRNA gene) of the Z153-2 strain was as shown in SEQ ID NO: 1 in the sequence listing.
上記のアポロンDB-BA9.0に対するBLAST相同性検索の結果、Z153-2株の16S rDNA塩基配列は、Exiguobacterium属の16S rDNA塩基配列に対し高い相同性を示し、エキシグオバクテリウム・プロファンダム（Exiguobacterium profundum)10C株に対し相同率99.7％の最も高い相同性を示した（表１）。なお、表中の「BSL」はバイオセーフティーレベルを意味しており、この欄が空欄のものはバイオセーフティーレベルが１の微生物である。以下、Exiguobacterium profundumをE. profundumと略称する場合がある。 As a result of the BLAST homology search against Apollon DB-BA9.0 above, the 16S rDNA base sequence of Z153-2 strain showed high homology to the 16S rDNA base sequence of the genus Exiguobacterium. ( Exiguobacterium profundum ) 10C strain showed the highest homology of 99.7% (Table 1). “BSL” in the table means a biosafety level, and a blank in this column is a microorganism with a biosafety level of 1. Hereinafter, Exiguobacterium profundum may be abbreviated as E. profundum .
Genbank/DDBJ/EMBLに対するBLAST相同性検索の結果においても、Z153-2株の16S rDNA塩基配列は、Exiguobacterium属の16S rDNA塩基配列に対し高い相同性を示したが、基準株に由来する16S rDNA塩基配列は検索されなかった（表２参照）。 In the results of BLAST homology search against Genbank / DDBJ / EMBL, the 16S rDNA base sequence of the Z153-2 strain showed high homology to the 16S rDNA base sequence of the genus Exiguobacterium , but the 16S rDNA derived from the reference strain The nucleotide sequence was not searched (see Table 2).
アポロンDB-BA9.0に対する相同性検索で得られた上位14塩基配列を用いた16S rDNA塩基配列に基づく簡易分子系統解析の結果を図１に示す。なお、図中において系統枝の分岐に位置する数字はブートストラップ値であり、左下の線はスケールバーを示している。また、株名の末尾のTはその種の基準株（Type strain）を示している。同図から明らかなように、Z153-2株はExiguobacterium属の種で形成されるクラスターに含まれた。また、Z153-2株はE. profundumとクラスターを形成し、このクラスターは98％の高いブートストラップ値で支持され近縁であることが示された。 FIG. 1 shows the results of a simple molecular phylogenetic analysis based on the 16S rDNA base sequence using the top 14 base sequences obtained by homology search for apolone DB-BA9.0. In the figure, the number located at the branch of the system branch is the bootstrap value, and the lower left line indicates the scale bar. Moreover, T at the end of the strain name indicates the reference strain (Type strain) of the species. As is clear from the figure, the Z153-2 strain was included in the cluster formed by the species of the genus Exiguobacterium . In addition, the Z153-2 strain formed a cluster with E. profundum, and this cluster was supported by a high bootstrap value of 98% and shown to be closely related.
以上のことから、Z153-2株はExiguobacterium属に含まれE. profundumに帰属する可能性が考えられたが、プライマー箇所を除くZ153-2株とE. profundumの16S rDNA塩基配列間には4塩基の相違点が確認された。このうち3塩基の相違はギャップ、混合塩基（IUBコード R=A又はG）及びE. profundumの登録配列におけるN（塩基の未決定）によることから、これらの相違を両者の明確な差として捉えることは難しいものの、残りの一塩基の相違は明確な差と捉えられる。
このことから、Z153-2株はE. profundumに近縁ではあるものの、両者が種として異なる可能性も否定できない。そのため、Z153-2株をE. profundumに近縁なExiguobacterium sp.と推定した。
Based on the above, it was considered that the Z153-2 strain was included in the genus Exiguobacterium and attributed to E. profundum , but there was a 4S rDNA base sequence between the Z153-2 strain and the E. profundum 16S rDNA base sequence excluding the primer site. Base differences were confirmed. Among these, the difference of 3 bases is due to gap, mixed base (IUB code R = A or G) and N (base undecided) in E. profundum registration sequence, so these differences are regarded as clear differences between the two Although difficult, the difference between the remaining single bases is regarded as a clear difference.
From this, although Z153-2 strain is closely related to E. profundum , it cannot be denied that they may be different species. Therefore, the Z153-2 strain was estimated to be an Exiguobacterium sp. Closely related to E. profundum .
1-B. 形態観察
光学顕微鏡による形態観察を行うと共に、BARROWらの方法（BARROW, (G.I.) and FELTHAM, (R.K.A）:Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edition. 1993, Cambridge University Press.)に基づき、カタラーゼ反応、オキシダーゼ反応、ブドウ糖からの酸／ガス産生、ブドウ糖の酸化／発酵（O/F）について試験を行った。なお、グラム染色にはフェイバーG「ニッスイ」（日水製薬製）を使用し、顕微鏡としては光学顕微鏡BX50F4（オリンパス製）を使用した。
1-B. Morphological observation In addition to morphological observation with an optical microscope, the method of BARROW et al. (BARROW, (GI) and FELTHAM, (RKA): Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edition. 1993, Cambridge University Press.), Catalase reaction, oxidase reaction, acid / gas production from glucose, glucose oxidation / fermentation (O / F) were tested. In addition, Faber G “Nissui” (manufactured by Nissui Pharmaceutical) was used for Gram staining, and an optical microscope BX50F4 (manufactured by Olympus) was used as a microscope.
その結果、Z153-2株は運動性を有するグラム陽性桿菌で、芽胞を形成せず、Nutrient agar培地上でコロニー色は黄色を呈し、グルコースを酸化せず、カタラーゼ反応は陽性、オキシダーゼ反応は陰性を示した（表３）。これらの性状は、16S rDNA塩基配列解析の結果において帰属が示唆されたExiguobacterium属の性状と一致した。 As a result, the Z153-2 strain is a gram-positive gonococci with motility, does not form spores, has a colony color of yellow on Nutrient agar medium, does not oxidize glucose, and catalase reaction is positive, oxidase reaction is negative (Table 3). These properties were consistent with the properties of the genus Exiguobacterium whose assignment was suggested in the results of 16S rDNA nucleotide sequence analysis.
1-C. 生理・生化学試験
細胞同定キット（APICORYNE, bioMerieux製, Lyon, France）による生理・生化学試験を行ったところ、Z153-2株は硝酸塩を還元し、α-グルコシダーゼ及びβ-グルコシダーゼ活性を示し、ゼラチンを加水分解し、グルコース、リボース、及びマンニトールなどを酸化し、キシロース及びラクトースを酸化しなかった（図２）。
1-C. Physiological and biochemical tests Physiological and biochemical tests were performed using a cell identification kit (APICORYNE, bioMerieux, Lyon, France). Z153-2 reduced nitrate, α-glucosidase and β-glucosidase. It showed activity, hydrolyzed gelatin, oxidized glucose, ribose, mannitol, etc. and did not oxidize xylose and lactose (FIG. 2).
また、英国NCIMB Ltd.の方法及び分類・同定の関連文献に従い、追加試験を実施したところ、Z153-2株は嫌気条件下で生育せず、15℃及び45℃で生育し、50℃で生育せず、7％NaClで生育し、10％NaClで生育せず、アミグダリン、アルブチン及びセロビオースなどを酸化し、D-キシロースを酸化しなかった（表４）。これらの性状は、16S rDNA塩基配列解析の結果において近縁性が示唆されたE. profundumの性状に類似点があるものの、相違点も確認された。特に10％NaClで生育せず、メリビオース及びDラフィノースを酸化し、D-キシロースを酸化しない点でE. profundumの性状と相違していた。 In addition, when additional tests were conducted in accordance with the NCIMB Ltd. method and classification / identification related literature, the Z153-2 strain did not grow under anaerobic conditions, but grew at 15 ° C and 45 ° C and grew at 50 ° C. Without growth, it grew with 7% NaCl, did not grow with 10% NaCl, oxidized amygdalin, arbutin and cellobiose, and did not oxidize D-xylose (Table 4). Although these properties were similar to the properties of E. profundum , which was suggested to be related in the results of 16S rDNA nucleotide sequence analysis, differences were also confirmed. In particular, it was different from the properties of E. profundum in that it did not grow with 10% NaCl, oxidized melibiose and D raffinose, and did not oxidize D-xylose.
以上の結果から、Z153-2株はExiguobacterium属に含まれ、既知種ではE. profundumに最も近縁と考えられる。しかし、上記の16S rDNA塩基配列解析及び生理・生化学性状試験の結果は、Z153-2株がE. profundumとはやや異なることを示唆していることから、種レベルでの帰属分類群を推定することは難しく、Z153-2株をE. profundumに近縁なExiguobacterium sp.とすることが妥当と判断した。なお、E. profundumはバイオセーフティーレベル１の微生物である。
From the above results, the Z153-2 strain is included in the genus Exiguobacterium , and it is considered that E. profundum is most closely related to known species. However, the results of the above 16S rDNA nucleotide sequence analysis and physiological and biochemical property tests suggest that the Z153-2 strain is slightly different from E. profundum, and therefore the taxonomic group at the species level is estimated. Therefore, it was judged appropriate to use Exiguobacterium sp. Closely related to E. profundum as the Z153-2 strain. E. profundum is a
2. Ｚ１５３−３株の分析
2-A. 16S rDNA部分塩基配列解析
上記1-Aと同様にしてZ153-3株の16S rDNA塩基配列解析を行った。但し、プライマーとしては、PCR増幅に9F, 1406Rを使用し、シークエンスに9F, 536Rを使用した。
2. Analysis of Z153-3 strain
2-A. 16S rDNA Partial Base Sequence Analysis 16S rDNA base sequence analysis of Z153-3 strain was performed in the same manner as in 1-A above. However, as primers, 9F and 1406R were used for PCR amplification, and 9F and 536R were used for sequencing.
上記シークエンスの結果、Z153-3株の16S rDNA（16S rRNA遺伝子）の部分塩基配列は、配列表の配列番号２に示す通りであった。 As a result of the above sequence, the partial base sequence of 16S rDNA (16S rRNA gene) of the Z153-3 strain was as shown in SEQ ID NO: 2 in the sequence listing.
アポロンDB-BA9.0に対するBLAST相同性検索の結果、Z153-3株の16S rDNA部分塩基配列は、Exiguobacterium属の16S rDNA塩基配列に対し高い相同性を示し、エキシグオバクテリウム・アセチリカム（Exiguobacterium acetylicum)DSM20416株に対し相同率99.6％の最も高い相同性を示した（表５）。以下、Exiguobacterium acetylicumをE. acetylicumと略称する場合がある。 As a result of BLAST homology search against Apollon DB-BA9.0, the 16S rDNA partial base sequence of Z153-3 strain showed high homology to the 16S rDNA base sequence of Exiguobacterium genus, and Exiguobacterium acetylicum ) It showed the highest homology with a homology rate of 99.6% with respect to the DSM20416 strain (Table 5). Hereinafter, Exiguobacterium acetylicum may be abbreviated as E. acetylicum .
Genbank/DDBJ/EMBLに対するBLAST相同性検索の結果においても、Z153-3株の16S rDNA部分塩基配列は、Exiguobacterium属の16S rDNA塩基配列に対し高い相同性を示し、基準株ではE. acetylicum DSM20416株及びNBRC12146株に対し、相同率99.6%の高い相同性を示した（表６）。 In the results of BLAST homology search against Genbank / DDBJ / EMBL, the 16S rDNA partial nucleotide sequence of the Z153-3 strain showed high homology to the 16S rDNA nucleotide sequence of the genus Exiguobacterium, and E. acetylicum DSM20416 strain was the reference strain And NBRC12146 strain showed a high homology of 99.6% (Table 6).
アポロンDB-BA9.0に対する相同性検索で得られた上位10塩基配列を用いた16S rDNA部分塩基配列に基づく簡易分子系統解析の結果、Z153-3株はExiguobacterium属の種で形成されるクラスターに含まれた（図３）。また、Z153-3株はE. acetylicumとクラスターを形成し、両者は同一の分子系統学的位置を示した。 As a result of simple molecular phylogenetic analysis based on the 16S rDNA partial base sequence using the top 10 base sequences obtained by homology search for Apollon DB-BA9.0, Z153-3 strain was found to be a cluster formed by species of the genus Exiguobacterium Was included (FIG. 3). The Z153-3 strain formed a cluster with E. acetylicum, and both showed the same molecular phylogenetic position.
以上のことから、Z153-3株はExiguobacterium属に含まれE. acetylicumに帰属する可能性が考えられた。Z153-3株とE. acetylicumのプライマー箇所を除く16S rDNA部分塩基配列間には2塩基の相違点が確認されたが、これらの相違はいずれも混合塩基（IUBコード R=G又はA、Y=T又はC）によることから、両者はほぼ一致すると捉えられる。 From the above, the Z153-3 strain likely attributable to E. Acetylicum included in Exiguobacterium genus were considered. A difference of 2 bases was confirmed between the 16S rDNA partial nucleotide sequences except the Z153-3 strain and the E. acetylicum primer, but these differences were all mixed bases (IUB code R = G or A, Y = T or C), it can be seen that the two are almost the same.
2-B. 形態観察
グラム染色にフェイバーG「ニッスイ」（日水製薬製）を使用すると共に、光学顕微鏡BX50F4（オリンパス製）及び実体顕微鏡SZH10（オリンパス製）を使用してZ153-3株の簡易形態観察を行った。
その結果、Z153-3株はグラム染色陽性、好気条件下での生育を示す桿菌で、Nutrient agar平板培地上でのコロニー色は淡黄色から黄色を呈した（表７）。この形態観察の結果は、Exiguobacterium属の一般的な性状と一致した。
2-B. Morphological observation Using Favor G “Nissui” (manufactured by Nissui Pharmaceutical Co., Ltd.) for Gram staining, using the optical microscope BX50F4 (manufactured by Olympus) and stereo microscope SZH10 (manufactured by Olympus) Morphological observation was performed.
As a result, the Z153-3 strain was a gonococcus showing positive Gram staining and growing under aerobic conditions, and the colony color on the Nutrient agar plate medium was pale yellow to yellow (Table 7). The results of this morphological observation were consistent with the general properties of the genus Exiguobacterium .
以上の結果から、Z153-3株はExiguobacterium acetylicumと推定した。なお、E. acetylicumはバイオセーフティーレベル１の微生物である。
From the above results, the Z153-3 strain was estimated to be Exiguobacterium acetylicum . E. acetylicum is a
上記のZ153-2株及びZ153-3株は、独立行政法人製品評価技術基盤機構特許微生物寄託センターに寄託され、それぞれ受託番号NITE P-02409及びNITE P-02410が付与されている（受託日はいずれも2017年1月24日）。 The above Z153-2 and Z153-3 strains have been deposited with the Patent Microorganisms Depositary Center for Product Evaluation and Technology, and have been assigned the deposit numbers NITE P-02409 and NITE P-02410, respectively (the deposit date is Both are January 24, 2017).
本発明に係る汚泥減容微生物資材は、上記の微生物（すなわちExiguobacterium sp. Z153-2株及びExiguobacterium acetylicum Z153-3株）のいずれか又は両方を主成分として含有するものである。該微生物の資材化方法としては、例えば、村尾澤夫他１名編「応用微生物学改訂版」（1993年、培風館）、上島孝之著「産業用酵素」（1995年、丸善）、又は微生物研究法懇談会編、「微生物学実験法」（1993年、講談社）などに記載されている方法を用いることができる。以下に具体的な資材化方法を列挙するが、本発明に係る汚泥減容微生物資材は下記の方法で製造された物に限定されるものではない。 The sludge-reducing microbial material according to the present invention contains one or both of the above microorganisms (namely, Exiguobacterium sp. Z153-2 and Exiguobacterium acetylicum Z153-3) as a main component. Examples of the materialization method of the microorganism include Sawao Murao et al., “Applied Microbiology Revised Edition” (1993, Baifukan), Takayuki Uejima “Industrial Enzyme” (1995, Maruzen), or Microbial Research Method The methods described in the round-table discussion, “Microbiology Experimental Method” (1993, Kodansha) can be used. Although the specific materialization method is enumerated below, the sludge volume reduction microbial material which concerns on this invention is not limited to the thing manufactured by the following method.
液状資材とする場合には、例えば、以下のいずれかの方法で資材化することができる。
（Ａ）上記微生物を肉汁培地（ブイヨン培地）などの一般栄養培地で所定の時間に亘って培養し、必要に応じてこれにｐＨ調整剤などを添加して資材とする。
（Ｂ）遠心分離等により上記（Ａ）の培養物から菌体を回収し、該菌体を生理食塩水等の媒体に適当な濃度となるように懸濁する。そして、必要に応じてこれにｐＨ調整剤などを加えて資材とする。
（Ｃ）凍結乾燥等により上記（Ａ）の培養物を適当な濃度に濃縮し、必要に応じてこれにｐＨ調整剤等を添加して資材とする。
（Ｄ）遠心分離等により上記（Ａ）の培養物から菌体を回収し、該菌体を肉汁培地等の培地に懸濁する。そして、必要に応じてこれにｐＨ調整剤などを加えて資材とする。
（Ｅ）上記（Ｄ）を更に凍結乾燥等によって適当な濃度に濃縮して資材とする。
In the case of a liquid material, for example, it can be made into a material by any of the following methods.
(A) The microorganism is cultured in a general nutrient medium such as a broth medium (bouillon medium) for a predetermined time, and a pH adjuster or the like is added to the medium as necessary to obtain a material.
(B) Bacteria are collected from the culture of (A) by centrifugation or the like, and suspended in a medium such as physiological saline to a suitable concentration. Then, if necessary, a pH adjuster or the like is added to make a material.
(C) The culture of (A) above is concentrated to an appropriate concentration by freeze-drying or the like, and a pH adjuster or the like is added thereto as necessary to obtain a material.
(D) The cells are collected from the culture of (A) by centrifugation or the like, and the cells are suspended in a medium such as a broth medium. Then, if necessary, a pH adjuster or the like is added to make a material.
(E) The above (D) is further concentrated to an appropriate concentration by freeze-drying or the like to obtain a material.
粉末資材とする場合には、例えば、以下のいずれかの方法で資材化することができる。
（ａ）上記微生物を肉汁培地などの一般栄養培地で所定の時間に亘って培養し、必要に応じてこれにｐＨ調整剤などを加え、凍結乾燥等によって乾燥させて資材とする。
（ｂ）遠心分離等により上記（ａ）の培養物から菌体を回収し、該菌体を生理食塩水又はスキムミルクとグルタミン酸ナトリウム等を含む溶液等の媒体に適当な濃度となるように懸濁し、必要に応じてこれにｐＨ調整剤などを加え、凍結乾燥等により乾燥させて資材とする。
（ｃ）遠心分離等により上記（ａ）の培養物から菌体を回収し、該菌体を肉汁培地等の培地に懸濁し、必要に応じてこれにｐＨ調整剤等を加え、凍結乾燥等によって乾燥させて資材とする。
（ｄ）上記（ａ）から（ｃ）のものに、繊維くず、おがくず、白土、ケイソウ土などの微粉体を加えて資材とする。
In the case of a powder material, for example, it can be made into a material by any of the following methods.
(A) The microorganism is cultured in a general nutrient medium such as a gravy medium for a predetermined time, and a pH adjuster or the like is added thereto as necessary, and dried by lyophilization or the like to obtain a material.
(B) The cells are collected from the culture of (a) by centrifugation or the like and suspended in a medium such as a solution containing physiological saline or skim milk and sodium glutamate to a suitable concentration. If necessary, a pH adjuster or the like is added to this and dried by lyophilization or the like to obtain a material.
(C) The cells are collected from the culture of (a) by centrifugation or the like, and the cells are suspended in a medium such as a gravy medium, and a pH adjuster is added thereto as necessary, followed by freeze-drying, etc. To dry the material.
(D) Fine powders such as fiber waste, sawdust, white clay, and diatomaceous earth are added to the above (a) to (c) to obtain materials.
また、上記の方法の他に、担体結合法、架橋法、包括法、複合法等の公知技術により、上記の微生物を種々の固定化用材料によって固定化してもよい。更に、上記微生物を他の公知の錠剤化技術によって錠剤化するようにしてもよい。 In addition to the above methods, the above microorganisms may be immobilized with various immobilization materials by a known technique such as a carrier binding method, a crosslinking method, an entrapment method, or a composite method. Further, the microorganisms may be tableted by other known tableting techniques.
本発明に係る余剰汚泥の減容化方法は、活性汚泥に上記の汚泥減容微生物資材を添加し、該活性汚泥を該資材中のExiguobacterium sp. Z153-2株及び／又はExiguobacterium acetylicum Z153-3株に分解させることで余剰汚泥の減容化を図るものである。例えば、上述の活性汚泥法を用いた有機性排水の処理施設における曝気槽に本発明に係る汚泥減容微生物資材を添加することにより、該曝気槽で発生する余剰汚泥を減容化することができる。このとき、前記微生物資材の添加量は、曝気槽における１日あたりの活性汚泥の発生量と、該微生物資材による活性汚泥の減少量とがほぼ同量となるように調整することが望ましい。これにより曝気槽内の活性汚泥の量を、排水中の有機性汚濁物質を十分に分解・除去可能であって、且つ余剰汚泥が発生しない程度の量に維持することが可能となる。 The method for reducing excess sludge according to the present invention comprises adding the above-mentioned sludge-reducing microorganism material to activated sludge, and adding the activated sludge to the Exiguobacterium sp. Z153-2 strain and / or Exiguobacterium acetylicum Z153-3 in the material. It is intended to reduce the volume of excess sludge by breaking it down into stock. For example, the volume of excess sludge generated in the aeration tank can be reduced by adding the sludge volume reducing microbial material according to the present invention to the aeration tank in the organic wastewater treatment facility using the activated sludge method described above. it can. At this time, it is desirable to adjust the addition amount of the microbial material so that the generation amount of activated sludge per day in the aeration tank and the decrease amount of the activated sludge by the microbial material become substantially the same amount. As a result, the amount of activated sludge in the aeration tank can be maintained at such an amount that the organic pollutants in the waste water can be sufficiently decomposed and removed and no excess sludge is generated.
本発明に係る余剰汚泥の減容化方法は、いわゆる標準活性汚泥法、すなわち上述のように、曝気槽で処理した水（処理水）を汚泥と共に沈殿槽に送り、汚泥を沈殿させることで処理水と汚泥を分離する方式の排水処理設備のほか、膜分離活性汚泥法（ＭＢＲ法, Membrane Bioreactor法）による排水処理設備にも同様に適用することができる。ＭＢＲ法は、活性汚泥法の一種であり、上記の沈殿槽に代えて、精密ろ過膜（ＭＦ膜）又は限外ろ過膜（UF膜）等のフィルターを用いて汚泥と処理水の分離を行うものである。 The surplus sludge volume reduction method according to the present invention is a so-called standard activated sludge method, that is, as described above, the water (treated water) treated in the aeration tank is sent to the sedimentation tank together with the sludge, and the sludge is precipitated. In addition to wastewater treatment equipment that separates water and sludge, wastewater treatment equipment using the membrane separation activated sludge method (MBR method, Membrane Bioreactor method) can also be applied. The MBR method is a kind of activated sludge method, and the sludge and treated water are separated using a filter such as a microfiltration membrane (MF membrane) or an ultrafiltration membrane (UF membrane) instead of the above-described sedimentation tank. Is.
以下、本発明に係る微生物であるエキシグオバクテリウム・エスピー（Exiguobacterium sp.)Z153-2株（受託番号NITE P-02409）及びエキシグオバクテリウム・アセチリカム（Exiguobacterium acetylicum)Z153-3株（受託番号NITE P-02410）の効果を確認するために行った試験例について説明する。 Hereinafter, the microorganisms according to the present invention, Exiguobacterium sp. Z153-2 strain (Accession No. NITE P-02409) and Exiguobacterium acetylicum Z153-3 strain (Accession No.) A test example conducted to confirm the effect of NITE P-02410) will be described.
（試験例１）
まず、以下の方法により本発明に係る微生物のプロテアーゼ活性を測定した。ここで、微生物のプロテアーゼ活性とは、当該微生物が最適環境下で培地中に分泌するタンパク質分解酵素の量（酵素活性の量）を意味する。
(Test Example 1)
First, the protease activity of the microorganism according to the present invention was measured by the following method. Here, the protease activity of a microorganism means the amount of proteolytic enzyme (amount of enzyme activity) secreted into the medium by the microorganism under an optimum environment.
プロテアーゼ活性測定に際しては、Exiguobacterium sp. Z153-2株及びExiguobacterium acetylicum Z153-3株を、それぞれスキムミルクを添加した人工下水培地で培養して使用した。
具体的には、人工下水培地100 ml（組成はYE（Yeast Extract）0.04％、バクトペプトン0.03％、牛肉エキス0.03％、リン酸水素二ナトリウム0.05％、尿素0.05％）にスキムミルクを1重量％添加したものを500 mL三角フラスコに入れ、pH 7.5程度に調整した上で汚泥を2〜3％加えた（これによりpH7程度となった）。これをオートクレーブ(121℃、15分)で滅菌した後、硫酸マグネシウム0.010％、塩化ナトリウム0.015％ 、塩化カルシウム0.009％、塩化カリウム0.007％を加え、前培養液を1体積％添加して25℃、120 rpmで振盪培養した。
For measuring protease activity, Exiguobacterium sp. Z153-2 and Exiguobacterium acetylicum Z153-3 were each cultured in an artificial sewage medium supplemented with skim milk.
Specifically, 100% artificial sewage medium (composition is YE (Yeast Extract) 0.04%, bactopeptone 0.03%, beef extract 0.03%, disodium hydrogen phosphate 0.05%, urea 0.05%) and 1% by weight skim milk. The resulting solution was placed in a 500 mL Erlenmeyer flask and adjusted to about pH 7.5, and then 2 to 3% of sludge was added (this resulted in a pH of about 7). After sterilizing this in an autoclave (121 ° C, 15 minutes), add magnesium sulfate 0.010%, sodium chloride 0.015%, calcium chloride 0.009%, potassium chloride 0.007%, and add 1% by volume of the pre-culture solution at 25 ° C. Cultured with shaking at 120 rpm.
培養初日（０日目）〜７日目の各日に培養液の一部を採取し、該培養液を遠心分離して得られた上清をプロテアーゼ活性測定に用いる試料とした。100 mM Tris-HCl（pH 7.2、終濃度76 mM）190 μLに3％アゾカゼイン（終濃度0.6％）50 μLを混合し、これに上述の試料10 μLを添加して37℃で5分間静置して反応させた。その後、20％トリクロロ酢酸（終濃度3.3％）を50 μL加えて反応を停止させ、反応液を遠心分離して上清の340 nm（アゾカゼインの分解で生じるアゾ色素の吸収波長）の吸光度を測定した。なお、検量線はProteinase K（40.1 U/mg）を標準とした。 A part of the culture solution was collected on each day of the first culture day (day 0) to the seventh day, and the supernatant obtained by centrifuging the culture solution was used as a sample for measuring protease activity. Mix 190 μL of 100 mM Tris-HCl (pH 7.2, final concentration 76 mM) with 50 μL of 3% azocasein (final concentration 0.6%), add 10 μL of the above sample to this, and allow to stand at 37 ° C. for 5 minutes. And reacted. Then, 50 μL of 20% trichloroacetic acid (final concentration: 3.3%) was added to stop the reaction, and the reaction mixture was centrifuged, and the absorbance of the supernatant was measured at 340 nm (absorption wavelength of the azo dye generated by azocasein degradation). did. Note that the standard curve was Proteinase K (40.1 U / mg).
以上の結果、培養０日目〜７日目におけるプロテアーゼ活性は図４に示す通りであった。なお、1 U（ユニット）は1分間にアゾ色素を1 μmol生成させることのできる酵素量である。同図から明らかなように、Exiguobacterium sp. Z153-2株のプロテアーゼ活性は約5 U/mlであり、Exiguobacterium acetylicum Z153-3株のプロテアーゼ活性は4 U/mlであった。
As a result of the above, the protease activity on
（試験例２）
続いて、本発明に係る微生物資材による汚泥の減容効果を確認した。この試験では、処理対象の汚泥を含む排水として標準活性汚泥法による排水処理設備の曝気槽から採取した水を使用した。本発明に係る微生物資材としては、Exiguobacterium sp. Z153-2株の培養液（以下「実施例資材１」とよぶ）及びExiguobacterium acetylicum Z153-3株の培養液（以下「実施例資材２」とよぶ）を使用した。なお、前記培養液は各微生物を乾燥ブイヨン培地（日水製薬株式会社製）を用いて、25℃、100 rpmで24時間振盪培養したものである。また、従来の微生物資材（以下「比較例資材」とよぶ）として、有限会社アースラブ・ニッポン製の有用微生物資材アースラブ（粉剤）を使用した。なお、各微生物資材 1 ml（比較例資材については 1 g）中に含まれる生菌数を蛍光標識法（CFDA染色法）で測定した結果、実施例資材１では3.6×108 cells、実施例資材２では2.4×108 cells、比較例資材では3.5×108 cellsとほぼ同程度であった。
(Test Example 2)
Then, the volume reduction effect of the sludge by the microorganism material which concerns on this invention was confirmed. In this test, water collected from an aeration tank of a wastewater treatment facility using a standard activated sludge method was used as wastewater containing sludge to be treated. The microorganism material according to the present invention includes a culture solution of Exiguobacterium sp. Z153-2 strain (hereinafter referred to as “
1 Lのガラスビーカーに処理対象とする汚泥を含んだ排水（食品工場の活性汚泥処理槽水）1 Lを入れ、そこに前記資材のいずれかを添加した。添加量は実施例資材１及び実施例資材２については1 ml、比較例資材については1 gとした。そして、エアレーションと撹拌により前記排水中の浮遊懸濁物質（Solid Suspension、SS）を完全に懸濁させ、水温25℃で4日間培養を行った。また対照として、前記排水1 Lにいずれの資材も添加しなかったものについても同様の条件で4日間に亘りエアレーションと撹拌を行った。そして、培養開始時（０日目）、24時間後（１日目）、及び96時間後（４日目）における排水中の汚泥量を以下の手順で測定した。
A 1 L glass beaker was charged with 1 L of waste water containing sludge to be treated (activated sludge treatment tank water in food factories), and any of the above materials was added thereto. The amount added was 1 ml for
すなわち、前記ビーカー内の排水を撹拌して汚泥を均一に懸濁させた状態で、該排水の一部を試料として採取し、該試料を予め質量を量っておいたフィルター（孔径1 μmのガラス繊維濾紙）で濾過した。そして、フィルター上に残った物質を高温で乾燥させてから全重量を量り、次の式により排水1 Lあたりの乾燥汚泥重量（MLSS[mg/L]）を算出した。なお、MLSS（Mixed Liquor Suspended Solids）は、活性汚泥処理における曝気槽内の汚泥混合液の浮遊物質を意味しており、以下の式においてaは乾燥後のフィルター及び該フィルター上の物質の質量[mg]、bはフィルターの質量[mg]である。
MLSS[mg/L]=((a-b)÷試料量[ml])×1000
That is, in a state where the waste water in the beaker is stirred and sludge is uniformly suspended, a part of the waste water is collected as a sample, and the sample is preliminarily weighed with a filter (having a pore diameter of 1 μm). Filtered through a glass fiber filter paper). Then, the substance remaining on the filter was dried at a high temperature and then weighed, and the dry sludge weight (MLSS [mg / L]) per 1 L of wastewater was calculated by the following formula. In addition, MLSS (Mixed Liquor Suspended Solids) means the suspended substance of the sludge mixed liquid in the aeration tank in the activated sludge treatment. In the following formula, a is the mass of the filter after drying and the substance on the filter [ mg], b is the mass [mg] of the filter.
MLSS [mg / L] = ((ab) ÷ sample amount [ml]) × 1000
上記測定の結果を表８及び図５に示す。なお、表８中の「汚泥分解量」は１日目又は４日目の汚泥量と０日目の汚泥量との差を意味している。また、「汚泥分解率」は（（４日目の汚泥量−０日目の汚泥量）÷０日目の汚泥量）×100を意味している。 The results of the above measurement are shown in Table 8 and FIG. The “sludge decomposition amount” in Table 8 means the difference between the sludge amount on the first or fourth day and the sludge amount on the zeroth day. The “sludge decomposition rate” means ((sludge amount on the fourth day−sludge amount on the zeroth day) ÷ sludge amount on the zeroth day) × 100.
一般に、標準活性汚泥法を用いた処理施設における汚泥発生量は１日あたり約200 mg/Lといわれており、そのうち約半分の100 mg/Lが余剰汚泥になるとされている。これに対し、上記の試験例において微生物資材を添加しなかった排水、すなわち通気撹拌のみを行った排水における汚泥の減少量は、表８に示す通り４日間で1 Lあたり150 mg、１日あたりに換算すると37.5 mg/Lであった。これは１日に生成される余剰汚泥の量を下回るため、通気撹拌を行っただけでは処理槽内の汚泥は増加し続けることになる。これが標準活性汚泥法における余剰汚泥発生の原因である。比較例資材では、４日間で1 Lあたり370mg(１日あたり92.5mg/L)であり、依然として分解力は満足するものではない。一方、実施例資材１を添加した排水における汚泥の減少量は４日間で1 Lあたり660 mg(１日にあたり165 mg/L)、実施例資材２を添加した排水における汚泥の減少量は４日間で１Lあたり710 mg（１日あたり177.5 mg/L）であり、上記１日に生成される余剰汚泥の量（100 mg/L）を上回った。このことから、本発明に係る微生物資材の添加により処理槽内における余剰汚泥の増加を抑えることが可能であると考えられる。また、上記の表８及び図５から明らかなように、実施例資材１又は実施例資材２を添加した排水では、比較例資材を添加した排水よりも高い汚泥減少効果が得られることが確認された。
Generally, the amount of sludge generated in a treatment facility using the standard activated sludge method is said to be about 200 mg / L per day, and about half of that amount is 100 mg / L. On the other hand, as shown in Table 8, the amount of sludge reduction in the wastewater to which no microbial material was added in the above test example, that is, wastewater with only aeration stirring, was 150 mg per liter per day for 4 days. Converted to 37.5 mg / L. Since this is less than the amount of surplus sludge produced on a single day, the sludge in the treatment tank will continue to increase only by aeration and stirring. This is the cause of excess sludge generation in the standard activated sludge method. The comparative material is 370 mg per liter for 4 days (92.5 mg / L per day), and the degradation power is still not satisfactory. On the other hand, the decrease in sludge in the wastewater to which
（試験例３）
更に、ＭＢＲ法による排水処理に対する本発明に係る微生物資材の有用性を確認した。本試験例では、処理対象の汚泥を含む排水として長野県の個人住宅に設置されたＭＢＲ処理方式の浄化槽から採取した排水を使用した。
該排水1 Lに上記試験例２と同様の「実施例資材２」を1 ml添加し、試験例２と同様にエアレーションと撹拌を行って25℃で４日間培養を行った。また、比較例として微生物資材を添加しない排水1 Lについても同様にエアレーションと撹拌を行った。
(Test Example 3)
Furthermore, the usefulness of the microbial material according to the present invention for wastewater treatment by the MBR method was confirmed. In this test example, wastewater collected from an MBR treatment septic tank installed in a private house in Nagano Prefecture was used as wastewater containing sludge to be treated.
1 ml of “
培養開始時（０日目）から４日目における汚泥量（MLSS）の推移を図６に示す。なお、汚泥量の測定は上記試験例２と同様の方法で行った。同図に示すように実施例資材１を添加したものでは、微生物資材を添加しなかったものに比べて、汚泥量が大きく減少していた。なお、ＭＢＲ法では汚泥量が15,000 mg/Lを超えると処理水と汚泥を分離するためのフィルターが目詰まりする危険性が出てくるが、実施例資材１の添加により、当初約16,000mg/Lであった汚泥量が１日で危険値である15,000 mg/L以下に低下し、４日目には13,100 mg/Lと危険値を大きく下回ることが確認された。
FIG. 6 shows the change in the amount of sludge (MLSS) from the start of culture (day 0) to the fourth day. The amount of sludge was measured by the same method as in Test Example 2 above. As shown in the figure, the amount of sludge was greatly reduced in the case where the
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| WO2021093290A1 (en) * | 2019-11-12 | 2021-05-20 | 青岛科技大学 | Protease-producing deep-sea exiguobacterium mutant strain and use thereof |
| WO2021193581A1 (en) * | 2020-03-24 | 2021-09-30 | 三菱ケミカル株式会社 | Method for treating drainage water |
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| WO2021093290A1 (en) * | 2019-11-12 | 2021-05-20 | 青岛科技大学 | Protease-producing deep-sea exiguobacterium mutant strain and use thereof |
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