JP4603880B2 - Novel microorganism BS-HA1 strain, method for microbial degradation of rubber - Google Patents
Novel microorganism BS-HA1 strain, method for microbial degradation of rubber Download PDFInfo
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- JP4603880B2 JP4603880B2 JP2004379885A JP2004379885A JP4603880B2 JP 4603880 B2 JP4603880 B2 JP 4603880B2 JP 2004379885 A JP2004379885 A JP 2004379885A JP 2004379885 A JP2004379885 A JP 2004379885A JP 4603880 B2 JP4603880 B2 JP 4603880B2
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J11/00—Recovery or working-up of waste materials
- C08J11/04—Recovery or working-up of waste materials of polymers
- C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
- C08J11/105—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation by treatment with enzymes
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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Description
本発明は、ポリイソプレン系ゴムを分解する作用を有する、ノカルディア属に属する放線菌ＢＳ−ＨＡ１株（ＦＥＲＭ Ｐ−１９３７８）に関する。更に本発明は、ポリイソプレン系ゴムを含有するゴム組成物を、ノカルディア属に属する放線菌であるＢＳ−ＨＡ１株（ＦＥＲＭ Ｐ−１９３７８）で微生物分解する方法に関する。 The present invention relates to an actinomycete BS-HA1 strain (FERM P-19378) belonging to the genus Nocardia, which has an action of degrading polyisoprene rubber. Furthermore, the present invention relates to a method for microbial degradation of a rubber composition containing a polyisoprene rubber with BS-HA1 strain (FERM P-19378) which is an actinomycete belonging to the genus Nocardia.
廃タイヤは一般のプラスチック製品と比較しても回収率は高く、特にセメント工場を中心として燃料として再利用されている。しかしながら近年環境問題の高まりとともに、廃タイヤを燃料として燃やすのではなく、コンポストのように低温で分解する省エネルギー型の方法や分解した材料を再利用するマテリアルリサイクルの方法の開発が求められている。 Waste tires have a higher recovery rate than ordinary plastic products, and are reused as fuel, especially in cement factories. However, with the recent increase in environmental problems, development of energy-saving methods that decompose at low temperatures, such as compost, and material recycling methods that recycle the decomposed material, is not required, instead of burning waste tires as fuel.
ゴム製品を分解する方法として、微生物による分解処理が考えられる。微生物分解は低温での処理なので、エネルギー消費は最も少ない処理法といえる。また、ゴム製品を微生物分解した後、分解物を再利用することも考えられる。例えば、分解されたモノマーやオリゴマーを再利用することや、粉ゴムの表面を微生物分解し、再度未加硫ゴムに混練り加硫することにより、新たな製品に利用することも考えられる。 As a method for decomposing a rubber product, a decomposing process using microorganisms can be considered. Microbial degradation is a low-temperature treatment, so energy consumption is the least treatment method. It is also conceivable to recycle the decomposition product after microbial decomposition of the rubber product. For example, it is conceivable to reuse the decomposed monomer or oligomer, or to microbially decompose the surface of the powdered rubber and to knead and vulcanize the unvulcanized rubber again for a new product.
ゴムの微生物分解については、従来様々な微生物がスクリーニングされ、検討されている。そのような微生物分解については、特開平９−１９４６２４号公報、特開平１１−６０７９３号公報等で報告されている。なおノカルディア属の放線菌は天然ゴムなどのポリイソプレン系ゴムを分解することが知られているが、通常のノカルディア属の菌ではカーボンブラックなどを配合した硬質ゴムを分解することは困難である。これまでに、カーボンブラックを含有したゴム製品を分解できる菌の例は非常に少なく、未だに実用化された例はない。 Various microorganisms have been screened and studied for microbial degradation of rubber. Such microbial degradation has been reported in JP-A-9-194624, JP-A-11-60793, and the like. Nocardia genus actinomycetes are known to degrade natural rubber and other polyisoprene-based rubbers, but normal Nocardia genus bacteria are difficult to degrade hard rubber compounded with carbon black. is there. To date, there are very few examples of bacteria capable of degrading rubber products containing carbon black, and no examples have yet been put to practical use.
そこで、ゴムの微生物分解に利用できる新規な微生物を得ること、特にカーボンブラックを含有したゴム製品を分解することができる微生物を得ることが本発明の課題である。 Accordingly, it is an object of the present invention to obtain a novel microorganism that can be used for microbial degradation of rubber, particularly to obtain a microorganism capable of decomposing a rubber product containing carbon black.
そこで、本発明者らは多くの土壌から天然ゴム分解菌の探索を行った結果、ゴム製品を分解できる微生物であるノカルディア属の放線菌ＢＳ−ＨＡ１株を見いだした。放線菌ＢＳ−ＨＡ１株によれば、従来困難であったカーボンブラックを含有するゴム製品を分解することが可能である。よって本発明により上記課題を解決することが可能となった。本発明の方法は、タイヤなどのゴム製品の廃棄及び再利用に有用である。 Accordingly, as a result of searching for natural rubber-degrading bacteria from many soils, the present inventors have found the actinomycete BS-HA1 belonging to the genus Nocardia, which is a microorganism capable of degrading rubber products. According to the actinomycete BS-HA1 strain, it is possible to decompose a rubber product containing carbon black, which has been difficult in the past. Therefore, the present invention can solve the above problems. The method of the present invention is useful for the disposal and reuse of rubber products such as tires.
本発明は次の（１）〜（７）からなる。
（１）ポリイソプレン系ゴムを分解する作用を有する、ノカルディア属に属する放線菌ＢＳ−ＨＡ１株（ＦＥＲＭ Ｐ−１９３７８）。
（２）ポリイソプレン系ゴムを含有するゴム組成物を、ノカルディア属に属する放線菌であるＢＳ−ＨＡ１株（ＦＥＲＭ Ｐ−１９３７８）により微生物分解する方法。
（３）前記ゴム組成物のポリイソプレン系ゴム含有量が１０重量％以上であることを特徴とする（２）の方法。
（４）前記ポリイソプレン系ゴムがシスポリイソプレンゴムである（２）の方法。
（５）前記ゴム組成物がカーボンブラックを含有とすることを特徴とする（２）の方法。
（６）前記ゴム組成物がゴム粉である（２）の方法。
（７）前記ゴム組成物がゴム製品を破砕したゴム片である（２）の方法。
The present invention comprises the following (1) to (7).
(1) Actinomyces BS-HA1 strain (FERM P-19378) belonging to the genus Nocardia, which has an action of decomposing polyisoprene rubber.
(2) A method in which a rubber composition containing a polyisoprene rubber is microbially decomposed by BS-HA1 strain (FERM P-19378) which is an actinomycete belonging to the genus Nocardia.
(3) The method according to (2), wherein the rubber composition has a polyisoprene rubber content of 10% by weight or more.
(4) The method according to (2), wherein the polyisoprene rubber is cis polyisoprene rubber.
(5) The method according to (2), wherein the rubber composition contains carbon black.
(6) The method according to (2), wherein the rubber composition is rubber powder.
(7) The method according to (2), wherein the rubber composition is a rubber piece obtained by crushing a rubber product.
本発明により、新規な微生物であるノカルディア属の放線菌ＢＳ−ＨＡ１株（ＦＥＲＭ Ｐ−１９３７８）が与えられた。放線菌ＢＳ−ＨＡ１株はポリイソプレン系ゴムを分解する活性を有する。よって本発明により、ポリイソプレン系ゴムを分解する新たな方法が与えられた。更に本発明により、カーボンブラックを含有するゴム製品の微生物分解が可能となった。 According to the present invention, a novel microorganism, the actinomycete BS-HA1 strain (FERM P-19378) of the genus Nocardia was provided. Streptomyces BS-HA1 strain has the activity of degrading polyisoprene rubber. Thus, the present invention provides a new method for decomposing polyisoprene rubber. Furthermore, the present invention has made it possible to microbially decompose rubber products containing carbon black.
以下、発明の実施の形態を具体的に説明する。
本発明は新規微生物であるノカルディア属の放線菌であるＢＳ−ＨＡ１株である。また本発明はポリイソプレン系ゴムを含有するゴム製品を上記微生物で分解する方法である。ノカルディア属の放線菌は天然ゴムなどのポリイソプレン系ゴムを分解することが知られているが、通常のノカルディア属の菌ではカーボンブラックなどを配合した硬質ゴムを分解することは困難である。発明者は多くの土壌から天然ゴム分解菌を鋭意スクリーニングした結果、タイヤなどのゴム製品に使用されるカーボン配合ゴムからなるゴム粉を分解できるＢＳ−ＨＡ１株を見出した。
Embodiments of the invention will be specifically described below.
This invention is BS-HA1 strain | stump | stock which is actinomycete of Nocardia genus which is a novel microorganism. The present invention is also a method for decomposing a rubber product containing a polyisoprene-based rubber with the above microorganisms. Nocardia genus actinomycetes are known to degrade natural rubber and other polyisoprene rubber, but normal Nocardia bacteria are difficult to decompose hard rubber compounded with carbon black and the like . As a result of intensive screening of natural rubber-degrading bacteria from many soils, the inventor found BS-HA1 strain capable of degrading rubber powder made of carbon-containing rubber used in rubber products such as tires.
ＢＳ−ＨＡ１株は独立行政法人 産業技術総合研究所 特許生物寄託センターにＦＥＲＭ Ｐ１９３７８株として２００３年６月５日に寄託されている。本発明においてＢＳ−ＨＡ１株は無機塩の液体培地中で粉ゴムを資化しながら増殖する。上記の無機塩としては窒素、リン、カリウム、カルシウム、マグネシウムなどが挙げられる。 The BS-HA1 strain was deposited on June 5, 2003 as FERM P19378 strain at the National Institute of Advanced Industrial Science and Technology (AIST). In the present invention, the BS-HA1 strain grows while assimilating the powdered rubber in a liquid medium of an inorganic salt. Examples of the inorganic salt include nitrogen, phosphorus, potassium, calcium, and magnesium.
菌株の固定は近年主流となっている１６ＳｒＲＮＡ遺伝子の比較に基づいて行った。以下方法の詳細について述べる。菌株をＹＭ寒天培地（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ ＮＪ，ＵＳＡ）に植菌し、３０℃で５日間培養した。その後、この菌体からＰｒｅｐＭａｎ Ｍｅｔｈｏｄ（Ａｐｐｌｉｅｄ Ｂｊｏｓｙｓｔｅｍｓ、ＣＡ，ＵＳＡ）により、ゲノムＤＮＡの抽出を行った。抽出したゲノムＤＮＡを鋳型として、ＰＣＲにより、１６Ｓ Ｒｉｂｏｓｏｍａｌ ＲＮＡ遺伝子（１６Ｓ ｒＤＮＡ）のうち、５末端側約５００ｂｐの領域を増幅した。その後、増幅した塩基配列をシーケンスし、検体の１６Ｓ ｒＤＮＡ部分塩基配列を得た。得られた１６Ｓ ｒＤＮＡの塩基配列から検体を近縁と考えられる種の相同性倹索を行い、下記の上位１０株を決定した。
相同性 菌株
９７．８％ Ｎｏｃａｒｄｉａ ｔｒａｎｓｖａｌｅｎｓｉｓ
９６．８％ Ｎｏｃａｒｄｉａ ｎｏｖａ
９６．６％ Ｎｕｃａｒｄｉａ ｂｒａｓｉｌｉｅｎｓｉｓ
９６．６％ Ｎｏｃａｒｄｉａ ａｓｔｅｒｏｉｄｅｓ
９６．４％ Ｎｏｃａｒｄｉａ ｆａｒｃｉｎｉａ
９５．８％ Ｎｏｃａｒｄｉａ ｐｓｅｕｄｏｂｒａｓｉｌｉｅｎｓｉｓ
９５．６％ Ｎｏｃａｒｄｉａ ｏｔｉｔｉｄｉｓｃａｖｉａｕｍ
９５．６％ Ｒｈｏｄｏｃｏｃｃｕｓ Ｇｌｏｂｅｒｕｌｕｓ
９５．４％ Ｎｏｃａｒｄｉａ ｂｒｅｖｉｃａｔｅｎａ
９５．４％ Ｔｓｕｋａｍｕｒｅｌｌａ ｗｒａｔｉｓｌａｖｉｅｎｓｉｓ
この結果から、ＢＳ−ＨＡＩ株はＮｏｃａｒｄｉａ ｓｐ．に属する菌株と同定した。
The strain was fixed based on the comparison of 16S rRNA genes, which has become the mainstream in recent years. Details of the method will be described below. The strain was inoculated into YM agar medium (Becton Dickinson NJ, USA) and cultured at 30 ° C. for 5 days. Thereafter, genomic DNA was extracted from the cells by PrepMan Methods (Applied Bjosystems, CA, USA). Using the extracted genomic DNA as a template, a region of about 500 bp on the 5 terminal side was amplified by PCR from the 16S Ribosomal RNA gene (16S rDNA). Thereafter, the amplified base sequence was sequenced to obtain a 16S rDNA partial base sequence of the specimen. From the obtained 16S rDNA base sequence, homology scrutiny of species considered to be closely related to the specimen was performed, and the following 10 top strains were determined.
Homology strain 97.8% Nocardia transvalensis
96.8% Nocardia nova
96.6% Nucardia brasiliensis
96.6% Nocardia asteroides
96.4% Nocardia farcinia
95.8% Nocardia pseudobrasiliensis
95.6% Nocardia otidiscaviaum
95.6% Rhodococcus Globerulus
95.4% Nocardia brevicatena
95.4% Tsukamurella wratislaviensis
From this result, the BS-HAI strain was obtained from Nocardia sp. And identified as a strain belonging to
本発明を適用する対象となるポリイソプレン系ゴムとしては、主鎖がシスポリイソプレン及びトランスポリイソプレンであるゴムまたはエラストマーを挙げることができる。本発明においてシスポリイソプレンであるゴムが最も好ましいが、イソプレンと他のモノマーとの共重合体を主鎖とするゴムまたはエラストマーにも本発明を適応することが可能である。具体的なシスポリイソプレンとしては、それに限定されるものではないが、天然ゴムやＮａｔｓｙｎなどの合成シスポリイソプレンが例示され、トランスポリイソプレンとしてはガッタパーチャなどの天然系ゴムが例示される。 Examples of the polyisoprene rubber to which the present invention is applied include rubbers or elastomers whose main chain is cis polyisoprene and trans polyisoprene. In the present invention, rubber which is cis polyisoprene is most preferable, but the present invention can also be applied to rubber or elastomer having a copolymer of isoprene and another monomer as a main chain. Specific examples of cis polyisoprene include, but are not limited to, natural rubber and synthetic cis polyisoprene such as Natsyn, and trans polyisoprene includes natural rubber such as gutta percha.
ゴム組成物を構成するゴムエラストマー中のポリイソプレン系ゴムの含有量が１０重量％以上であれば本方法の適応が可能である。ゴムエラストマー中のポリイソプレンは５０重量％以上が好ましく、さらに好ましくは８０重量％以上であるが、その範囲に限定されるものではない。 If the content of the polyisoprene rubber in the rubber elastomer constituting the rubber composition is 10% by weight or more, the present method can be applied. The polyisoprene in the rubber elastomer is preferably 50% by weight or more, more preferably 80% by weight or more, but is not limited to this range.
また本発明で対象となるゴム製品はカーボンブラックを含有することができる。カーボンブラックの含有量は通常の廃タイヤから採取された粉ゴムであれば３０〜７０ｐｈｒであるが、その範囲内に限定されるものではなく、カーボンブラックの含有量が３０ｐｈｒ以下及び７０ｐｈｒ以上でも本発明の方法は適応可能である。 In addition, the rubber product targeted by the present invention can contain carbon black. The carbon black content is 30 to 70 phr if it is a powder rubber collected from a normal waste tire, but is not limited to that range, and even if the carbon black content is 30 phr or less and 70 phr or more, The inventive method is adaptable.
本発明で分解の対象となるゴムは、イソプレン系ゴムであれば、ゴム工業で通常使用されているシランカップリング剤、硫黄、加硫剤、加硫促進剤、加硫促進助剤、酸化防止剤、オゾン劣化防止剤、老化防止剤、プロセス油、亜鉛華（ＺｎＯ）、ステアリン酸、過酸化物等の通常の配合剤が配合されたものでよい。ただし、ゴム配合剤の中にＢＳ−ＨＡ１株の増殖を阻害する配合剤が配合されている場合は、粉ゴムを溶剤抽出し、これを除去する必要がある。 If the rubber to be decomposed in the present invention is an isoprene-based rubber, a silane coupling agent, sulfur, vulcanizing agent, vulcanization accelerator, vulcanization accelerating agent, antioxidant that are usually used in the rubber industry Ordinary compounding agents such as an agent, an ozone deterioration preventing agent, an antiaging agent, a process oil, zinc white (ZnO), stearic acid, and peroxide may be blended. However, when a compounding agent that inhibits the growth of the BS-HA1 strain is blended in the rubber compounding agent, it is necessary to solvent-extract the powdered rubber and remove it.
本発明ではゴム組成物をさらに微粉砕したゴム粉であれば微生物の分解効率がさらに向上する。ここにおけるゴム粉が、ゴム製品をロール粉砕法などの常温で機械的に破砕する方法や液体窒素などにより冷凍してから破砕する冷凍破砕法や、超高圧水を用いる水撃粉砕法で微粉砕したゴム粉であれば、微生物の分解効率がさらに向上する。 In the present invention, if the rubber composition is a finely pulverized rubber powder, the decomposition efficiency of microorganisms is further improved. The rubber powder here is finely pulverized by a method of mechanically crushing rubber products at room temperature such as a roll crushing method, a freezing crushing method of freezing and crushing with liquid nitrogen, or a water hammer crushing method using ultra-high pressure water If the rubber powder is used, the decomposition efficiency of microorganisms is further improved.
また、粉ゴムを完全に分解するのではなく、粉ゴムの表面を微生物分解し、再度未加硫ゴムに混練り加硫することにより、新たな製品に利用することも可能である。ここ用いる粉ゴムは、廃タイヤ・チューブを破砕して得られる粉末ゴムに限らず、タイヤ製造時に発生する未加硫スクラップ物、タイヤ加硫時に発生するスピュー片などを粉砕したものでもよい。また、ゴム粉でなく、ゴム製品を破砕したゴム片でも本発明による微生物分解は可能である。以下実施例で本発明を具体的に説明するが、本発明はこれらの実施例により限定されるものではない。 Further, instead of completely decomposing the powder rubber, the surface of the powder rubber can be microbially decomposed and kneaded with the unvulcanized rubber again and vulcanized to be used for a new product. The powder rubber used here is not limited to powder rubber obtained by crushing waste tires and tubes, but may be one obtained by pulverizing unvulcanized scrap generated during tire production, spew pieces generated during tire vulcanization, and the like. In addition, microbial degradation according to the present invention is possible even with a rubber piece obtained by crushing a rubber product instead of rubber powder. EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
シリコーン栓付きのガラス容器に準備した表１の組成の培地に、アセトン抽出済みの０．５ｍｍ角のゴム片１０個を入れ、９０℃でオートクレーブ中で滅菌処理した。そのゴム片は表２の組成で天然ゴムを配合したものである。これに天然手袋ゴムを唯一の炭素源として前培養したＢＳ−ＨＡ１株の菌液を加え、温度３０℃においてマグネティックスターラーで６０日間回転培養を行った後、１２１℃でオートクレーブ滅菌し、ゴム片をろ紙でろ過した。ろ過したゴム片を純水で数回洗浄し、十分に乾燥したことを確認後、ゴム片の重量減少を測定した。 Ten pieces of 0.5 mm square rubber pieces already extracted with acetone were placed in a medium having the composition shown in Table 1 prepared in a glass container with a silicone stopper, and sterilized at 90 ° C. in an autoclave. The rubber piece is a blend of natural rubber with the composition shown in Table 2. To this was added BS-HA1 strain pre-cultured with natural glove rubber as the only carbon source, and after 60 days of rotation with a magnetic stirrer at 30 ° C., autoclaved at 121 ° C. It filtered with the filter paper. The filtered rubber piece was washed several times with pure water, and after confirming that it was sufficiently dried, the weight loss of the rubber piece was measured.
（比較例）
実施例と同様の実験を、ＢＳ−ＨＡ１株の代わりに土壌から分離した放線菌を用いて行った。実施例のＢＳ−ＨＡ１株では重量減少は３５％であったが、比較例では重量減少は０％であった。よってＢＳ−ＨＡ１株がカーボンブラックを配合したゴムでも分解できることが確認された。
(Comparative example)
An experiment similar to the example was performed using actinomycetes isolated from soil instead of the BS-HA1 strain. In the BS-HA1 strain of the example, the weight reduction was 35%, but in the comparative example, the weight reduction was 0%. Therefore, it was confirmed that the BS-HA1 strain can be decomposed even with rubber containing carbon black.
本発明の新規の微生物であるノカルディア属の放線菌ＢＳ−ＨＡ１株（ＦＥＲＭ Ｐ−１９３７８）はポリイソプレン系ゴムを分解する作用を有する。また該微生物により、これまでは困難であったカーボンブラックを含有するゴム製品の微生物分解が可能である。よって本発明の微生物を用いた微生物分解方法は、タイヤなどのゴム製品の廃棄及び再利用に有用である。
Nocardia genus actinomycete BS-HA1 strain (FERM P-19378), which is a novel microorganism of the present invention, has an action of degrading polyisoprene rubber. Moreover, the microorganisms can microbially decompose rubber products containing carbon black, which has been difficult until now. Therefore, the microbial decomposition method using the microorganism of the present invention is useful for the disposal and reuse of rubber products such as tires.
Claims (7)
The method according to claim 2, wherein the rubber composition is a rubber piece obtained by crushing a rubber product.
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