JP5509471B2 - Novel microorganism and method for producing carbonate - Google Patents
Novel microorganism and method for producing carbonate Download PDFInfo
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- JP5509471B2 JP5509471B2 JP2009198357A JP2009198357A JP5509471B2 JP 5509471 B2 JP5509471 B2 JP 5509471B2 JP 2009198357 A JP2009198357 A JP 2009198357A JP 2009198357 A JP2009198357 A JP 2009198357A JP 5509471 B2 JP5509471 B2 JP 5509471B2
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- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 title claims description 53
- 244000005700 microbiome Species 0.000 title claims description 43
- 238000004519 manufacturing process Methods 0.000 title claims description 21
- 108010046334 Urease Proteins 0.000 claims description 51
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 41
- 239000004202 carbamide Substances 0.000 claims description 41
- 239000011575 calcium Substances 0.000 claims description 36
- 238000006243 chemical reaction Methods 0.000 claims description 36
- 229910021645 metal ion Inorganic materials 0.000 claims description 23
- 229910001424 calcium ion Inorganic materials 0.000 claims description 18
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 17
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 7
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 6
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 6
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 5
- 229910052791 calcium Inorganic materials 0.000 claims description 5
- 150000002500 ions Chemical class 0.000 claims description 5
- 229910052742 iron Inorganic materials 0.000 claims description 5
- -1 iron ions Chemical class 0.000 claims description 4
- 230000003301 hydrolyzing effect Effects 0.000 claims description 3
- 229910001427 strontium ion Inorganic materials 0.000 claims description 3
- 239000000243 solution Substances 0.000 description 45
- 230000000694 effects Effects 0.000 description 39
- 241000186547 Sporosarcina Species 0.000 description 28
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- 239000011777 magnesium Substances 0.000 description 24
- 241000246348 Sporosarcina sp. Species 0.000 description 21
- 241000894006 Bacteria Species 0.000 description 17
- 238000001556 precipitation Methods 0.000 description 17
- 229910000019 calcium carbonate Inorganic materials 0.000 description 13
- 239000002609 medium Substances 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 12
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 10
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 10
- 238000002156 mixing Methods 0.000 description 9
- 239000002689 soil Substances 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000008272 agar Substances 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 8
- 239000002244 precipitate Substances 0.000 description 8
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- 239000000203 mixture Substances 0.000 description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 238000000635 electron micrograph Methods 0.000 description 6
- 238000006460 hydrolysis reaction Methods 0.000 description 6
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 5
- 238000012300 Sequence Analysis Methods 0.000 description 5
- 229910021529 ammonia Inorganic materials 0.000 description 5
- 235000019270 ammonium chloride Nutrition 0.000 description 5
- 239000000908 ammonium hydroxide Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000001095 magnesium carbonate Substances 0.000 description 5
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
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- 239000007853 buffer solution Substances 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 4
- 235000014380 magnesium carbonate Nutrition 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
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- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 239000011435 rock Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- 241000304886 Bacilli Species 0.000 description 2
- 229910021532 Calcite Inorganic materials 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
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- 241000193395 Sporosarcina pasteurii Species 0.000 description 2
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- MFEVGQHCNVXMER-UHFFFAOYSA-L 1,3,2$l^{2}-dioxaplumbetan-4-one Chemical compound [Pb+2].[O-]C([O-])=O MFEVGQHCNVXMER-UHFFFAOYSA-L 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 229910000003 Lead carbonate Inorganic materials 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- LFYJSSARVMHQJB-QIXNEVBVSA-N bakuchiol Chemical compound CC(C)=CCC[C@@](C)(C=C)\C=C\C1=CC=C(O)C=C1 LFYJSSARVMHQJB-QIXNEVBVSA-N 0.000 description 1
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 1
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- 229940116318 copper carbonate Drugs 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- GEZOTWYUIKXWOA-UHFFFAOYSA-L copper;carbonate Chemical compound [Cu+2].[O-]C([O-])=O GEZOTWYUIKXWOA-UHFFFAOYSA-L 0.000 description 1
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- 229910052712 strontium Inorganic materials 0.000 description 1
- CIOAGBVUUVVLOB-UHFFFAOYSA-N strontium atom Chemical compound [Sr] CIOAGBVUUVVLOB-UHFFFAOYSA-N 0.000 description 1
- IATRAKWUXMZMIY-UHFFFAOYSA-N strontium oxide Chemical class [O-2].[Sr+2] IATRAKWUXMZMIY-UHFFFAOYSA-N 0.000 description 1
- PWYYWQHXAPXYMF-UHFFFAOYSA-N strontium(2+) Chemical compound [Sr+2] PWYYWQHXAPXYMF-UHFFFAOYSA-N 0.000 description 1
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Images
Description
本発明は、新規微生物及び炭酸塩の生成方法に関し、より詳細には、強いウレアーゼ活性を有し、且つカルシウムイオン耐性に優れるのみならず、アルカリ条件下で培養可能である新規微生物及びこの新規微生物のウレアーゼ活性を利用した炭酸塩の生成方法に関する。 The present invention relates to a novel microorganism and a method for producing carbonate, and more specifically, a novel microorganism that has strong urease activity and is excellent not only in calcium ion resistance but also capable of being cultured under alkaline conditions, and the novel microorganism. The present invention relates to a method for producing carbonates using the urease activity of
従来より、例えば、特許文献１に記載されているように、Ｂａｃｉｌｌｕｓ ｐａｓｔｅｕｒｉｉ（バチラス・パステウリ）などがウレアーゼ活性を有することが知られている。また、ウレアーゼ活性による尿素の加水分解反応は、反応液が酸性であるか、アルカリ性であるかによって、反応式が異なり、炭酸塩を生成する場合には、アルカリ条件下において、下記反応式によって発生する炭酸イオン（ＣＯ３ ２−）と金属イオンとが反応して炭酸塩が生成される。
（ＮＨ２）２ＣＯ ＋ ２Ｈ２Ｏ → ＣＯ３ ２− ＋ ２ＮＨ３
Conventionally, for example, as described in
(NH 2) 2 CO + 2H 2 O → CO 3 2- + 2NH 3
しかしながら、このように微生物のウレアーゼ活性を利用して、例えば、炭酸カルシウムを生成しようとした場合、従来のＢａｃｉｌｌｕｓ ｐａｓｔｅｕｒｉｉ（バチラス・パステウリ）などは、カルシウムイオンの存在によって、そのウレアーゼ活性が変動し、安定した炭酸カルシウム生成をすることが困難となったりする可能性があった。また、微生物を上記の反応系において生存させた状態で炭酸塩を継続的に生成させるには、その微生物がアルカリ条件下で培養可能であることが望ましい。更に、微生物のウレアーゼ活性を利用した炭酸塩の生成を種々の分野において実用化するに当たっては、強いウレアーゼ活性を有する微生物の開発が望まれていた。また、一般に微生物の生育温度の最適温度は、例えば、３０℃以上というようにやや高温であることが多いが、炭酸塩の生成を実用化する分野によっては、例えば、２０℃程度の比較的低温で炭酸塩を生成する必要が生じる場合もある。 However, using the urease activity of microorganisms in this way, for example, when trying to produce calcium carbonate, the conventional Bacillus pasteurii (Bacillus pasturei) and the like have their urease activity changed due to the presence of calcium ions, It may be difficult to produce stable calcium carbonate. Further, in order to continuously produce carbonate in a state where the microorganism is alive in the above reaction system, it is desirable that the microorganism can be cultured under alkaline conditions. Furthermore, in order to put the production of carbonates utilizing the urease activity of microorganisms into various fields, it has been desired to develop microorganisms having strong urease activity. In general, the optimum temperature for the growth temperature of microorganisms is often a little high, for example, 30 ° C. or more. However, depending on the field where the production of carbonate is put into practical use, for example, a relatively low temperature of about 20 ° C. In some cases, it may be necessary to produce carbonate.
本発明は、上記事情に鑑みなされたもので、カルシウムイオン耐性に優れるのみならず、アルカリ条件下で培養可能であり、比較的低温でも好適に生育し、且つ強ウレアーゼ活性を有する新規微生物及びこのような微生物を利用した炭酸塩の生成方法を提供することを目的とする。 The present invention has been made in view of the above circumstances, and is not only excellent in calcium ion resistance, but also a novel microorganism that can be cultured under alkaline conditions, grows favorably even at relatively low temperatures, and has strong urease activity. An object of the present invention is to provide a method for producing a carbonate using such microorganisms.
本発明者らは、上記目的を達成するため、強ウレアーゼ活性を有する微生物を自然界より広く検索した結果、土壌から強いウレアーゼ活性を有するバクテリアが抽出され、更に、そのバクテリアからより強いウレアーゼ活性を有するのみならず、カルシウムイオンに対する耐性にも優れるバクテリアを発現させるに至り、そのバクテリアがアルカリ条件下で培養可能であり、比較的低温でも好適に生育し得ることを見出し、本発明をなすに至った。 In order to achieve the above object, the present inventors have extensively searched for microorganisms having strong urease activity from the natural world. As a result, bacteria having strong urease activity are extracted from the soil, and further, have a stronger urease activity from the bacteria. As a result, the present inventors have found that bacteria having excellent resistance to calcium ions can be expressed, the bacteria can be cultured under alkaline conditions, and can grow well even at relatively low temperatures, and the present invention has been made. .
即ち、本発明は、（１）カルシウムイオン耐性を有するウレアーゼ産生微生物であるスポロサルシナ（Ｓｐｏｒｏｓａｒｃｉｎａ）属ＮＯ−Ａ１０株（受託番号 ＮＩＴＥ Ｐ−７９１）及び（２）ウレアーゼ産生微生物により尿素を加水分解する反応を利用して炭酸塩を生成する炭酸塩の生成方法であって、ウレアーゼ産生微生物として上記（１）に記載のスポロサルシナ属ＮＯ−Ａ１０株と、尿素と、カルシウムイオン、マグネシウムイオン、鉄イオン及びストロンチウムイオンから選ばれる１種又は２種以上の金属イオンと、を反応液中で反応させて炭酸塩を生成することを特徴とする炭酸塩の生成方法を提供する。 That is, the present invention relates to (1) Sporosarcina sp. NO-A10 strain (accession number NITE P-791), which is a urease-producing microorganism having resistance to calcium ions, and (2) a reaction for hydrolyzing urea by a urease-producing microorganism. Is a method for producing a carbonate that produces carbonate using urea, and as a urease-producing microorganism, the sporosarcinae NO-A10 strain described in (1) above, urea, calcium ion, magnesium ion, iron ion, and strontium Provided is a method for producing a carbonate, characterized by reacting one or more metal ions selected from ions in a reaction solution to produce a carbonate.
本発明によれば、カルシウムイオン耐性に優れるのみならず、アルカリ条件下で培養可能であり、比較的低温でも好適に生育し、且つ強ウレアーゼ活性を有する新規微生物が得られ、その微生物を利用することにより、炭酸塩を効率的に生成することができるのみならず、例えば、安定した炭酸カルシウム生成をしたり、アルカリ条件下、比較的低温条件下であっても、好適に炭酸塩を生成することが可能となる。 According to the present invention, a novel microorganism that is not only excellent in resistance to calcium ions but can be cultured under alkaline conditions, grows favorably even at a relatively low temperature, and has strong urease activity, is utilized. In addition to being able to efficiently produce carbonate, for example, it can stably produce calcium carbonate, or suitably produce carbonate even under alkaline conditions and relatively low temperature conditions. It becomes possible.
以下、本発明につき更に詳細に説明する。本発明の新規微生物であるスポロサルシナ属ＮＯ−Ａ１０株は、以下のようにして得られたものである。本発明者らは、炭酸塩を生成させるための強いウレアーゼ活性を有するバクテリアを次のように抽出、また新株を開発した。中国地方、関越地方、東海地方の複数のサイトにおいて、ボーリングを行って土壌サンプルを採取した。サンプリング深さはおおよそ０〜６．５ｍであった。このほか、静岡県において露頭の土壌サンプルを採取した。ボーリングで得たコアサンプルを観察しながら、１ｍごとに約１０ｇの土壌をガラス瓶に取り、それぞれ約２０ｍＬの石灰水（Ｃａ（ＯＨ）２）および濃度の異なる（０．５〜２０ｇ／リットル）ＣａＣｌ２溶液を加えて２日間放置した。これは炭酸塩の生成に使用するカルシウムイオンの耐性バクテリアを抽出するためである。これらのガラス瓶中で生存しているバクテリアを抽出し、そのうちで特に培養速度の速いバクテリアをコロニーの大きさで判断し、１サンプルから１０株の菌を得た。この段階でおよそ１５０株が得られた。それぞれの菌株を２０ミリリットルのペプトン液（濃度１０ｇ／リットル）で培養し、それを遠心分離してバクテリアを採取した。得られたバクテリアを試験管に移し、その中に１Ｍの尿素２０ミリリットルを入れてウレアーゼ活性を次の簡易方法によって調べた。菌がウレアーゼ活性を示す場合には、尿素の加水分解に伴うアンモニア発生によってｐＨが上昇する。従って、ｐＨが上昇するかどうかを調べることによってウレアーゼ活性が起こるかどうか調べることが可能である。ｐＨが上昇したものについては、１ＭのＣａＣｌ２溶液を加えて炭酸塩が生成されるか（白濁するか）調べた。 Hereinafter, the present invention will be described in more detail. Sporosarcina NO-A10 strain, which is a novel microorganism of the present invention, was obtained as follows. The present inventors have extracted bacteria having strong urease activity for producing carbonate as follows, and have developed a new strain. Soil samples were collected by drilling at multiple sites in Chugoku, Kanetsu and Tokai regions. The sampling depth was approximately 0 to 6.5 m. In addition, an outcrop soil sample was collected in Shizuoka Prefecture. While observing the core sample obtained by boring, about 10 g of soil is taken into a glass bottle every 1 m, and each of about 20 mL of lime water (Ca (OH) 2 ) and CaCl of different concentration (0.5 to 20 g / liter) CaCl Two solutions were added and left for 2 days. This is to extract calcium ion resistant bacteria used for the production of carbonate. Bacteria surviving in these glass bottles were extracted, and among them, bacteria having a particularly high culture rate were judged by the size of the colonies, and 10 strains were obtained from one sample. Approximately 150 strains were obtained at this stage. Each strain was cultured in 20 ml of peptone solution (concentration: 10 g / liter), and centrifuged to collect bacteria. The obtained bacteria were transferred to a test tube, 20 ml of 1M urea was put therein, and urease activity was examined by the following simple method. When the bacterium exhibits urease activity, the pH rises due to the generation of ammonia accompanying the hydrolysis of urea. Therefore, it is possible to determine whether urease activity occurs by examining whether the pH increases. For what pH rises, carbonate added CaCl 2 solution 1M are examined either generated (or cloudy).
その結果、全く沈殿が発生しないもの、またウレアーゼ活性が起こる結果として白濁が沈殿するものに分かれ、いずれもグラム陽性菌である３つの菌株が顕著なウレアーゼ活性を起こすことが分かった。これらの菌を再度培養して、それに１Ｍの尿素と１Ｍの塩化カルシウムを混合した液を加えて、実際に炭酸塩が生成するかどうかを調べた。さらに、これら３つの菌株について、白濁の量を目視により観察して、その結果から最も炭酸塩を多く沈殿させた菌株としてスポロサルシナ属ＮＯ−Ｎ１０株（中国地方から採取）の優位性が分かった。酸性土壌の場合、炭酸塩は生成しないので、アルカリ条件で反応時のｐＨを一定に保つため緩衝液（１００ｍＭの水酸化アンモニウム＋塩化アンモニウム溶液、ｐＨ９．２５に調整）を１０分の１に薄めて使用した。それらを用いて、１．０Ｍ尿素と１．０Ｍ塩化カルシウム溶液を１０ｍＭアンモニウムバッファーでｐＨを９．２前後に上げてウレアーゼ活性（炭酸塩の沈殿）を調べたところ、短時間（３０分ほど）で沈殿が起こる試験管があり、これからスポロサルシナ属ＮＯ−Ａ１０株菌が再分離できた。このようにして、極めてスポロサルシナ属ＮＯ−Ｎ１０株とは異なる強いウレアーゼ活性を示すような、菌の変質が起こりアルカリ条件で培養可能な菌株として、本発明の新規微生物であるスポロサルシナ属ＮＯ−Ａ１０株が現れた。本発明のスポロサルシナ属ＮＯ−Ａ１０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（千葉県木更津市かずさ鎌足２−５−８）に２００９年８月６日付けで受託番号ＮＩＴＥ Ｐ−７９１（Ｓｐｏｒｏｓａｒｃｉｎａ ｓｐ． ＮＯ−Ａ１０）として寄託されており、スポロサルシナ属ＮＯ−Ｎ１０も同様に受託番号ＮＩＴＥ Ｐ−７９２（Ｓｐｏｒｏｓａｒｃｉｎａ ｓｐ． ＮＯ−Ｎ１０株）として寄託されている。なお、バクテリアの培養に一般的に使われるペプトンやポリペプトンは高価で、大量の菌を生産するには不適である。そこで安価なＥＤＣ（ｅｌｅｃｔｒｏｎ ｄｏｎｏｒ ｃｏｍｐｏｕｎｄｓ）が使用可能かどうか調べたところ、スポロサルシナ属ＮＯ−Ｎ１０株は問題なく、また、スポロサルシナ属ＮＯ−Ａ１０株についても培養可能であることが分かった。図１に、ＥＤＣ培地（ＥＤＣ １０ｇ/リットル，ｐＨ８．２（緩衝液で調整））を使用してスポロサルシナ属ＮＯ−Ａ１０株及びスポロサルシナ属ＮＯ−Ｎ１０株を２０〜２３℃で培養した場合の培養曲線を示す。図１において、横軸は時間、縦軸はバクテリア濃度を調べるため波長６００ｎｍを使用したｏｐｔｉｃａｌ ｄｅｎｓｉｔｙ（ＯＤ）で表してある。 As a result, it was divided into those in which precipitation did not occur at all, and those in which white turbidity was precipitated as a result of urease activity, and it was found that all three strains, which are Gram-positive bacteria, caused remarkable urease activity. These bacteria were cultured again, and a mixture of 1M urea and 1M calcium chloride was added thereto to examine whether or not carbonates were actually formed. Furthermore, the amount of white turbidity was visually observed for these three strains, and from the results, the superiority of Sporosarcina genus NO-N10 strain (collected from the Chugoku region) as a strain with the highest carbonate precipitation was found. In the case of acidic soil, carbonate is not generated, so the buffer solution (100 mM ammonium hydroxide + ammonium chloride solution, adjusted to pH 9.25) is diluted to 1/10 to keep the pH constant during the reaction under alkaline conditions. Used. Using them, 1.0M urea and 1.0M calcium chloride solution was raised to about 9.2 with 10 mM ammonium buffer and urease activity (precipitation of carbonate) was examined. A short time (about 30 minutes) There was a test tube in which precipitation occurred, and from this, Sporosarcina sp. NO-A10 strain was re-isolated. In this way, as a strain capable of culturing under alkaline conditions in which the bacteria are altered and exhibit strong urease activity that is extremely different from that of Sporosarcinia sp. NO-N10 strain, Sporosarcinia sp. NO-A10 strain which is the novel microorganism of the present invention Appeared. The Sporosarsina genus NO-A10 strain of the present invention is registered with the National Institute of Technology and Evaluation Patent Microorganisms Deposit Center (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture) with the accession number NITE P as of August 6, 2009. -791 (Sporosarcina sp. NO-A10), and Sporosarcina sp. NO-N10 is also deposited under the same accession number NITE P-792 (Sporosarcina sp. NO-N10 strain). In addition, peptone and polypeptone generally used for bacterial culture are expensive and are not suitable for producing a large amount of bacteria. Therefore, when it was examined whether or not inexpensive EDC (electron donor compounds) can be used, it was found that the Sporosarcina genus NO-N10 strain has no problem and that the Sporosarcina genus NO-A10 strain can also be cultured. FIG. 1 shows the culture in the case of culturing Sporosarcina sp. NO-A10 strain and Sporosarcina sp. NO-N10 strain at 20-23 ° C. using EDC medium (EDC 10 g / liter, pH 8.2 (adjusted with buffer)). The curve is shown. In FIG. 1, the horizontal axis represents time, and the vertical axis represents optical density (OD) using a wavelength of 600 nm for examining the bacterial concentration.
スポロサルシナ属ＮＯ−Ａ１０株の同定を細菌 １６Ｓ ｒＲＮＡ部分配列解析により行った。即ち、菌体サンプルを０．５ｍｌの滅菌水に懸濁し、ビーズビーディング法により破砕処理し、タンパク質を除去した後、磁性ビーズを用いて精製を行い、５０μｌのＤＮＡを得た。この精製ＤＮＡをＰＣＲの鋳型ＤＮＡとして用いた。ＰＣＲ増幅のＰＣＲ反応条件は、反応液組成は、１０Ｆ（１０ｐｍｏｌ／μｌ）１μｌ、８００Ｒ（１０ｐｍｏｌ／μｌ）１μｌ、×１０ ＫＯＤ−Ｐｌｕｓ ｂｕｆｆｅｒ（ＴＯＹＯＢＯ）５μｌ、２ｍＭ Ｎｕｃｌｅｏｔｉｄｅ Ｍｉｘ ＰＬＵＳ（Ｒｏｃｈｅ）５μｌ、２５ｍＭ ＭｇＳＯ４（ＴＯＹＯＢＯ）２μｌ、ＫＯＤ−Ｐｌｕｓ（ＴＯＹＯＢＯ）１μｌ、（Ｔｅｍｐｌａｔｅ １μｌ）、滅菌ｍｉｌｌｉＱ水３４μｌ、計５０μｌ、反応温度サイクルは、９４℃２分 １サイクル、９４℃１５秒 ６２℃３０秒 ７２℃５０秒 ２５サイクル、７２℃５分 １サイクルとした。ＰＣＲプライマー配列は、１０Ｆ（５’−ＧＴＴＴＧＡＴＣＣＴＧＧＣＴＣＡ−３’、配列番号１）、８００Ｒ（５’−ＴＡＣＣＡＧＧＧＴＡＴＣＴＡＡＴＣＣ−３’、配列番号２）であり、使用装置は、ｉＣｙｃｌｅｒ（Ｂｉｏ−Ｒａｄ）である。
Sporosarcina genus NO-A10 strain was identified by bacterial 16S rRNA partial sequence analysis. That is, the bacterial cell sample was suspended in 0.5 ml of sterilized water, crushed by a bead beading method, protein was removed, and purification was performed using magnetic beads to obtain 50 μl of DNA. This purified DNA was used as a template DNA for PCR. PCR reaction conditions for PCR amplification are: reaction solution composition: 1 μl of 10F (10 pmol / μl), 1 μl of 800R (10 pmol / μl), 5 μl of 10 KOD-Plus buffer (TOYOBO), 2 μm of Nucleotide Mix PLUS (Roche), 5 μl, 25 mM MgSO 4 (TOYOBO) 2 μl, KOD-Plus (TOYOBO) 1 μl, (
そして、下記の試薬及び機器を使用して、精製・シークエンス解析を行った。
＜ＰＣＲ産物精製＞
Ｍｏｎｔａｇｅ ＰＣＲ Ｃｅｎｔｒｉｆｕｇａｌ Ｆｉｌｔｅｒ Ｄｅｖｉｃｅｓ（ＭＩＬＬＩＰＯＲＥ）
＜シークエンス試薬＞
ＢｉｏＤｙｅＲ Ｔｅｒｍｉｎａｔｏｒｓ ｖｌ．１ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）
＜シークエンス反応物精製＞
ｉｌｌｕｓｔｒａＴＭ ＡｕｔｏＳｅｑ Ｇ−５０ Ｄｙｅ Ｔｅｒｍｉｎａｔｏｒ Ｒｅｍｏｖａｌ Ｋｉｔ（ＧＥ Ｈｅａｌｔｈｃａｒｅ）
＜シークエンサー＞
ＡＢＩ Ｐｒｉｓｍ ３１３０ Ｇｅｎｅｔｉｃ Ａｎａｌｙｚｅｒ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）
＜シークエンスプライマー＞
ＰＣＲプライマーをシークエンスプライマーとして用いて両鎖解析した。
Then, purification and sequence analysis were performed using the following reagents and equipment.
<PCR product purification>
Montage PCR Centrifugal Filter Devices (MILLIPORE)
<Sequence Reagent>
BioDye R Terminators vl. 1 Cycle Sequencing Kit (Applied Biosystems)
<Sequence reaction product purification>
illustra ™ AutoSeq G-50 Dye Terminator Removal Kit (GE Healthcare)
<Sequencer>
ABI Prism 3130 Genetic Analyzer (Applied Biosystems)
<Sequence primer>
Both strands were analyzed using PCR primers as sequence primers.
得られたＤＮＡ配列を公共のデーターベースと照合して相同性検索（Ｂｌａｓｔ検索）を行い、近縁種を予測した。その結果、Ｓｐｏｒｏｓａｒｃｉｎａ属に分類されるが、Ｓｐｏｒｏｓａｒｃｉｎａ属に属する他の菌種とは配列を異にし、これらの新菌種に最も近縁な種は、Ｓｐｏｒｏｓａｒｃｉｎａ ｇｉｎｓｅｎｇｉｓｏｉｌであるが、塩基配列の一部を異にすることが認められた。なお、図２にＳｐｏｒｏｓａｒｃｉｎａ属ＮＯ−Ａ１０の塩基配列（配列番号３）、下記表１に相同性検索結果上位データを示す。 Homology search (Blast search) was performed by comparing the obtained DNA sequence with a public database, and related species were predicted. As a result, it is classified into the genus Sporosarcina, but the sequence is different from other bacterium species belonging to the genus Sporosarcina, and the species most closely related to these new bacterium species is Sporosarcina ginsengisoil, but part of the nucleotide sequence It was recognized that they were different. FIG. 2 shows the base sequence (SEQ ID NO: 3) of Sporosarcina sp. NO-A10, and Table 1 below shows the high-order data of the homology search results.
以下、本発明の新規微生物であるスポロサルシナ（Ｓｐｏｒｏｓａｒｃｉｎａ）属ＮＯ−Ａ１０の菌学的性質を詳述する。 Hereinafter, the mycological properties of the sporosarcina genus NO-A10, which is a novel microorganism of the present invention, will be described in detail.
＜分類学中の位置＞
Ｓｐｏｒｏｓａｒｃｉｎａ ｓｐ．ＮＯ−Ａ１０株（１６Ｓ ｒＲＮＡ部分塩基配列解析）
<Location in taxonomy>
Sporosarcina sp. NO-A10 strain (16S rRNA partial nucleotide sequence analysis)
＜科学的性質＞
・好気性
・グラム陽性の桿菌
・芽胞形成
・白色の光沢コロニーを形成する。
・強いウレアーゼ活性を有する。ウレアーゼ活性については、後述する実施例において詳述する。
・カタラーゼ陽性
・オキシターゼ陰性
・ＯＦテスト陰性
・カルシウムイオン耐性
・尿素耐性
<Scientific properties>
・ Aerobic ・ Gram-positive bacilli ・ Spore formation ・ White glossy colonies are formed.
・ Has strong urease activity. The urease activity will be described in detail in Examples described later.
Catalase positive, oxidase negative, OF test negative, calcium ion resistance, urea resistance
＜培地条件＞
・普通寒天培地
・普通寒天培地をオートクレイブした後、濾過滅菌した１／１０量の１００ｍＭのアンモニウムバファー（ＮＨ４ＯＨとＮＨ４Ｃｌを最終濃度１０ｍＭ）を無菌的に加えｐＨを８．２に調整して培養する。
・温度は１４〜３７℃で生育（至適温度２５〜３２℃）
・至適ｐＨは８〜９
＜培地の組成＞
市販の普通寒天培地
（培地１０００ｍｌあたり）
肉エキス ５ｇ
ペプトン １０ｇ
塩化ナトリウム ５ｇ
寒天 １５ｇ
<Medium conditions>
-Normal agar medium-After autoclaving the normal agar medium, 1/10 volume of 100 mM ammonium buffer (NH 4 OH and NH 4 Cl in a final concentration of 10 mM) aseptically sterilized by filtration and aseptically added to pH 8.2 Adjust and incubate.
・ Growth at 14-37 ° C (optimum temperature 25-32 ° C)
・ The optimum pH is 8-9
<Composition of medium>
Commercially available ordinary agar medium (per 1000 ml of medium)
5g meat extract
Peptone 10g
Sodium chloride 5g
Agar 15g
なお、本発明の新規微生物を発現した菌株であるスポロサルシナ属ＮＯ−Ｎ１０株の菌株の菌学的性質は、以下の通りである。 The mycological properties of the strain of the genus Sporosarcinia NO-N10, which is a strain expressing the novel microorganism of the present invention, are as follows.
＜分類学中の位置＞
Ｓｐｏｒｏｓａｒｃｉｎａ ｓｐ．ＮＯ−Ｎ１０株（１６Ｓ ｒＲＮＡ部分塩基配列解析）
<Location in taxonomy>
Sporosarcina sp. NO-N10 strain (16S rRNA partial nucleotide sequence analysis)
＜科学的性質＞
・好気性
・グラム陽性の桿菌
・芽胞形成
・白色の光沢コロニーを形成する。
・ウレアーゼ活性を有する。ウレアーゼ活性については、後述する実施例において詳述する。
・スポロサルシナ属ＮＯ−Ｎ１０株と同種
・カタラーゼ陽性
・オキシターゼ陰性
・ＯＦテスト陰性
<Scientific properties>
・ Aerobic ・ Gram-positive bacilli ・ Spore formation ・ White glossy colonies are formed.
-Has urease activity. The urease activity will be described in detail in Examples described later.
・ Same as Sporosarcina sp. NO-N10 ・ Calase positive ・ Oxidase negative ・ OF test negative
＜培地条件＞
・普通寒天培地
・普通寒天培地で温度１４〜３２℃で生育（至適温度２０〜２８℃）
・至適ｐＨは６．５〜８
＜培地の組成＞
市販の普通寒天培地
（培地１０００ｍｌあたり）
肉エキス ５ｇ
ペプトン １０ｇ
塩化ナトリウム ５ｇ
寒天 １５ｇ
<Medium conditions>
・ Ordinary agar medium ・ Growth on ordinary agar medium at a temperature of 14 to 32 ° C. (optimum temperature 20 to 28 ° C.)
・ The optimum pH is 6.5-8.
<Composition of medium>
Commercially available ordinary agar medium (per 1000 ml of medium)
5g meat extract
Peptone 10g
Sodium chloride 5g
Agar 15g
本発明の炭酸塩の生成方法は、上述したように、ウレアーゼ産生微生物により尿素を加水分解する反応を利用して炭酸塩を生成するに当たり、上記スポロサルシナ属ＮＯ−Ａ１０株をウレアーゼ産生微生物として利用するものであり、より具体的には、スポロサルシナ属ＮＯ−Ａ１０株と、尿素と、カルシウムイオン、マグネシウムイオン、鉄イオン及びストロンチウムイオンから選ばれる１種又は２種以上の金属イオンと、を反応液中で反応させて炭酸塩を生成するものである。このほか、銅や鉛イオンなどの炭酸塩を生成できるので、それらのイオンが多く含まれている土壌中では、金属イオンを除いた反応液を浸透させることによって、炭酸銅や炭酸鉛などを生成させることができ、銅イオンや鉛イオンなどを固定（不動化）できる。ここで、本発明の炭酸塩の生成方法における反応液とは、スポロサルシナ属ＮＯ−Ａ１０株による尿素の加水分解反応、該加水分解反応によって発生した炭酸イオンと金属イオンとの反応による炭酸塩の生成反応という一連の反応が行われる反応系（液）であり、より具体的には、スポロサルシナ属ＮＯ−Ａ１０株、尿素、金属イオン、必要に応じて使用される適宜緩衝液、更に、スポロサルシナ属ＮＯ−Ａ１０株を培養液中で培養した状態で配合する場合は培養液、尿素を水溶液又は懸濁液として配合する場合は尿素水溶液又は懸濁液、金属イオンを金属塩の水溶液として配合する場合は金属塩水溶液からなるものである。 As described above, the method for producing carbonate according to the present invention utilizes the above-mentioned sporosarcina genus NO-A10 strain as a urease-producing microorganism when producing a carbonate by utilizing a reaction of hydrolyzing urea by a urease-producing microorganism. More specifically, Sporosarsina genus NO-A10, urea, and one or more metal ions selected from calcium ion, magnesium ion, iron ion and strontium ion in the reaction solution To produce a carbonate. In addition, since carbonates such as copper and lead ions can be generated, copper carbonate and lead carbonate are generated by infiltrating the reaction solution excluding metal ions in the soil that contains a large amount of those ions. It is possible to fix (immobilize) copper ions, lead ions, and the like. Here, the reaction solution in the carbonate production method of the present invention is a hydrolysis reaction of urea by Sporosarcina sp. NO-A10, and the production of carbonate by the reaction of carbonate ions and metal ions generated by the hydrolysis reaction. It is a reaction system (liquid) in which a series of reactions is performed. More specifically, Sporosarcinia NO-A10 strain, urea, metal ions, an appropriate buffer used as necessary, and Sporosarcinia NO -When blending A10 strain cultured in culture medium, when blending culture solution, urea as aqueous solution or suspension, urea aqueous solution or suspension, when blending metal ions as aqueous metal salt solution It consists of a metal salt aqueous solution.
本発明の炭酸塩の生成方法において、スポロサルシナ属ＮＯ−Ａ１０株の配合割合は、特に制限されるものではないが、例えば、スポロサルシナ属ＮＯ−Ａ１０株を培養液中で培養した状態又は懸濁液とした状態で反応系に配合する場合、該培養液（又は懸濁液）を反応液全量に対して５０％（体積比）となる標準状態に配合するのであれば、培養液濃度０．４Ｏ．Ｄ．６００（ｎｍ）以上が好ましく、より好適には培養液濃度０．６Ｏ．Ｄ．６００（ｎｍ）以上、更に好適には培養液濃度０．８Ｏ．Ｄ．６００（ｎｍ）以上である。スポロサルシナ属ＮＯ−Ａ１０株の配合割合が小さすぎると、ウレアーゼ産生が少なすぎる場合がある。なお、スポロサルシナ属ＮＯ−Ａ１０株の配合割合の上限は、特に制限されるものではないが、大きすぎると１個体当たりのウレアーゼ活性が低くなる場合があることを考慮すれば、１．６Ｏ．Ｄ．６００（ｎｍ）以下であることが望ましい。 In the method for producing carbonate of the present invention, the mixing ratio of Sporosarcina NO-A10 strain is not particularly limited, but for example, a state or suspension in which Sporosarcina NO-A10 strain is cultured in a culture solution If the culture solution (or suspension) is added to the standard state of 50% (volume ratio) with respect to the total amount of the reaction solution, the culture solution concentration is 0.4O. . D. 600 (nm) or more is preferable, and more preferably the culture solution concentration is 0.6 O.D. D. 600 (nm) or more, more preferably culture medium concentration 0.8O. D. 600 (nm) or more. If the proportion of Sporosarcinia NO-A10 strain is too small, urease production may be too small. In addition, although the upper limit of the mixing | blending ratio of Sporosarcina genus NO-A10 strain | stump | stock is not restrict | limited especially, when it considers that the urease activity per 1 person may become low when too large, 1.6O. D. It is desirable that it is 600 (nm) or less.
本発明の炭酸塩の生成方法において使用する尿素としては、市販の尿素を使用することができる。尿素の反応液中における濃度は、特に制限されるものではないが、０．５〜２２００ｍＭが好適であり、より好ましくは５０〜１５００ｍＭ、更に好ましくは５００〜１０００ｍＭである。尿素の反応液中における濃度が低すぎると、加水分解が不足し、ｐＨが減少したり、炭酸イオンが不足したりする場合があり、高すぎるとアンモニアが過剰に発生してアンモニア臭が出る場合がある。 As urea used in the method for producing carbonate of the present invention, commercially available urea can be used. Although the density | concentration in the reaction liquid of urea is not restrict | limited in particular, 0.5-2200 mM is suitable, More preferably, it is 50-1500 mM, More preferably, it is 500-1000 mM. If the urea concentration in the reaction solution is too low, hydrolysis may be insufficient, pH may decrease, or carbonate ions may be insufficient. If it is too high, ammonia may be generated excessively, resulting in an ammonia odor. There is.
本発明の炭酸塩の生成方法は金属イオンとして、カルシウムイオン、マグネシウムイオン、鉄イオン、ストロンチウムイオンを１種単独で又は２種以上を適宜組み合わせて添加するものである。これらの中でも、特に、各金属イオン単独、カルシウムイオンとマグネシウムイオンとの併用がより好適である。これらの金属イオンは、それぞれ、これらの金属イオンを含む金属イオン源を後述するように液中に配合することによって好適に供給される。このような金属イオン源は、その種類が特に制限されるものではないが、例えば、カルシウム、マグネシウム、鉄、ストロンチウムの酸化物、水酸化物および塩化物で、特に塩化カルシウム、塩化マグネシウムなどが挙げられる。 In the method for producing a carbonate of the present invention, calcium ions, magnesium ions, iron ions, and strontium ions are added alone or in combination of two or more as metal ions. Among these, each metal ion alone and combined use of calcium ion and magnesium ion are more preferable. Each of these metal ions is suitably supplied by blending a metal ion source containing these metal ions into the liquid as described later. The type of such metal ion source is not particularly limited, and examples thereof include calcium, magnesium, iron, strontium oxides, hydroxides and chlorides, and particularly calcium chloride and magnesium chloride. It is done.
本発明の反応液中における金属イオンの配合割合は、特に制限されるものではないが、０．５〜２２００ｍＭが好適であり、より好ましくは５０〜１５００ｍＭ、更に好ましくは５００〜１０００ｍＭである。金属イオンの反応液中における濃度が低すぎると生成炭酸塩の量が不足する場合があり、高すぎるとスポロサルシナ属ＮＯ−Ａ１０株の失活が起き易くなる場合がある。 The compounding ratio of the metal ions in the reaction solution of the present invention is not particularly limited, but is preferably 0.5 to 2200 mM, more preferably 50 to 1500 mM, still more preferably 500 to 1000 mM. If the concentration of the metal ion in the reaction solution is too low, the amount of the produced carbonate may be insufficient, and if it is too high, the sporosarcinia NO-A10 strain may be easily deactivated.
本発明の炭酸塩の生成方法において、スポロサルシナ属ＮＯ−Ａ１０株、尿素、金属イオンを反応液中に添加する手段、手順は、特に制限されないが、例えば、尿素、金属イオンを所定の濃度になるように適宜緩衝液に加え、それに培養液（懸濁液）から遠心分離したスポロサルシナ属ＮＯ−Ａ１０株を加えて反応させる方法が挙げられる。なお、スポロサルシナ属ＮＯ−Ａ１０株を培養液（懸濁液）から遠心分離せずにスポロサルシナ属ＮＯ−Ａ１０株の培養液（懸濁液）を加える場合には、培養液（懸濁液）の分を加えても尿素、金属イオンが所期の濃度になるように計算した量を予め加えておく。又は、一度にすべてを混合して反応させてもよい。但し、本発明の場合、スポロサルシナ属ＮＯ−Ａ１０株と尿素との混合によって、反応が開始することを考慮すると、スポロサルシナ属ＮＯ−Ａ１０株の配合は、尿素と金属イオンとの混合後、又は同時とすることが好ましい。 In the method for producing carbonate of the present invention, means and procedures for adding Sporosarcinia NO-A10 strain, urea, and metal ions to the reaction solution are not particularly limited, but, for example, urea and metal ions have a predetermined concentration. Thus, in addition to the buffer solution as appropriate, there may be mentioned a method of adding and reacting Sporosarcina sp. NO-A10 strain centrifuged from the culture solution (suspension). In addition, when adding the culture solution (suspension) of Sporosarcina genus NO-A10 strain without centrifuging Sporosarcina genus NO-A10 strain from the culture solution (suspension), The calculated amount is added in advance so that urea and metal ions have the desired concentration even if the minute is added. Alternatively, all may be mixed and reacted at once. However, in the case of the present invention, considering that the reaction starts by mixing Sporosarcinia NO-A10 strain and urea, the compounding of Sporosarcinia NO-A10 strain may be performed after mixing urea and metal ions or simultaneously. It is preferable that
本発明において炭酸塩を生成する際の反応条件は、温度及びｐＨに制限されるが、ｐＨは緩衝剤（例えば、アンモニア緩衝液（初期ｐＨ９．０、１０ｍＭ水酸化アンモニウム＋塩化アンモニウム））によって管理することができる。なお、金属イオンとしてカルシウムのみが存在する場合、ｐＨが７以上、カルシウムイオン以外またはカルシウムイオンとそれ以外の金属イオンが混在する場合はｐＨ８以上が望ましい。また、反応温度も特に制限されないが、本発明の場合、スポロサルシナ属ＮＯ−Ａ１０株が反応系の中で生存している限り、尿素の供給量に合わせて経時的に炭酸イオンが生成されることを考慮すれば、上述したスポロサルシナ属ＮＯ−Ａ１０株の生育温度、より好ましくは至適温度、至適ｐＨで反応させると、より効果的である。また、なお、上述したように、一般に微生物の生存の適温はやや高温であることが多いが、スポロサルシナ属ＮＯ−Ａ１０株は、およそ少なくとも１４℃〜３７℃の範囲では培養可能で、約２５〜３２℃が適温であるので、比較的低温の条件下で炭酸塩を生成する場合に、特に効果的である。 The reaction conditions for producing carbonate in the present invention are limited to temperature and pH, but the pH is controlled by a buffer (for example, ammonia buffer (initial pH 9.0, 10 mM ammonium hydroxide + ammonium chloride)). can do. In addition, when only calcium exists as a metal ion, pH is 7 or more, and when calcium ion and other metal ions are mixed except calcium ion, pH 8 or more is desirable. In addition, although the reaction temperature is not particularly limited, in the case of the present invention, as long as the Sporosarcinia NO-A10 strain is alive in the reaction system, carbonate ions are generated over time according to the amount of urea supplied. In view of the above, it is more effective to react at the growth temperature, more preferably the optimum temperature and the optimum pH of the aforementioned Sporosarcina genus NO-A10 strain. In addition, as described above, the appropriate temperature for the survival of microorganisms is generally slightly high, but Sporosarcinia NO-A10 strain can be cultured in the range of at least about 14 ° C. to 37 ° C. Since 32 ° C. is a suitable temperature, it is particularly effective when producing carbonates under relatively low temperature conditions.
以下、実施例を示し、本発明をより詳細に説明するが、本発明は下記実施例に何ら限定されるものではない。 EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated in detail, this invention is not limited to the following Example at all.
［実施例１〜５及び実験例１、２］
本発明の新規微生物であるスポロサルシナ属ＮＯ−Ａ１０株のウレアーゼ活性を以下のように調べた。また、本発明の微生物であるスポロサルシナ属ＮＯ−Ａ１０株のウレアーゼ活性を他のウレアーゼ産生微生物と比較するために、上述したように多数の土壌菌の中で特に顕著なウレアーゼ活性を示す３つの菌株の中で最も炭酸塩を多く沈殿させた菌株として優位性を示し、スポロサルシナ属ＮＯ−Ａ１０株を発現させた菌株であるスポロサルシナ属ＮＯ−Ｎ１０株についても同様にウレアーゼ活性を調べた。ウレアーゼ活性を調べるには尿素の加水分解のみでことは足りるが、ウレアーゼ産生微生物の中にはカルシウムイオンによってウレアーゼ活性が阻害されるものも少なくないので、本発明では尿素と同じモル濃度の塩化カルシウムの存在下でウレアーゼ活性を調べた。
[Examples 1 to 5 and Experimental Examples 1 and 2]
The urease activity of the sporosarcina genus NO-A10 strain, which is a novel microorganism of the present invention, was examined as follows. In addition, in order to compare the urease activity of the sporosarcinia NO-A10 strain, which is the microorganism of the present invention, with other urease-producing microorganisms, three strains exhibiting particularly remarkable urease activity among a number of soil fungi as described above. Among these, the superiority as a strain in which a large amount of carbonate was precipitated was shown, and the urease activity was also examined in the same manner for the Sporosarcina sp. NO-N10 strain, which is a strain expressing Sporosarcina sp. NO-A10. In order to investigate urease activity, only hydrolysis of urea is sufficient. However, since urease producing microorganisms often inhibit urease activity by calcium ions, in the present invention, calcium chloride having the same molar concentration as urea is used. The urease activity was examined in the presence of.
なお、カルシウムによる失活が無い場合、１Ｍの尿素が加水分解されると１Ｍの炭酸カルシウムが生成するので、炭酸カルシウム（カルサイト）の生成量がウレアーゼ活性を示すことになる。例えば、アンモニア緩衝液（１０ｍＭ水酸化アンモニウム＋塩化アンモニウム）をベースに尿素と塩化カルシウム（ＣａＣｌ２）を同じモル濃度になるように加えて２００ミリリットルとして、それを薄めて所定の濃度にする（実施例１及び実験例１：各０．４Ｍ、実施例２：各０．６Ｍ、実施例３及び実験例２：各１．０Ｍ、実施例４：各１．２Ｍ，実施例５：各１．５Ｍ）。また、培養微生物については、２００ミリリットルのＥＤＣ培養液（ＥＤＣ １０ｇ／リットル）で培養した微生物を遠心分離（５０ミリリットルずつ容器に分け、４５００ｒｐｍ、１５分間）して、上澄み液を取り除き、その中にそれぞれ５％ＮａＣｌ溶液５０ミリリットルを加える。反応液を作ってからは反応が進むので、測定時間を考慮して量を決めて、最終的に所定の濃度になるように、（尿素＋金属イオン）液と微生物液を混合した。これをピペットで５ミリリットルをとり検体とした。試験管は約２０℃で振盪させて、所定の時間に炭酸塩の沈殿量を測定した。なお、以下の実施例及び実験例では、全実験を通してウレアーゼ産生微生物は最終溶液の半分の体積の培養液で培養した量を使った。 When there is no inactivation due to calcium, 1M urea is hydrolyzed to produce 1M calcium carbonate. Therefore, the amount of calcium carbonate (calcite) produced indicates urease activity. For example, based on an ammonia buffer (10 mM ammonium hydroxide + ammonium chloride), urea and calcium chloride (CaCl 2 ) are added to the same molar concentration to make 200 ml, and then diluted to a predetermined concentration (implementation) Example 1 and Experimental Example 1: 0.4M each, Example 2: 0.6M each, Example 3 and Experimental Example 2: 1.0M each, Example 4: 1.2M each, Example 5: 1. 5M). As for the cultured microorganisms, the microorganisms cultured in 200 ml of EDC medium (EDC 10 g / liter) are centrifuged (divided into containers of 50 ml each, 4500 rpm, 15 minutes), and the supernatant is removed. Add 50 ml each of 5% NaCl solution. Since the reaction proceeds after preparing the reaction solution, the amount was determined in consideration of the measurement time, and the (urea + metal ion) solution and the microbial solution were mixed so that the final concentration was reached. This was taken as a sample by pipetting 5 ml. The test tube was shaken at about 20 ° C., and the amount of carbonate precipitated was measured at a predetermined time. In the following Examples and Experimental Examples, urease-producing microorganisms were used in an amount cultured in a culture solution having a half volume of the final solution throughout the entire experiment.
なお、この場合の実験では、試験管に入れたバクテリア培養液とそれと同等体積の反応液を入れたものを複数本準備しておいて、所定の時間になったとき、液体中の炭酸塩をろ紙で濾し、ろ過された炭酸塩を測った。また、ガラス壁面に着いたものは、試験管を炉乾燥（１１０℃）させた後に質量を測定して、予め測定しておいた試験管質量をそれより差し引いて求めた。なお、時間が経つほど、液体中には炭酸塩は含まれておらず、試験管壁面に付着および底へ沈殿した。 In this case, in the experiment, prepare a plurality of bacteria culture solutions in a test tube and a reaction solution of the same volume, and when the prescribed time is reached, the carbonate in the liquid is removed. It filtered with the filter paper and the filtered carbonate was measured. Moreover, what arrived at the glass wall surface measured the mass after drying a test tube in a furnace (110 degreeC), and calculated | required the test tube mass previously measured by subtracting from it. As time passed, the liquid did not contain carbonate, and adhered to the test tube wall surface and settled to the bottom.
結果を下記表２及び図３に示す。表２は、各反応時間における実験例１、２と実施例１〜５の炭酸カルシウムの沈殿量（Ｍ）を記載したものである。図３のグラフの横軸は時間、縦軸は生成した炭酸塩の質量から変換した濃度を示している。なお、表２の濃度及び図３に示した凡例中の濃度は加えた塩化カルシウムの濃度を示す。すなわち、１Ｍの塩化カルシウム液を使用した場合、最終的には縦軸（炭酸カルシウム）が約１Ｍになるまで反応が進んでいる（反応式で１ＭのＣａＣｌ２から１ＭのＣａＣＯ３が生成）。この実験から明らかなように加えた尿素の全てが加水分解され、尿素の分解によって生成されたＣＯ３は、加えたＣａ全てと反応していることが分かる。その反応が終了するには、実施例１〜５（スポロサルシナ属ＮＯ−Ａ１０株）ではおよそ１０〜２５時間、実験例１、２（スポロサルシナ属ＮＯ−Ｎ１０株）では１２５時間以上要すると思われる。また、実施例５のように塩化カルシウム、尿素の濃度が１．５Ｍというように、高濃度の塩化カルシウム、尿素の存在下においても反応が短期間に起こるのは、炭酸塩生成時間の短縮化においてきわめて優位であると言える。更に、上記実施例１〜５と実験例１、２の結果によれば、本発明のスポロサルシナ属ＮＯ−Ａ１０株は、Ｓｐｏｒｏｓａｒｃｉｎａ属ＮＯ−Ｎ１０株と比較しても、格段に強いウレアーゼ活性を有することが認められた。そして、上記実験例１、２に使用したスポロサルシナ属ＮＯ−Ｎ１０株は、上述したように、サンプリングした多数の土壌菌の中で特に顕著なウレアーゼ活性を起こす３つの菌株の中で最も炭酸塩を多く沈殿させた菌株として優位性を示したものである。従って、本発明のスポロサルシナ属ＮＯ−Ａ１０株は、従来のウレアーゼ産生微生物と比較すると、格段に強いウレアーゼ活性を有することが認められる。また、本発明のスポロサルシナ属ＮＯ−Ａ１０株によれば、炭酸カルシウムを少なくとも１．５Ｍまでの炭酸塩濃度まで失活が無く、速くしかも効率よく生成できることが認められた。なお、実施例３（スポロサルシナ属ＮＯ−Ａ１０株、尿素、塩化カルシウム１．０Ｍ）において、反応時間１時間後に生成された炭酸カルシウムの電子顕微鏡写真を図４に、反応時間７２時間後に生成された炭酸カルシウムの電子顕微鏡写真を図５に示す。 The results are shown in Table 2 below and FIG. Table 2 describes the precipitation amount (M) of calcium carbonate in Experimental Examples 1 and 2 and Examples 1 to 5 in each reaction time. In the graph of FIG. 3, the horizontal axis represents time, and the vertical axis represents the concentration converted from the mass of the carbonate produced. In addition, the density | concentration of Table 2 and the density | concentration in the legend shown in FIG. 3 show the density | concentration of the added calcium chloride. That is, when a 1M calcium chloride solution is used, the reaction proceeds until the vertical axis (calcium carbonate) reaches about 1M in the end (1M CaCl 3 is generated from 1M CaCl 2 in the reaction formula). As is clear from this experiment, it is understood that all of the added urea is hydrolyzed, and CO 3 produced by the decomposition of urea reacts with all of the added Ca. The reaction is considered to take about 10 to 25 hours in Examples 1 to 5 (Sporosarcina sp. NO-A10 strain) and 125 hours or more in Experimental Examples 1 and 2 (Sporosarcina sp. NO-N10 strain). In addition, as in Example 5, the concentration of calcium chloride and urea is 1.5M, and the reaction occurs in a short time even in the presence of high concentrations of calcium chloride and urea. It can be said that it is extremely advantageous. Furthermore, according to the results of Examples 1 to 5 and Experimental Examples 1 and 2, the Sporosarcina genus NO-A10 strain of the present invention has a much stronger urease activity than the Sporosarcina genus NO-N10 strain. It was recognized that And as mentioned above, the Sporosarcina genus NO-N10 strain used in Experimental Examples 1 and 2 has the highest carbonate among the three strains that cause particularly remarkable urease activity among a large number of sampled soil fungi. It shows superiority as a strain with many precipitates. Therefore, it is recognized that the Sporosarcinina NO-A10 strain of the present invention has a much stronger urease activity compared to conventional urease-producing microorganisms. In addition, according to the Sporosarcina sp. NO-A10 strain of the present invention, it was confirmed that calcium carbonate was not inactivated up to a carbonate concentration of at least 1.5 M, and could be generated quickly and efficiently. In Example 3 (Sporosarcina NO-A10 strain, urea, calcium chloride 1.0 M), an electron micrograph of calcium carbonate produced after 1 hour of reaction time is shown in FIG. 4, and produced after 72 hours of reaction time. An electron micrograph of calcium carbonate is shown in FIG.
［実施例６及び実施例７］
上記実施例３において、１Ｍの塩化カルシウム液に代えて、０．５Ｍの塩化カルシウム液と０．５Ｍの塩化マグネシウム液を加えた以外は、実施例３と同様にして本発明の新規微生物であるスポロサルシナ属ＮＯ−Ａ１０株を利用して炭酸塩（カルシウムイオンとマグネシウムイオンの併用）を生成して、実施例６とした。また、上記実施例１において、尿素液を０．５Ｍ、塩化マグネシウム液を０．５Ｍとした以外は、実施例１と同様にして本発明の新規微生物であるスポロサルシナ属ＮＯ−Ａ１０株を利用して炭酸塩（炭酸マグネシウム）を生成して、実施例７とした。これらの生成量を実施例１と同様にして調べた。また、各時間における反応液のｐＨを測定した。これらの結果を下記表３及び図６に示す。実施例６の場合、およそ２０時間後には加えた量の約６５％の沈殿が生成した。その後徐々に沈殿量は増えて最終的には８０％程度まで増加すると思われる。このとき（約９時間後）の電子顕微鏡写真を図７に示す。
[Example 6 and Example 7]
In Example 3 above, the microorganism of the present invention is the same as in Example 3 except that 0.5 M calcium chloride solution and 0.5 M magnesium chloride solution are added instead of 1 M calcium chloride solution. Carbonate (combined use of calcium ion and magnesium ion) was produced using Sporosarcinia NO-A10 strain, and it was set as Example 6. Further, in Example 1 above, the Sporosarcina sp. NO-A10 strain, which is a novel microorganism of the present invention, was used in the same manner as in Example 1 except that the urea solution was 0.5 M and the magnesium chloride solution was 0.5 M. Thus, a carbonate (magnesium carbonate) was produced, and Example 7 was obtained. These production amounts were examined in the same manner as in Example 1. Further, the pH of the reaction solution at each time was measured. These results are shown in Table 3 below and FIG. In the case of Example 6, approximately 65% of the added amount was formed after approximately 20 hours. After that, the amount of precipitation gradually increases and seems to finally increase to about 80%. The electron micrograph at this time (after about 9 hours) is shown in FIG.
［実施例８〜実施例１７］
次に、Ｃａイオン／Ｍｇイオン（モル比）の割合を表４及び表５に示すように変えて、スポロサルシナ属ＮＯ−Ａ１０株、尿素を加えて炭酸塩を以下のように生成し、得られた炭酸塩の量を以下の実験方法、評価方法によって評価した。結果を下記表４及び表５に併記すると共に、図８に示す。
[Examples 8 to 17]
Next, the ratio of Ca ions / Mg ions (molar ratio) is changed as shown in Tables 4 and 5, and Sporosarcina NO-A10 strain, urea is added to produce carbonate as follows. The amount of carbonate was evaluated by the following experimental method and evaluation method. The results are shown in Table 4 and Table 5 below, and are shown in FIG.
＜実験方法＞
緩衝溶液（１０ｍＭ水酸化アンモニウム＋塩化アンモニウム）をベースに１ＭＣａＣｌ２と１Ｍ尿素を加えて溶液２００ミリリットル（Ｃａ液）を準備した。また、緩衝溶液（１０ｍＭ水酸化アンモニウム＋塩化アンモニウム）に１Ｍ ＭｇＣｌ２と１Ｍ尿素を加えた溶液２００ミリリットル（Ｍｇ液）を準備した。Ｃａ液とＭｇ液を所定の割合で混ぜて１０ミリリットルに分取した。その割合はＭｇ／Ｃａ比で１０：０，９：１，８：２，７：３，５：５，４：６，３：７，２：８，１：９，０：１０である。また、培養した菌体溶液２００ミリリットルを５０ミリリットルずつ分けて、４５００ｒｐｍで１５分間遠心分離した後、上澄み液を除き、沈殿した菌体に０．５％ＮａＣｌ溶液をそれぞれ５０ミリリットル加えた。これらから１０ミリリットルをメスピペットで取り、Ｃａ液とＭｇ液との混合液１０ミリリットルに加えた。これから５ミリリットルをメスピペットで取り分け検体とした。その後、約２０℃で振盪させて、７２時間及び１週間後に沈殿物の質量を測った。沈殿物は溶液中と試験管ガラス壁に付着したものに分けて測定した。溶液中の沈殿はろ過して乾燥質量を、試験管に付着したものは乾燥して試験管と沈殿物の質量を測定した。ろ紙および試験管の質量をそれぞれの測定値から差し引いて、それらを加えたものを全沈殿物とした。
<Experiment method>
Based on a buffer solution (10 mM ammonium hydroxide + ammonium chloride), 1 M CaCl 2 and 1 M urea were added to prepare 200 ml of a solution (Ca solution). In addition, a 200 ml solution (Mg solution) in which 1 M MgCl 2 and 1 M urea were added to a buffer solution (10 mM ammonium hydroxide + ammonium chloride) was prepared. Ca liquid and Mg liquid were mixed at a predetermined ratio and dispensed into 10 ml. The ratio of Mg / Ca is 10: 0, 9: 1, 8: 2, 7: 3, 5: 5, 4: 6, 3: 7, 2: 8, 1: 9, 0:10. Further, 200 ml of the cultured cell solution was divided into 50 ml, centrifuged at 4500 rpm for 15 minutes, the supernatant was removed, and 50 ml of each 0.5% NaCl solution was added to the precipitated cells. 10 ml of these were taken with a measuring pipette and added to 10 ml of a mixture of Ca solution and Mg solution. From this, 5 milliliters was separated with a female pipette and used as a specimen. Thereafter, the mixture was shaken at about 20 ° C., and the mass of the precipitate was measured after 72 hours and 1 week. Precipitates were measured separately in the solution and those adhered to the test tube glass wall. The precipitate in the solution was filtered to obtain a dry mass, and the deposit on the test tube was dried to measure the mass of the test tube and the precipitate. The mass of the filter paper and the test tube was subtracted from the respective measured values, and the sum of them was defined as the total precipitate.
Ｍｇ／Ｃａモル比ではなく、Ｃａ／(Ｃａ＋Ｍｇ）を考えれば、（Ｃａ＋Ｍｇ）は１Ｍとしたので分母を１とでき、図８のように使用したＭｇのモル濃度で表すことができる。図８でＭｇが１（すなわちＣａ／（Ｃａ＋Ｍｇ）＝０）の場合、Ｃａの沈殿量はゼロでなければならない。一方、Ｍｇが０Ｍ（すなわちＣａ／（Ｃａ＋Ｍｇ）＝１）の場合、Ｃａの沈殿量は１Ｍである。従って、０＜Ｍｇ＜１Ｍ（即ち、０＜Ｃａ／（Ｃａ＋Ｍｇ）＜１）において、カルシウムの沈殿がＭｇに優先すると仮定すれば、Ｃａの沈殿は、Ｃａ（Ｍ）＝Ｃａ／（Ｃａ＋Ｍｇ）×１（Ｍ）で表せる。従って、Ｍｇの沈殿量は、次のようになる。Ｍｇ（Ｍ）＝測定値−Ｃａ（Ｍ）。従って、図８から、次のことがわかる。Ｃａ／（Ｃａ＋Ｍｇ）が０．２以下の場合、炭酸マグネシウム（マグネサイト）又は炭酸マグネシウム又はＣａ−炭酸マグネシウムが生成し、７２時間以降も沈殿が起こり続ける。Ｃａ／（Ｃａ＋Ｍｇ）＝０．３のとき、沈殿は７２時間で終了していると思われ、そのときの沈殿物のＭｇ／Ｃａ比は１：１である。沈殿速度の点からは安定していると考えられる。Ｃａ／（Ｃａ＋Ｍｇ）＝０．５のとき、１週間後に沈殿物のＭｇ／Ｃａ比は０．５となっている。上記実施例８〜１７では沈殿量を時間で示してあるが、この場合も１６９時間後に０．７４３Ｍの沈殿が起こっており、この結果と一致する。沈殿速度は比較的速く、７２時間でほぼ安定している。 Considering Ca / (Ca + Mg) instead of Mg / Ca molar ratio, since (Ca + Mg) is 1M, the denominator can be 1, and can be expressed by the molar concentration of Mg used as shown in FIG. In FIG. 8, when Mg is 1 (that is, Ca / (Ca + Mg) = 0), the precipitation amount of Ca must be zero. On the other hand, when Mg is 0M (that is, Ca / (Ca + Mg) = 1), the precipitation amount of Ca is 1M. Therefore, assuming that the precipitation of calcium prevails over Mg in 0 <Mg <1M (ie, 0 <Ca / (Ca + Mg) <1), the precipitation of Ca is Ca (M) = Ca / (Ca + Mg) × 1 (M). Therefore, the precipitation amount of Mg is as follows. Mg (M) = measured value−Ca (M). Therefore, the following can be understood from FIG. When Ca / (Ca + Mg) is 0.2 or less, magnesium carbonate (magnesite) or magnesium carbonate or Ca-magnesium carbonate is generated, and precipitation continues to occur after 72 hours. When Ca / (Ca + Mg) = 0.3, the precipitation seems to be completed in 72 hours, and the Mg / Ca ratio of the precipitate at that time is 1: 1. It is considered stable from the viewpoint of the precipitation rate. When Ca / (Ca + Mg) = 0.5, the Mg / Ca ratio of the precipitate is 0.5 after one week. In Examples 8 to 17 above, the amount of precipitation is shown by time. In this case, 0.743 M of precipitation occurs after 169 hours, which is consistent with this result. The precipitation rate is relatively fast and is almost stable in 72 hours.
また、下記表６に、スポロサルシナ属ＮＯ−Ａ１０株による反応終了までのカルサイト生成速度を示す。 In addition, Table 6 below shows the calcite production rate until the end of the reaction by the Sporosarcina NO-A10 strain.
炭酸塩は自然界で岩盤、岩石、ノジュールなどとして形成されているように、硬い固体物質である。したがって、砂などのような集合体や多孔体などの間隙で形成されると粒子を結合して岩化を起こすので、土木工学における地盤の強度不足の解消に応用できる。具体的には、地すべり、地盤の崩壊、土石流、液状化、などの対策工事に応用できるばかりでなく、構造物の基礎の安定化（破壊や沈下）にも対策として適用できる。また間隙中やクラック内で炭酸塩を生成させることにより、止水効果が得られる。この応用は、地中止水壁の構築、アースダムのコアの止水性の向上、廃棄物処分場からの漏れの防止などに利用できる。さらには、地盤環境工学において、重金属を取り込んだ炭酸塩の形成によって、汚染土壌内における有害物質の不動化が可能である。 Carbonate is a hard solid material, as it is formed in nature as rock, rock, nodule and the like. Therefore, when formed in a gap such as an aggregate such as sand or a porous body, the particles are combined to cause rock formation, which can be applied to solve the lack of ground strength in civil engineering. Specifically, it can be applied not only to countermeasures such as landslides, ground collapse, debris flow, and liquefaction, but also to stabilization (destruction and settlement) of the foundation of structures. Moreover, the water-stopping effect can be obtained by generating carbonates in the gaps and cracks. This application can be used for construction of a water wall for earth suspension, improvement of the water stoppage of the core of the earth dam, and prevention of leakage from the waste disposal site. Furthermore, in geotechnical engineering, it is possible to immobilize harmful substances in contaminated soil by forming carbonates that incorporate heavy metals.
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| CN101119942A (en) * | 2004-12-20 | 2008-02-06 | 墨道什大学 | Microbial biocementation |
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