JP2015521533A - バイオメタン製造のための方法および組成物 - Google Patents
バイオメタン製造のための方法および組成物 Download PDFInfo
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- JP2015521533A JP2015521533A JP2015516468A JP2015516468A JP2015521533A JP 2015521533 A JP2015521533 A JP 2015521533A JP 2015516468 A JP2015516468 A JP 2015516468A JP 2015516468 A JP2015516468 A JP 2015516468A JP 2015521533 A JP2015521533 A JP 2015521533A
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- waste
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- biomethane
- enzymatic hydrolysis
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Abstract
Description
(i) 5〜40%の非水含量(non-water content)、かつ45〜75℃の温度にてMSWを準備するステップ、
(ii)廃棄物の生分解可能な部分の液化および微生物代謝生成物の蓄積をもたらす45〜75℃の温度での、微生物発酵と同時に行なわれるMSWの生分解可能な部分の酵素的加水分解ステップ、それに続く
(iii)廃棄物の液化された生分解可能な部分を生分解可能でない固形物から分類して、そのうちの少なくとも25重量%が酢酸塩、酪酸塩、エタノール、ギ酸塩、乳酸塩および/またはプロピオン酸塩のいずれかの組みわせを含む、溶存揮発性固形物を含むことを特徴とするバイオ液体を生成させるステップ、それに続く
(iv)バイオ液体の嫌気的消化によりバイオメタンを生成させるステップ。
非水含量の少なくとも40重量%が溶存揮発性固形物として存在し、該溶存揮発性固形物は、少なくとも25重量%の酢酸塩、酪酸塩、エタノール、ギ酸塩、乳酸塩および/またはプロピオン酸塩のいずれかの組みわせを含む
ことを特徴とする、都市固形廃棄物(MSW)の酵素的加水分解および微生物発酵により生成される有機液体バイオガス基質を提供する。
(i)微生物発酵により予備調整され、それにより非水含量の少なくとも40重量%が溶存揮発性固形物として存在し、該溶存揮発性固形物は少なくとも25重量%の酢酸塩、酪酸塩、エタノール、ギ酸塩、乳酸塩および/またはプロピオン酸塩のいずれかの組みわせを含む、有機液体バイオガス基質を準備するステップ、
(ii)該液体基質を、嫌気的消化システムに移すステップ、それに続く
(iii)該液体基質の嫌気的消化を行い、バイオメタンを生成させるステップ。
(i)微生物発酵により予備調整され、それにより非水含量の少なくとも40重量%が溶存揮発性固形物として存在し、該溶存揮発性固形物は少なくとも25重量%の酢酸塩、酪酸塩、エタノール、ギ酸塩、乳酸塩および/またはプロピオン酸塩のいずれかの組みわせを含む、有機液体バイオメタン基質を準備するステップ、
(ii)該液体基質を嫌気的消化システムに移すステップ、それに続く
(iii)該液体基質の嫌気的消化を行い、バイオメタンを生成させるステップ。
実施例5に記載の試験からのバイオ液体サンプルを用いて、実験台規模の反応を行なった。
固形物の変換率は、総乾物の割合(%)として上清中に見出される固形物の含有量として測定した。図1は、MSWブランク、単離された酵素調製物、微生物接種物のみ、および微生物接種物と酵素との組み合わせについての変換率を示す。結果は、実施例5からのEC12Bの添加が、反応ブランク(MSWブランク)中の乾物のバックグラウンド放出と比較して、有意に高い乾物の変換率をもたらしたことを示す(スチューデントt検定、p<0.0001)。EC12Bサンプルの添加により誘導される同時に行なわれる微生物発酵およびCTec3を用いた酵素的加水分解は、CTec3のみを用いて加水分解された反応物およびEC12Bのみを添加された反応物と比較して、有意に高い乾物の変換率をもたらした(p<0.003)。
上清中で測定されたグルコースおよび微生物代謝生成物(乳酸塩、酢酸塩およびエタノール)の濃度は、図2に示される。示されている通り、モデルMSWブランク中にはこれらの低いバックグラウンド濃度があり、乳酸含量はおそらくは、基質を作製するために用いた材料がまったく無菌でなく、または細菌を殺すために加熱されていないので、モデルMSWに元々含まれる細菌により生じている。CTec3の添加の効果は、上清中のグルコースおよび乳酸の増加をもたらした。グルコースおよび細菌代謝生成物の最高濃度は、実施例5からのEC12Bバイオ液体をCtec3と同時に加えた場合の反応物中で見出された。つまり、同時に行なわれる発酵および加水分解は、モデルMSWでの乾物の変換率を改善し、液体中の細菌代謝生成物の濃度を増大させる。
参考文献:Jacob Wagner Jensen, Claus Felby, Henning Jorgensen, Georg Ornskov Ronsch, Nanna Dreyer Norholm. Enzymatic processing of municipal solid waste. Waste Management. 12/2010; 30(12):2497-503.
Riber, C., Petersen, C., Christensen, T.H., 2009. Chemical composition of material fractions in Danish household waste. Waste Management 29, 1251-1257。
試験は、実験室規模実験で得られた結果を確認するために、未分類MSWを用いて、図3に示される特別に設計したバッチ反応器中で行なった。実験は、同時に行なわれる微生物発酵および酵素的加水分解を達成するために、実施例3の細菌から得られたバイオ液体を含む微生物の接種物を添加することの効果を試験した。試験は、未分類MSWを用いて行なった。
サンプルを60℃にて48時間乾燥させた。乾燥の前後でのサンプルの重量を用いて、DMパーセンテージを算出した。
揮発性固形物を算出し、灰分含量を差し引いたDMパーセンテージとして表わす。サンプルの灰分含量は、最小で4時間、炉内で550℃にて予め乾燥させたサンプルを燃焼させることにより見出された。次に、灰分は以下の通りに算出した:
実験は、Amager ressource center(ARC, Copenhagen, Denmark)に置かれたREnescience実証用プラントにて行なわれた。プラントの主要な特徴を示す模式図を、図4に示す。ARC REnescience Waste Refineryのコンセプトは、MSWを4種類の生成物に分類することである。バイオガス製造のためのバイオ液体、リサイクル用の不活性材料(ガラスおよび砂)ならびに金属、プラスチックおよび樹木のRDF製造またはリサイクルに好適な無機材料の2D区分および3D区分。
・「EC12B」と名付けられた、目の細かい篩から離れるバイオ液体
・貯蔵タンク中のバイオ液体
・ホエー篩後の洗浄水
・2D区分
・3D区分
・両方の洗浄ユニットからの不活性な底部分
バイオ液体の生成は、貯蔵タンク上のロードセルを用いて測定した。新鮮な水の入力流を流量計を用いて測定し、再利用または排出された洗浄廃棄物をロードセルを用いて測定した。
実施例3から取得されたバイオ液体のサンプルを、微生物組成に関して分析した。
実施例1に記載されるREnescience実証用プラントを用いて、未分類MSWの、同時に行なわれる細菌発酵および酵素的加水分解を用いた総有機物捕捉の詳細研究を行なった。
バイオ液体「EC12B」のサンプルは、2012年12月15日および16日に実施例5に記載される試験中に回収され、サンプル中の微生物を同定するために16S rDNA分析を行なう目的で、−20℃にて保存した。16S rDNA分析は、リボソーム小サブユニットの16S成分に基づいて原核生物の同定および系統発生学分析に対して広く用いられる。凍結サンプルはドライアイス上でGATC Biotech AB(Solna, SE)に輸送され、ここで16S rDNA分析が行なわれた(GATC_Biotech)。分析は、以下のステップを含んだ:ゲノムDNAの抽出、ユニバーサルプライマー(超可変領域V1〜V3にまたがるプライマー対、27F:AGAGTTTGATCCTGGCTCAG/534R:ATTACCGCGGCTGCTGG;507bp長)を用いるアンプリコンライブラリー調製、GS FLXアダプターを用いたPCRタグ付け、サンプル当たり104,000〜160,000の読み出し数を与えるGenome Sequencer FLX装置での配列決定。得られた配列を次に、リボソームデータベースプロジェクトからのrDNAデータベースに対してBlastNで問い合わせた(Cole et al., 2009)。データベースは、少なくとも1200bp長およびNCBI分類学的関連付けを有する良質の配列を含む。現行リリース(RDPリリース10、2012年9月19日に更新)は、9,162種の細菌および375種の古細菌配列を含む。BLAST結果をフィルタリングして、短く低い品質のヒットを除いた(配列同一性90%以上、アライメント範囲90%以上)。
実施例3に記載されたREnescience実証用プラントを用いて、オランダから輸入されたMSWを処理した。MSWは以下の組成を有することが見出された:
実施例5に記載された実験で得られたバイオ液体を20リットルバケツ中で凍結させ、後の使用のために−18℃で保存した。この材料を、フィルター支持体上に固定化されたバイオフィルム内の嫌気的消化集団を含む、2つの同一の十分調整された固定化フィルター嫌気的消化システムを用いて、バイオメタン製造に関して試験した。
実施例2で得られた「高乳酸塩」バイオ液体および「低乳酸塩」バイオ液体を、実施例8に記載された固定化フィルター嫌気的消化システムを用いてバイオメタン生成について比較した。測定値が取得され、実施例8に記載された通りに「上昇」時間および「下降」時間が決定された。
コムギワラを、前処理し、繊維画分および液体画分へと分離し、続いて繊維画分を別個に洗浄した。続いて、5kgの洗浄された繊維を、実施例3から得られたバイオ液体(biovaeske)からなる発酵性微生物の接種を伴って、Cellic CTEC3の投入と共に、水平回転ドラム反応器中でインキュベートした。コムギワラを、50度にて3日間、同時の加水分解および微生物発酵に供した。
特定の単独培養細菌を用いる、同時に行なわれる微生物および酵素的加水分解反応を、モデルMSW(実施例1に記載される)を用いて、実施例1に記載された手順に従って、実験室規模で行なった。反応条件および酵素投入量を表4に特定する。
バイオ液体「EC12B」および再循環水「EA02」のサンプルを、実施例7に記載された試験の間に採取した(サンプリングは、3月21日および22日に行なわれた)。リボソーム小サブユニットの16S成分に基づく原核生物の同定および系統発生学分析に対して広く用いられる、微生物を同定するための16S rDNA分析を行なう目的のために、液体サンプルを10%グリセロール中で凍結し、−20℃で保存した。凍結サンプルはドライアイス上でGATC Biotech AB(Solna, SE)に輸送され、ここで16S rDNA分析が行なわれた(GATC_Biotech)。分析は、以下のステップを含んだ:ゲノムDNAの抽出、ユニバーサルプライマー(超可変領域V1〜V3にまたがるプライマー対、27F:AGAGTTTGATCCTGGCTCAG/534R:ATTACCGCGGCTGCTGG;507bp長)を用いるアンプリコンライブラリー調製、GS FLXアダプターを用いたPCRタグ付け、サンプル当たり104,000〜160,000の読み出し数を与えるGenome Sequencer FLX装置での配列決定。得られた配列を次に、リボソームデータベースプロジェクトからのrDNAデータベースに対してBlastNで問い合わせた(Cole et al., 2009)。データベースは、少なくとも1200bp長およびNCBI分類学的関連付けを有する良質の配列を含む。現行リリース(RDPリリース10、2012年9月19日に更新)は、9,162種の細菌および375種の古細菌配列を含む。BLAST結果をフィルタリングして、短く低い品質のヒットを除いた(配列同一性90%以上、アライメント範囲90%以上)。
実施例7に記載された試験から回収された3月21日および22日からのEA02のサンプルを、同定および単離された細菌の単独培養物を取得する目的のために、Novo Nordic Centre for Biosustainability(NNセンター)(Hoersholm, Denmark)でのプレート播種のために送付した。NNセンターに到着すると、サンプルを50℃にて一晩インキュベートし、続いて種々のプレート上に撒き(GM17、トリプシンダイズ培地(tryptic soy broth)、および牛肉エキス(GM17寒天:48.25g/L m17アガー、20分間のオートクレーブ後、グルコースを0.5%の最終濃度まで添加する、トリプシンダイズ寒天(Tryptic soy agar):30g/Lトリプシンダイズ培地(Tryptic soy broth)、15g/L寒天、牛肉培地(Beef broth)(Statens Serum Institute, Copenhagen, Denmark)、15g/Lアガロースを添加)、50℃にて好気的に増殖させた。
同定されたサンプル
サンプルID:13-349(バチルス・サフェンシス(Bacillus safensis))(EA02-21/3)由来、DSM 27312
サンプルID:13-352(ブレビバチルス・ブレビス(Brevibacillus brevis))(EA02-22/3)由来、DSM 27314
サンプルID:13-353(バチルス・サブティリスsp.サブティリス(Bacillus subtilis sp. subtilis))(EA02-22/3)由来、DSM 27315
サンプルID:13-355(バチルス・リケニフォルミス(Bacillus licheniformis))(EA02-21/3)由来、DSM 27316
サンプルID:13-357(アクチノミセス・ボビス(Actinomyces bovis))(EA02-22/3)由来、DSM 27317
同定されなかったサンプル
サンプルID:13-351(EA02-22/3)由来、DSM 27313
サンプルID:13-362A(EA02-22/3)由来、DSM 27318
サンプルID:13-365(EA02-22/3)由来、DSM 27319
サンプルID:13-367(EA02-22/3)由来、 DSM 27320。
Cole, J. R., Wang, Q., Cardenas, E., Fish, J., Chai, B., Farris, R. J., & Tiedje, J. M. (2009). The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic acids research, 37 (suppl 1), (D141-D145).
GATC_Biotech supporting material. Defining the Microbial Composition of Environmental Samples Using Next Generation Sequencing. Version 1.
Tribelli, P. M., Iustman, L. J. R., Catone, M. V., Di Martino, C., Reyale, S., Mendez, B. S., Lopez, N. I. (2012). Genome Sequence of the Polyhydroxybutyrate Producer Pseudomonas extremaustralis, a Highly Stress-Resistant Antarctic Bacterium. J. Bacteriol. 194(9):2381.
Nancy I. Lopez, N. I., Pettinari, J. M., Stackebrandt, E., Paula M. Tribelli, P. M., Potter, M., Steinbuchel, A., Mendez, B. S. (2009). Pseudomonas extremaustralis sp. nov., a Poly(3-hydroxybutyrate) Producer Isolated from an Antarctic Environment. Cur. Microbiol. 59(5):514-519。
Claims (11)
- 以下のステップ:
(i)微生物発酵により予備調整され、それにより非水含量の少なくとも40重量%が溶存揮発性固形物として存在し、該溶存揮発性固形物は少なくとも25重量%の酢酸塩、酪酸塩、エタノール、ギ酸塩、乳酸塩および/またはプロピオン酸塩のいずれかの組みわせを含む、有機液体バイオメタン基質を準備するステップ、
(ii)該液体基質を、嫌気的消化システムに移すステップ、それに続く
(iii)該液体基質の嫌気的消化を行い、バイオメタンを生成させるステップ
を含む、バイオメタンを製造する方法。 - 前記微生物発酵が、pH5.5より低い条件で行なわれる、請求項1に記載の方法。
- 前記有機液体バイオメタン基質が、未分類MSWの、同時に行なわれる酵素的加水分解および微生物発酵により生成される、請求項1に記載の方法。
- 前記有機液体バイオメタン基質が、前処理されたリグノセルロース系バイオマスの、同時に行なわれる酵素的加水分解および微生物発酵により生成される、請求項1に記載の方法。
- 前記有機液体バイオメタン基質が、オートクレーブ処理により未分類MSWから得られる液化された有機材料の、同時に行なわれる酵素的加水分解および微生物発酵により生成される、請求項1に記載の方法。
- 前記有機液体バイオメタン基質が、少なくとも8%の総固形物を含む、請求項1に記載の方法。
- 前記嫌気的消化システムが、固定化フィルター嫌気的消化装置である、請求項1に記載の方法。
- 前処理されたリグノセルロース系バイオマスの酵素的加水分解および微生物発酵により生成される有機液体バイオメタン基質であって、
非水含量の少なくとも40重量%が溶存揮発性固形物として存在し、該溶存揮発性固形物は少なくとも25重量%の酢酸塩、酪酸塩、エタノール、ギ酸塩、乳酸塩および/またはプロピオン酸塩のいずれかの組みわせを含む
ことを特徴とする、上記液体バイオメタン基質。 - 少なくとも8%の総固形物含量を有する、請求項7に記載の液体バイオメタン基質。
- 25℃にて15mg/L未満の溶存メタン含量を有する、請求項7に記載の液体バイオメタン基質。
- 少なくとも10%の総固形物含量を有する、請求項7に記載の液体バイオメタン基質。
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JP2009509522A (ja) * | 2005-09-30 | 2009-03-12 | エルサム・エンジニアリング・アクティーゼルスカブ | 廃棄物画分の非−加圧前−処理、酵素加水分解および発酵 |
JP2007275889A (ja) * | 2007-04-27 | 2007-10-25 | Masahiro Izutsu | リグノセルロース系バイオマスの利用方法 |
JP2010531668A (ja) * | 2007-06-27 | 2010-09-30 | ノボザイムス アクティーゼルスカブ | 発酵製品を生産するための方法 |
JP2010536558A (ja) * | 2007-08-22 | 2010-12-02 | イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー | バイオマス処理方法 |
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JP2021513499A (ja) * | 2018-02-13 | 2021-05-27 | レネサイエンス エー/エス | 消化物を含む建築材料 |
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