JP3896564B2 - Novel bacterial cellulose-producing microorganism and enhanced oil recovery method using the same - Google Patents

Description

【００５６】
また、上記培養の嫌気環境は嫌気培養システム(Becton Dickinson and Company社製)および添付のプロトコルに従って構築した。
【００５７】
以上の分離手順で、菌分離用試料から約47株の微生物が分離され、そのうちの２株がコロニー周辺に顕著な粘性物質を生産するのが確認された。次にこれらの分離株を、４％（v/v）中国産ビートモラセス含有水溶液を高圧蒸気滅菌して調製した液体培地に接種し、気相部を窒素で３分間置換して培養系内を嫌気環境とした後、30℃で１日間静置培養して、培養液中への不溶性ポリマーの生産状況を指標として（目視）、不溶性ポリマーを形成する菌株を単離した。当該菌は顕微鏡観察やその菌体の大きさから、細菌であると認められた。
【００５８】
この菌株を、後述する菌学的特徴に基づいてEnterobacter属細菌（Enterobacter sp. CJF-002）と命名し、平成12年3月29日に茨城県つくば市東町１丁目１番３号に所在の通商産業省工業技術院生命工学工業技術研究所に寄託した（受託番号 FERM P-17799）。以下実施例において当該菌を単に「CJF-002株」と称する。
【００５９】
実施例２：菌学的性質（形態、生理学的性質）
実施例１にて単離されたCJF-002株の菌学的位置づけを行うために、CJF-002株の形態的性質、培地における生育状態および生理学的性質について分析した。
Ａ．形態的性質
寒天培地を用いて、３０℃で２４時間の培養条件下で生育した細菌について、次の形態学的性質が認められた。
【００６０】
(1) 細胞の形：桿状
(2) 細胞の大きさ：幅0.5-1.0μm、長さ1.5-3.0μm（光学顕微鏡観察）
(3) 運動性の有無：有
(4) コロニーの色：白色
(5) コロニーの形状：皺状の歪つな形状
Ｂ．培養的性質（培地における生育状態）
(1) 肉汁液体培養：（＋）
(2) 好気性条件下での生育：（＋）
(3) 嫌気性条件下での生育：（＋）。
【００６１】
Ｃ．生理学的性質
CJF-002株の生理学的性質の決定には、ブドウ糖発酵性桿菌の同定キットであるIDテスト・EB-20（日水製薬製）を用いた。具体的には、高圧蒸気滅菌したNutrient broth（Difco社製）液体培地に１白金耳のCJF-002株を接種した後、30℃で一夜静置培養して前培養液を得た。この前培養液をキットに添付のプロトコールに従って処理し、生理学的性質に関する種々の測定を行った。結果を以下に示す。
【００６２】
(1) グラム染色性： 陰性
(2) O-F試験［Hugh leifson法］：（F）
(3) グルコースからのガスの生産：（＋）
(4) インドールの生成：（-）
(5) 硫化水素の生成：（-）
(6) 有機酸の利用： クエン酸 資化性（＋）
(7) 色素の生産： （-）
(8) ウレアーゼ： （-）
(9) オキシダーゼ： （-）［シトクロムｃオキシダーゼ］
(10) カタラーゼ：（＋）
(11) アルギニンジヒドロラーゼ：（＋）
(12) リジンジカルボキシラーゼ： （-）
(13) オルニチンデカルボキシラーゼ：（＋）
(14) フェニルアラニンジアミナーゼ：（-）
(15) ゼラチン分解性：（-）
(16) マロン酸分解性：（-）
(17) ONPG：（＋）
(18) 生育の範囲： 温度；4-45℃／pH；2.2-9.53
(19) Ｌ-アラビノースを利用した発酵性：（＋）
(20) イノシトールを利用した発酵性：（-）
(21) Ｄ-ソルビトールを利用した発酵性：（-）
(22) Ｄ-ラムノースからの酸の生成：（＋）
(23) Ｄ-マンニトールからの酸の生成：（＋）
(24) Ｄ-アドニトールからの酸の生成：（-）
(25) Ｄ-ラフィノースからの酸の生成：（-）
(26) Ｄ-スクロースからの酸の生成：（＋）
Ｄ．その他の特徴的性質： エスクリンの分解 （＋）。
【００６３】
上記で得られたCJF-002株の生理学的性質をBergey's Manual of Systematic Bacteriology[Vol.1(1984), Vol.2(1986), Vol.3(1989), Vol.4(1989), Williams & Wilkins, Baltimore]に記載される種々の微生物の諸性質と対比したところ、本発明のCJF-002株は、これまでに報告されているEnterobacter 属に属する細菌の生理学的性質と最もよく一致していた。
【００６４】
実施例３：菌学的性質（化学的性質）
実施例１で得られたCJF-002株の菌学的位置づけを遺伝子工学的側面から特徴付けるために、CJF-002株の16S-rRNA遺伝子のクローニング、及びその塩基配列の決定（解析）を行った。
（１）CJF-002株が保有する遺伝子のクローニング
鋳型DNAには、InstaGene Matrix法[Lamballerie et al., A one-step microbial DNA extraction method using "Chelex 100" suitable for gene amplification. Res Microbiol 143: 785-790 (1992)] を用いて調製したCJF-002株染色体DNAを用いた。そして、上流側プライマーに、5'-AGAGTTTGATCCTGGCTCAG-3'(配列番号：３）を、下流側プライマーに、5'- AAAGGAGGTGATCCAGCC-3'(配列番号：４）のプライマーを用い、Taq DNA ポリメラーゼ（宝酒造社製）でPCR増幅した。PCRにおける変性反応、アニーリング反応及び伸長反応はそれぞれ94℃で1分、52℃で2分及び72℃で2分の条件で行い、かかる反応サイクルを30回繰り返した。PCR増幅断片をpMOS Blue T-vector Kit （Amersham International plc, 製）を用いてプラスミドベクターにクローニングした。当該組み換えプラスミドは、QIAquick Gel Extraction Kit（QIAGEN製）を用いて大量調製した。宿主菌として、E. coli JM109[recA1, endA1, gryA96, thi, hsdR17, supE44, relA1, △(lac-proAB),F'(traD36, proAB+, lac1y, lacZ △ M15)]を用いた。当該宿主大腸菌の形質転換は、Sambrookら[Molecular Cloning, A Laboratory Manual 2nd ed. (1989)]の方法に従った。なお、培地はＬ培地（トリプトン10 g/l、酵母エキス 5 g/l、NaCl 5 g/l、グルコース 1 g/l、pH 7.2）を用い、抗生物質として100μg/mlアンピシリンを用いた。
（２）CJF-002株が保有する遺伝子の解析
CJF-002株のクローンが保有する遺伝子の塩基配列を、自動解析装置ABI DNA Sequencer （Perkin-Elmer Co.製）とABI PRISM Dye Cycle Sequencing Kit（Perkin-Elmer Co.製）を用いて解析し、微生物分類の重要な指標の一つである、16S-rRNA遺伝子の塩基配列を解読した。当該遺伝子配列を配列番号：２に示す。この配列を基に、Gene Bank等の遺伝子データベースに登録された他の微生物が保有する16S-rRNA遺伝子の配列と比較して両者の相同性をみた。結果を表２に示す。なお、これらの相同性解析にはBLAST search program[Altschul et al., Basic local alignment search tool. J. Mol. Biol., 215, 403-410 (1990)]を使用した。
【００６５】
【表２】

【００６６】
この表からわかるように、CJF-002株の16S-rRNA遺伝子の塩基配列はEnterobacter属細菌の16S-rRNA遺伝子の塩基配列と98％以上の高い割合で一致していた。特にEnterobacter cloacaeの16S-rRNA遺伝子の塩基配列と極めて高い割合で一致していた。
【００６７】
以上の実施例２及び３で示す菌学的性質（生理学的性質等、化学的性質）から、本発明のCJF-002株はEnterobacter属に最も近縁にある細菌であると判断された。
【００６８】
実施例４：微生物産生バクテリアセルロースの評価
実施例１で単離したCJF-002株に関して、当該微生物が生産する不溶性ポリマー物質を以下の手順に従って評価した。
【００６９】
まず、水で４％(v/v)に希釈した中国産ビートモラセス液体培地と２.０％のスクロースを添加したPolysaccharide-production medium [Akihiko Shimada, Viva Origino, 23, 1, 52-53, 1995]（以下「ＰＰＭ培地」と称す）の２種類の培地を基本培地とし、これらを高圧蒸気滅菌処理した培地30mlをそれぞれ50ml容のバイアル瓶に分注して、CJF-002株を接種し、30℃で1日間静置培養した。なお、上記CJF-002株には、予め高圧蒸気滅菌したNutrient broth（Difco社製）液体培地を用いて30℃で一夜静置培養したもの（前培養物）を用いた。
【００７０】
次いで、上記２種類の基本培地中でCJF-002株が生産する不溶性ポリマーについてそれぞれ(1)糖成分の組成、及び(2)糖成分の結合様式を分析した。なお、これらの分析は、前記培養により得られた不溶性ポリマーを500mlの滅菌蒸留水で数回水洗し、次いで加熱洗浄して得られた不溶性ポリマーの凍結乾燥品を用いて行った。
【００７１】
（１）糖成分の組成、
糖成分の組成分析は、前記不溶性ポリマーの凍結乾燥品を糖成分が含まれていないことを予め確認した市販のセルラーゼで加水分解し、次いでこれらの加水分解物について、以下に示す条件で中性糖およびウロン酸の分析を行うことにより実施した。尚、上記不溶性ポリマーは、トリフルオロ酢酸（TFA）、塩酸及び硫酸などの有機酸や無機酸では加水分解され難く、加水分解率はそれぞれ12-13％、5-9％及び20-33％程度であった。一方、セルラーゼ等による酵素分解処理では65-75％の分解率が得られた。なお、当該ポリマーの分解性はその生産条件、すなわち、生産に使用した培地の違いより影響を受けなかった。
【００７２】
(i) 中性糖の分析条件
ＨＰＬＣ装置 ： LC-9A（島津製作所製）
カラム ： TSK-gel Sugar AXG（φ4.6 mm×150 mm、東ソー社製）
使用溶離液 ： 0.5 mMのホウ酸カリウム緩衝液（溶離液Ａ）
流速 ： 0.4 ml/min
ポストカラム標識： 1％アルギニン及び３％ホウ酸を用いて、流速を0.5ml/min、反応温度を150℃とした。
【００７３】
(ii) ウロン酸の分析条件
ＨＰＬＣ装置 ： LC-9A（島津製作所製）
カラム ： Shimpal ISA-07（φ4.6 mm×250 mm、島津製作所製）
使用溶離液 ： 0.5 mMのホウ酸カリウム緩衝液（溶離液Ａ）
流速 ： 0.8 ml/min
ポストカラム標識： 1％アルギニンおよび3％ホウ酸を用い、流速を 0.8ml/min、反応温度を150℃とした。
【００７４】
４％(v/v)モラセス培地で得られた不溶性ポリマーとＰＰＭ培地（2%スクロース含有）で得られた不溶性ポリマーについて、各測定条件によって得られた糖成分の組成比を表３に示す。
【００７５】
【表３】

【００７６】
中性糖の分析において、主たるピークの保持時間は市販のグルコースの標品の保持時間と一致し、その組成比は前記２種の液体培地で生産された不溶性ポリマーはいずれも97％以上であった。またグルコース以外の糖成分としては、マンノース、アラビノース、ガラクトースが１％程度かそれ以下の濃度で検出された。一方、グルクロン酸やガラクチュロン酸などのウロン酸はいずれも検出されなかった。
【００７７】
（２）糖成分の結合様式
不溶性ポリマーの糖成分の結合様式は、ガスクロマトグラフィー（GC）およびガスクロマトグラフィー質量分析装置（GC-MS）を用いたメチル化分析で得られた結果から推定した。
【００７８】
具体的には、まず前記不溶性ポリマーの凍結乾燥品について、粉末NaOH法によってその糖成分を完全メチル化し、生成したメチル化多糖をTFAにより加水分解して単糖にした後、無水酢酸-ピリジン溶液にて還元アセチル化して部分メチル化糖アルコールのアセチル誘導体（部分メチル化アルジトールアセテート）の形にした。これを下記の条件によるＧＣ分析およびＧＣ−ＭＳ測定に供した。
【００７９】
(i) ＧＣ分析条件
ＧＣ装置 ： HP5890A（Hewlett-Packerd社製）
カラム ： SPB-5（fused silica capillary、25m×0.25mm I.D. スペルゴジャパン社製）
キャリアーガス： Ｈｅ
検出モード ： ＦＩＤ。
【００８０】
(ii) ＧＣ−ＭＳ測定条件
ＧＣ装置および質量分析部分： JMS DX-303（日本電子社製）
イオン化 ： ＥＩ法。
【００８１】
中国産ビートモラセス液体培地で生産された不溶性ポリマーのメチル化分析の結果を表４に示す。
【００８２】
【表４】

【００８３】
試料の部分メチル化アルジトールアセテートのガスクロマトグラムにおいて、主たるピークのマススペクトルは、1,4,5-トリ-O-アセチル-2,3,6-トリ-O-メチルグルシトールの標準マススペクトルと一致し、1,4結合の結合様式が推定された。さらに、ガスクロマトグラムにおいて得られた各ピークとの面積比から、主たるピークの組成比は、非還元末端グルコース（1,5-ジ-O-アセチル-2,3,4,6-テトラ-O-メチルグルシトール）を１とした場合29.22であり、かつ、他の1,3結合や1,6結合などの組成比が0.5以下であったことから、前記不溶性ポリマーの主要な結合様式は1,4結合であると推定された。以上のことから、前記不溶性ポリマーは1,4結合のグルコースが主成分であることが明らかになった。
【００８４】
さらに、前述するように、当該ポリマーは有機酸や無機酸では加水分解され難いが、グルコースのβ1,4結合を選択的に分解する基質特異性を有するセルラーゼによっては比較的容易に分解されるといった性質を有しており、かかる性質を考え合わせると、前記不溶性ポリマーはβ1,4結合のグルコースを主骨格とするセルロースかあるいはそれに極めて類似した物質であると判断された。
【００８５】
なお、当該ポリマーの酸やセルラーゼによる分解性、および糖成分の組成などは、当該ポリマーの生産条件、すなわち生産に使用した培地の違いによって影響を受けない。このことから、ＰＰＭ培地や中国産ビートモラセス液体培地で生産された不溶性ポリマーは同一かあるいは極めて類似した物質と推定される。これらのことから、当該不溶性ポリマーの生産にはＰＰＭ培地の様な人工培地だけでなく、製糖工場からの廃棄物であるモラセスなどの安価な天然物質を培地として利用できるものと考えられる。
【００８６】
実施例５：微生物のモラセス培地での増殖性及びバクテリアセルロースの生産性
実施例１で単離したCJF-002株に関して、安価なモラセス培地での増殖性およびバクテリアセルロースの生産性を以下の手順に従って評価した。
【００８７】
まず、前培養として、CJF-002株を、高圧蒸気滅菌した2mlの4％(v/v)中国産ビートモラセス液体培地で30℃で一夜静置培養した。続いて本培養として、高圧蒸気滅菌した0.1および1％(v/v)中国産ビートモラセス液体培地30mlを用いて、CJF-002株の初期濃度を2.2×10⁴ および2.2×10² CFU/mlとして30℃で72時間静置培養を行った。培養期間中、培養から4、6、8、10、12、14、16、20、24、28、32、36、40、44、48、56、64及び72時間後に培養物を採取し、各採取ポイントでのCJF-002株の増殖度を下記(1)に示す直接計数法を用いて評価した。また同時に、培地中のバクテリアセルロース濃度を下記の(2)の手順に従って測定して、バクテリアセルロースの生産性を評価した。
【００８８】
(1) CJF-002株の増殖度の測定
CJF-002株は不溶性のバクテリアセルロースを生産し、またそのバクテリアセルロース内にも多数の菌体が存在している。このため、次の分析方法により系内のCJF-002株の総菌数を測定した。すなわち、まず、採取した培養液に500mMリン酸緩衝液（pH5.5）、0.2％ナリジクス酸、およびセルラーゼ（糖成分不含）（1％溶液）を添加し、30℃で10時間インキュベートする。ポリマーの分解を目視で確認したら、その溶液に8％パラホルムアルデヒドを添加して、菌体細胞を固定化する。次いで、固定化菌体溶液をブラックフィルターでろ過し、2mlのPBS（20mMリン酸buffer+130mM NaCl, pH7.4）で洗浄した後、0.01％アクリジンオレンジ溶液（0.01M Tris-HCl, pH7.4）で5分間染色し、検鏡する。
(2) バクテリアセルロース濃度の測定
採取した培養液に３倍量のアセトンを加えて、ポリマー成分を析出させ（アセトン抽出）、遠心処理によってポリマー成分を回収した後、常温で一晩乾燥し、常法のphenol-sulfuric acid reaction法にて糖濃度を測定する。
【００８９】
図１に、CJF-002株の増殖性（図Ａ）およびバクテリアセルロースの生産性（図Ｂ）の経時変化を示す。これから、CJF-002株の初期濃度が10⁴ CFU/mlの場合、培養10時間後に対数増殖期から定常期に移行し、CJF-002株は極めて早期に増殖することが判明した。増殖速度はモラセス濃度の違いによって左右されないが、最大増殖濃度はモラセス濃度に依存し、中国産ビートモラセスの濃度が1％の場合には約10⁹ CFU/ml、0.1％の場合には約10⁸ CFU/mlであった。CJF-002株の初期濃度が10² CFU/mlの場合は、培養14時間後に対数増殖期から定常期に移行し、初期濃度が10⁴ CFU/mlの場合よりも４時間程度後期にシフトした。この場合の増殖速度も中国産ビートモラセスの濃度の違いによって左右されず、最大増殖濃度も初期濃度が10⁴ CFU/mlの場合と同様な結果となった。これらの結果から、CJF-002株の増殖速度を計算すると対数増殖期の倍加時間（Doubling time）はおよそ40分であることが判明した。
【００９０】
バクテリアセルロースの生産性については、CJF-002株の初期濃度を10⁴ CFU/ml、中国産ビートモラセスの濃度を1％とした場合、培養開始後約14時間までに0.2-0.25 g/lものバクテリアセルロースが生産され、その後はほぼ一定になることがわかった。また、CJF-002株の初期濃度が10² CFU/mlの場合には、培養開始後約16-20時間までに同濃度のバクテリアセルロースが生産された。中国産ビートモラセスの濃度が0.1％の場合も、前記の結果と同様な傾向が見られたが、バクテリアセルロースの生産量は大きく低下し、最大でも0.05g/l以下であった。
【００９１】
得られたCJF-002株の増殖曲線とバクテリアセルロースの生産曲線との対比により、CJF-002株によるバクテリアセルロースの生産は、その増殖に約４時間遅れて進行することが判明した。
【００９２】
実施例６：バクテリアセルロース生産性に対するモラセス濃度の影響
次に、CJF-002株のバクテリアセルロースの生産性に対するモラセス濃度の影響について評価した。まず、CJF-002株を高圧蒸気滅菌した2mlの4％(v/v)中国産ビートモラセス液体培地を利用して30℃で一夜静置培養して前培養液を調製し、該前培養液を用いて本培養を行った。本培養には、高圧蒸気滅菌した0.001、0.01、0.1、1、5および10％の中国産ビートモラセス液体培地30mlを用い、CJF-002株の初期濃度を1.0×10⁶ CFU/mlとして30℃で24時間、静置培養した。培養終了後、CJF-002株の生菌数をNutrient broth（Difco社製）寒天培地を用いたＣＦＵ法（colony forming unit）により測定し、バクテリアセルロースの生産性を、培養液から取り出したバクテリアセルロースの乾燥重量を測定することにより評価した。
【００９３】
結果を図２に示す。図２に示すように、CJF-002株は中国産ビートモラセスの濃度が0.1％(v/v)以上で増殖するが、バクテリアセルロースの生産は1％以上の中国産ビートモラセス培地を用いた場合において活発化し、10％濃度の液体培地を用いた場合には、0.4g/l以上ものバクテリアセルロースが生産された。これらの結果から、バクテリアセルロースの生産性はモラセス濃度の上昇に依存して増大すること、並びにCJF-002株は安価なモラセスを利用して極めて短時間に大量のバクテリアセルロースを生産できることが判明した。
【００９４】
実施例７：バイオフィルムの形成能力
実施例１で単離したCJF-002株について、油層岩に対するバイオフィルムの形成能力を評価した。
【００９５】
具体的には、前記扶余油田から採取した後、高圧蒸気滅菌した油層岩小片（厚さ3mm、直径37mm、浸透率[Kair]607md）を１Lビーカー内に垂直に固定し、そこに4％(v/v)の中国産ビートモラセスと合成油層水成分を混和した液体培地500mlを注ぎ込んで、ここに実施例６と同様な手法により調製したCJF-002株の前培養液を初期濃度が1.0×10⁶ CFU/mlになるように接種した。
【００９６】
なお、上記合成油層水は、中国石油天然気株式有限公司吉林石油分公司の扶余油田に位置する生産井から採取した、原油とともに生産される産出流体の化学成分分析の結果に基づいて決定された、NaCl；1210 mg/l、KCl；23 mg/l、NaHCO₃；2820 mg/l、CaCl₂；140 mg/l、MgCl₂；253 mg/l、FeCl₃；2 mg/l、KH₂PO₄；10 mg/l、NaHSO₄；3 mg/lの組成からなるものである [米林英治, 田口充, 藤原和弘, 吉田信一郎, 榎本兵治, 微生物攻法に関する研究の現状とフィールドテストへの展望, 石油技術協会誌, Vol. 61 （6）, 487-493（1996）、Yonebayashi, H., Ono, K., Enomoto, H., Chida, T., Hong, C. X., Fujiwara, K., Microbial Enhanced Oil Recovery Field Pilot in a Waterflooded Reservoir, Society of Petroleum Engineers, 38070（1997）なども参照されたい]。
【００９７】
また、より油層環境に近い条件下でCJF-002株のバイオフィルムの形成能力を把握するため、およびCJF-002株を接種した前記液体培地の入った１Ｌビーカーを嫌気ジャー（Becton Dickinson and Company,社製）内に静置して油層内に類似した嫌気性の環境を構築するとともに、１Lビーカー内に撹拌子を入れ、約1.7cm/secの回転流速で培地を撹拌することにより、油層内の流体の流れを加速試験的に再現した。さらに培養温度は前記扶余油田の油層温度である30℃とし、36時間培養を行った。
【００９８】
培養終了後、培養液中のCJF-002株の生菌数をNutrient broth(Difco社製)寒天培地を用いたＣＦＵ法により測定し、油層岩表面へのバイオフィルムの形成状況を目視観察により評価した。
【００９９】
図３のＢは、油層岩表面にバイオフィルムが形成されている状態を示す図面であり、この図からわかるようにバイオフィルムは油層岩全体を覆い尽くしていた。また培養液中のCJF-002株の濃度は2.2×10⁸ CFU/mlであり、初期濃度よりも2オーダー以上高濃度であったことから、油層岩表面に形成されたバイオフィルムはCJF-002株が増殖することにより生産したバクテリアセルロースに由来するものと推定された。これらの結果から、CJF-002株は油層環境条件下で増殖し、強固なバイオフィルムを油層岩表面に形成することが判明した。また、バイオフィルムの形成に要する時間も短時間であることから、CJF-002株は油層内で早期に高浸透性領域を閉塞しうるものと考えられた。このことからCJF-002株は、微生物攻法の短時間処理化乃至は効率化に多いに寄与するものであり、微生物攻法の原料微生物として使用することにより圧入操業コストを低減し得ることを示唆するものである。
【０１００】
実施例８：原油増進回収能力
実施例１で単離したCJF-002株に関して、原油を飽和させた油層岩を用いて原油増進回収能力を評価した。まず、中国石油天然気株式有限公司吉林石油分公司の扶余油田から油層コアを採取し、深度359.2mのコア（直径約11cm、長さ約17cm）からプラグコア（直径約3.7cm、長さ約11cm、孔隙容積[PV]；23.7ml、孔隙率[φ]；29.4％、浸透率[Kair]；1051md）を準備した。次いでプラグコアを180℃で3日間乾熱滅菌し、実施例７に記載の合成油層水に浸漬して、真空ポンプを用いて一昼夜脱気することにより、プラグコア内の孔隙部分を合成油層水で飽和した。続いて、当該プラグコアをコアホルダーに装着し、121℃で240分滅菌した後、水浸透率を測定し、約5.3PVの扶余油田産の原油を60 ml/minの流速でプラグコア内に送液して、プラグコアの孔隙部分を原油で飽和させた。そして、前記合成油層水を40 ml/minの流速で再度送液して、水攻法により回収されうるプラグコア内の原油を全て回収することにより、水攻末期の油層を再現した（水飽和率[％PV]；56.9％、油飽和率[％PV]；31.6％、浸透率[Kwater]；208.0md）。
【０１０１】
次に、これに引き続いて、CJF-002株の圧入を行った。まず、CJF-002株を高圧蒸気滅菌した0.1％(v/v)の中国産ビートモラセス、合成油層水成分および0.005％のセルラーゼ（糖成分不含）をそれぞれ混和した液体培地を使用して、30℃で24 時間培養した。次いで、当該培養液を合成油層水に1：100の割合で混和し、プラグコア内に10ml/minの流速で1日間圧入した。このときのCJF-002株の圧入濃度はおよそ10⁶ CFU/mlであった。なお、CJF-002株の圧入にあたっては、CJF-002株の圧入溶液を適当容積のボトルに入れておき、これを送液ポンプで順次プラグコア内に送液するという手順で行ったが、その際、CJF-002株がプラグコア内に圧入される前にボトル内あるいは送液ライン内でバイオフィルムを生産するのを抑止するため、ボトルおよび送液ラインの温度を15℃に保持した。そして、前記手順で調製した新鮮なCJF-002株の培養液に、高圧蒸気滅菌した0.1％(v/v)の中国産ビートモラセス、合成油層水成分および0.01％のセルラーゼ（糖成分不含）をそれぞれ混和したものを栄養源溶液として、前記CJF-002株の圧入と同条件でプラグコア内に圧入し、さらに同条件で合成油層水を圧入した。
【０１０２】
本実験では、実験期間を通じて回収された溶出液について、総菌体濃度をNutrient broth(Difco社製)寒天培地を用いたＣＦＵ法により測定し、さらにCJF-002株の生存濃度を本発明者らが以前に確立した遺伝子工学的手法を用いてモニタリングした [Fujiwara, K., Tanaka, S., Ohtsuka, M., Ichimura, N., Yonebayashi, H., Hong, C. X., Enomoto, H., Evaluation of the Use of Amplified 16S rRNA Gene-Restriction Fragment Length Polymorphism Analysis to Detect Enterobacter cloacae and Bacillus licheniformis for Microbial Enhanced Oil Recovery Field Pilot, Sekiyu Gakkaishi, 42 (5), 342-351 (1999)も参照されたい]。また実験期間を通じて、回収された原油量およびプラグコア内の圧力変化（各種溶液の圧入圧力と溶出圧力との差圧）を評価するとともに、実験終了後のプラグコア内に生存するCJF-002株の濃度をCFU法および上記遺伝子工学的手法を用いて測定し、またバイオフィルム形成の有無を実施例５に記載の方法により評価した。
【０１０３】
結果を図４に示す。図４に示すように、栄養源溶液の圧入直後から、溶出液中のCJF-002株の濃度、並びに圧入圧力と溶出圧力との差圧が上昇した。これは、栄養源溶液の圧入によりCJF-002株がプラグコア内で増殖し、それに伴ってバイオフィルムが形成されたことを示唆する結果と考えられる。また、前記差圧の上昇に少し遅れて原油回収率が上昇し、最高で25％もの原油回収率が観測されたが、この結果は、プラグコアのなかでも浸透性の高い領域がバイオフィルムにより閉塞され圧入流体の流路が変化して、圧入流体の水/油の置換効率が改善されたことによると考えられる。さらに、圧入実験終了後、プラグコア内に生存するCJF-002株の濃度を測定したところ、プラグコア１ｇ（乾燥重量）あたり10⁷ CFUのCJF-002株がプラグコア内にほぼ均一に分布していることが判明し、またバイオフィルムの形成も確認された。
【０１０４】
以上のことから、CJF-002株によれば、嫌気性環境である油層環境下で増殖し、バイオフィルムを形成することができ、よって形成されたバイオフィルムが油層の高浸透性領域を閉塞して、優れた原油増進回収効果をもたらしうることが示唆された。
【０１０５】
実施例９：実油層における原油増進回収効果
実施例１で単離したCJF-002株に関して、実油層における原油増進回収効果を評価した。実験は中国石油天然気株式有限公司吉林石油分公司の扶余油田に位置する生産井#22-264（実油層）に対して行い、圧入方法として「圧入→密閉（生産中止）→生産再開」のプロセスを一巡させる、１サイクルハフ＆パフ攻法を採用した。以下にCJF-002株の圧入実験の手順を示す。
【０１０６】
まず、高圧蒸気滅菌した液体培地（１Ｌ）（１％中国産ビートモラセスおよび0.01％のセルラーゼ含有）を用いてCJF-002株を30℃で一夜静置培養し、得られた前培養液を前記と同様な培地１０Ｌ（８本）に接種して、さらに30℃で一夜静置培養することにより、圧入に使用するCJF-002株の培養液を得た。
【０１０７】
次に、４台の15klタンクローリー（積載容量約12kL）を用意し、これらのタンク内部を蒸気滅菌処理するとともに、１台目のタンクローリーに地上施設の地下水貯蔵タンクから地下水をタンクローリーに積み込み、そこに前記CJF-002株の１０Ｌ培養液８本（平均9.1×10⁸ CFU/ml ＝ 7.3×10¹⁰ CFU/80L）を混合して坑井元まで運搬した。坑井元までの道路は悪路で、運搬には約30分を要し、この間にタンク内の圧入溶液を撹拌していたことになる。坑井元では、タンクローリーと同様な手順で加熱滅菌処理したポンプ車によって、坑井のアニュラス部から圧入を行った。続いて２台目はスペーサーとして地下水のみを、３台目以降は、地下水で5％程度を目処として希釈した中国産ビートモラセス溶液を前記と同様な手順で順次圧入した（圧入総量：130kl）。これらの圧入における圧入レートは平均するとおよそ0.5kl/min（30 kl/h）であった。そして所定量の前記圧入溶液を圧入後、アニュラス部に残留している当該溶液を全て油層内に圧入するため、タンクローリー半載分に相当する約６klの地下水を後押し水として圧入した。
【０１０８】
圧入終了後、油層内に圧入したCJF-002株がモラセスを利用して油層内で増殖し、バイオフィルムを形成するのに要する期間を考慮して、坑井を10日間密閉（生産中止）した。
【０１０９】
坑井を所定期間密閉した後、生産を再開し、生産井から産出される流体の産液量および含水率（Water cut）の変化を測定することにより、石油増進回収効果を評価した。さらに、生産再開後、チュービング内に停滞している油層水を排出した後に、坑口装置のドレイン部から滅菌ボトル内に前記産出流体を経時的に採取し、実施例８に記載の手法を用いて前記産出流体中に生存するCJF-002株の濃度をモニタリングした。
【０１１０】
図５に、当該圧入実験における前記産出流体および原油の生産挙動を示した。CJF-002株の圧入により、前記産出流体の産液量がおよそ35％低下し、さらに、4-5倍もの原油回収量の増加が認められた。また、経時的に採取した前記産出流体中には、10⁴-10⁵ CFU/ml程度のCJF-002株が高頻度に検出された。これらの結果から、生産井#22-264の近傍油層に存在する高浸透性領域が、CJF-002株の生産するバイオフィルムにより閉塞されたことによって、産出流体の産液量の低下がもたらされ、さらにそれと同時に、油層内の流体の流路が変化たことにより、未回収の原油が掃攻されて、原油回収量が増加したものと考えられる。
【０１１１】
以上の実施例８及び９におけるCJF-002株を用いた原油掃攻撃実験及びハフ＆パフテストの結果から、CJF-002株は石油増進回収効果をもたらすことが確認でき、当該菌が微生物攻法において極めて有効に利用できる菌であることが判明した。
【０１１２】
【発明の効果】
このように、本発明によると、初期の目的であった、バクテリアセルロースを産生する新規微生物、その微生物が生産するバクテリアセルロース、バクテリアセルロースの製造方法、およびその微生物ならびにバクテリアセルロースの石油の増進回収法への利用方法が提供される。
【０１１３】
本発明の微生物は、簡易な装置で安価な培地を用いて大量培養が可能であり、かつ嫌気性条件下で活発に増殖してバイオフィルムなどの不溶性ポリマーを生産しうる微生物であることから、微生物攻法による石油回収を低コストで且つ効率よく実施するための微生物として有用である。
【０１１４】
本発明によれば、地球環境問題に対して環境適合性材料の開発が急がれている昨今、天然材料として注目されているバクテリアセルロースが安価に供給できるものと期待される。また、本発明において、微生物攻法における微生物の効果的な利用手段の一つである、微生物産生不溶性ポリマーによる高浸透性領域の選択的なプラッギングに対して、本発明の微生物は効果的に利用できることが判明し、多様な油田における石油の増進回収も大いに期待される。
【０１１５】
【配列表】

【図面の簡単な説明】
【図１】本発明のバクテリアセルロース産生微生物（CJF-002株）をビートモラセス培地で培養した場合の、菌の増殖性（図Ａ）及びセルロースの生産性（図Ｂ）を示す図である（実施例５）。
【図２】本発明のバクテリアセルロース産生微生物（CJF-002株）の増殖性及びバクテリアセルロースの生産性に対するビートモラセス濃度の影響を示す図である（実施例６）。
【図３】油層岩表面にバイオフィルムが形成されている状況を示す図である（実施例７）。尚、図Ａは実験に使用する前の油層岩小片の様子を示す図であり、図Ｂは油層岩小片にCJF-002株を接種して培養した後の油層岩小片の様子を示す図である（実施例７）。
【図４】実施例８において、原油を飽和させた油層岩を用いて原油の増進回収能力を調べた結果を示す図である。
【図５】実施例９において行ったハフ＆パフテストの結果を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel microorganism producing bacterial cellulose, a method for producing bacterial cellulose using the microorganism, and an enhanced oil recovery method using the microorganism or bacterial cellulose produced by the microorganism.
[0002]
[Prior art]
As a bacterium producing cellulose, a certain kind of microorganism has been conventionally known. Examples of the microorganism include Acetobacter genus, Alkaligenes genus, Pseudomonas genus, Agrobacterium genus, Rhizobium genus, Sphaerotilus genus, Sarsina genus, Acrocinia, There are various bacteria belonging to the genus Mobacter, Aerobacter, Azotobacter and Zogrea. Among them, Acetobacter xylinum is particularly prominent, and since microorganisms belonging to the genus Acetobacter have been known to have high cellulose-producing ability, Used in industrial production [Biochem. J., 158, 345 (1954)].
[0003]
Bacterial cellulose is characterized in that the fragment width of fibrils is about 2 digits smaller than cellulose produced from wood pulp or the like. Therefore, the material obtained by solidifying the disaggregated product of bacterial cellulose into a paper or solid form exhibits a high tensile elastic modulus, and therefore excellent mechanical properties based on the structural characteristics of fibrils are expected. Applied. For example, it is known that sheet-like bacterial cellulose produced by Acetobacter xylinum ATCC 23769 can be used for a medical pad (Japanese Patent Laid-Open No. 59-120159).
[0004]
Therefore, conventionally, for the purpose of improving the productivity of bacterial cellulose, attempts have been made to optimize culture conditions and search for a factor for promoting cellulose production. For example, JP-A-62-265990, JP-A-63-202394, and JP-B-6-43443 describe a method for producing bacterial cellulose. Suitable nutrient media for culturing bacterial cellulose producing bacteria include Schramm / Hestrin media consisting of carbon source, peptone, yeast extract, sodium phosphate and citric acid (Schramm et al., J. General Biology, ll, pp. 123-129, l954) is known. In addition, inositol, phytic acid and pyrroloquinoline quinone (PQQ) (Japanese Patent Publication No. 5-1718), which are cellulose production promoting factors by specific nutrients in the medium, are added to such a nutrient medium. By adding carboxylic acid or a salt thereof (Japanese Patent Application No. 5-191467), invertase (Japanese Patent Application No. 5-331491) and methionine (Japanese Patent Application No. 5-335664), Increased productivity has been found. Further, an example in which the yield is increased by adding a small amount of a cellulase preparation while retaining the cellulase activity (Japanese Patent Laid-Open No. 63-74490), or a method for containing a deactivated cellulase preparation in the production medium Have also been reported (Japanese Patent Laid-Open No. 2-238888).
[0005]
Microorganisms belonging to the genus Acetobacter are aerobic bacteria. For this reason, it is better to supply oxygen sufficiently for the growth of the bacteria, and since it is several times faster than stationary culture, aeration and agitation culture is generally performed in acetic acid fermentation and the like. However, it is known that stationary culture is more efficient for producing bacterial cellulose. With respect to a method for producing bacterial cellulose using such properties of bacteria, for example, JP-A-1-243997 discloses a method for producing bacterial cellulose using microorganisms capable of producing cellulosic substances. A method is described in which bubble culture is performed while ventilating an oxygen-containing gas, and then this is dispensed into a shelf culture vessel to expand the culture area and perform stationary culture.
[0006]
Thus, efficient production of bacterial cellulose using microorganisms, especially aerobic microorganisms such as Acetobacteria, requires a two-step process under different conditions such as cultivation (growth) of microorganisms and production of bacterial cellulose. As a result, the production process becomes complicated as a result, and the actual situation is that the production cost has not been greatly reduced as expected. Despite the various possibilities described above for bacterial cellulose, the background of the lack of progress in application development to date is the production cost that has not been found to meet such production costs. If this problem can be solved, it is expected that a wider variety of applications can be developed.
[0007]
On the other hand, enhanced oil recovery technology using microorganisms (hereinafter referred to as “microorganism attack”) uses self-injection oil (primary recovery) using self-injection energy accumulated in the oil reservoir and water injection into the oil reservoir, After water flooding (secondary recovery) that restores the production capacity that has been attenuated by pushing oil into production wells, the third collection technology forcibly recovers more than 65-70% of the oil remaining in the oil reservoir. One.
[0008]
Microbial attack is a method that uses microorganisms that live in the general natural environment or oil reservoir environment, so it is economical, and depending on the use of microorganisms, it is possible to improve all the causes of sluggish recovery of crude oil. This is an oil extraction technology that has been attracting attention in recent years because of its characteristics.
[0009]
One of the effective ways to use microorganisms in microbial attack is to grow microorganisms in situ in an oil reservoir, and to produce microorganisms and other insoluble polymers such as biofilms. There is an attack method that selectively plugs a flow path that is preferentially swept with water to improve the sweep efficiency of the water flood method and to expect an oil recovery effect.
[0010]
In particular, this flooding causes a channeling phenomenon (a phenomenon in which the injected water passes only through a specific high permeability area in the oil reservoir) during the oil recovery process by the water flooding method, which significantly reduces the efficiency of oil scavenging. It is considered effective against existing oil reservoirs.
[0011]
As a major research result so far, Jack et al. Have conducted detailed plugging experiments using Leukonostoc mesenteroides that produce insoluble dextran using an oil reservoir model, and observed clear plugging on porous media. [Jack, TR and Diblasio, E., Thompson, BG, and Ward, V., 1983: Bacterial Systems for Selective plugging in Secondary Oil Production, Preprints, Symposia, Division of Petroleum Chemistry, American Chemical Society, 27, 773- 784; Jack, TR and Diblasio, E., 1985: Selective Plugging For Heavy Oil Recovery, In "Microbial Enhanced Oil Recovery", Vol. 1, Int'l. Bioresources Journal, ed. By Zajic JE and Donaldson EC, Bioresource Publications , E1 Paso, TX, 213-225; Jack, TR and Diblasio, E., 1985: Microbes and Oil Recovery, Proceedings of the International Conference on Microbial Enhancement of Oil Recovery, Fountainhead, Oklahoma, May 20-25, 1984. ed. by Zajic JE a nd Donaldson EC, Petroleum Bioresource, El Paso, pp. 205-212; Jack, TR and Stehmeier, L., 1988: Selective plugging in Watered Out Wells, In the proceedings of the Symposium on Application if Microorganisms to Petroleum Technology, Bartlesville, Oklahoma, August 12-13, 1987, ed. By Linville, W., US Department of Energy, Bartlesville; Jack, TR, Shaw, JC, Wardlaw, NC, and Costerton, JW, 1989; Microbial Plugging in Enhanced Oil Recovery, See also the literature in Chapter 7 in Microbial Enhancement of Oil Recovery, ed. By Donaldson EC, Chilingarian, GV, and Yen, TF, Elsevier, Amsterdam, pp. 125-149].
[0012]
However, in general, the optimal growth temperature range of Leuconostoc mesenteroides is relatively low, 20-30 ° C, and the dextran production temperature range is 10-25 ° C, and the optimal production temperature range is as low as 10-20 ° C. Therefore, effective plugging in the highly permeable region cannot be expected for an oil layer having an oil layer temperature of 30 ° C. or higher.
[0013]
In addition, Leuconostoc mesenteroides are heterozygous lactic acid bacteria (CO₂And microorganisms that produce ethanol and acetic acid), and they are subject to significant growth inhibition by organic acids such as lactic acid and acetic acid produced by them. A device is required.
[0014]
Furthermore, since this strain requires amino acids such as glutamic acid and valine for growth, the medium used for mass culture also has a problem of becoming expensive.
[0015]
From the above, the microorganisms that can be used in the microbial attack are microorganisms that can reduce the operating cost as much as possible, that is, they can be cultured in large quantities using an inexpensive medium in a simple ground facility, and the oil reservoir environment Microorganisms that can proliferate and produce insoluble polymers such as biofilms are preferred. However, no microorganism satisfying these conditions has been discovered so far.
[0016]
[Problems to be solved by the invention]
The present invention has been made in view of the above-described conventional problems, and an object thereof is to provide a novel bacterial cellulose-producing microorganism that can be effectively used in the above-described microbial attack for oil extraction at a lower cost. To do. It is another object of the present invention to provide a method for producing bacterial cellulose using the microorganism, and an enhanced oil recovery method using the microorganism and bacterial cellulose produced by the microorganism.
[0017]
[Means for Solving the Problems]
The inventors of the present invention have been intensively researched day and night in order to solve the above problems, and found a facultative anaerobic bacterium that can produce bacterial cellulose that is insoluble in water. We found that it was possible to culture in large quantities by using inexpensive molasses, etc., and from these properties, it was confirmed that the bacteria were extremely useful microorganisms suitable for microbial attack of oil extraction. The present invention has been developed based on such knowledge.
[0018]
  That is, the present invention firstly(1) InThe following are novel bacterial cellulose-producing microorganisms:
(1) Enterobacter sp. CJF-002 ( FERM BP-8227 ) A bacterial cellulose-producing microorganism that is a strain, or a progeny or variant thereof having the bacteriological properties and bacterial cellulose-producing ability of the strain.
[0019]
  In addition, the present invention secondly,(2) ~ (Four)A method for producing bacterial cellulose using the above-mentioned microorganisms:
(2) the above (1) Described inA method for producing bacterial cellulose, comprising culturing a bacterial cellulose-producing microorganism and collecting and recovering bacterial cellulose from the culture.
(3) the above (1) Described inBacterial cellulose-producing microorganisms are cultured in a liquid medium containing at least one monosaccharide or disaccharide(2)A method for producing the bacterial cellulose as described.
(Four) It is characterized by culturing by static culture method(2) Or (3)A method for producing bacterial cellulose as described in 1. above.
[0020]
  Thirdly, the present invention provides a use of the microorganisms in the enhanced recovery of petroleum. That is, the third aspect of the present invention is the following:(Five) ~ (6)The following is an enhanced oil recovery method using the microorganisms and bacterial cellulose produced by the microorganisms:
(Five) the above (1) Described inA method for enhanced recovery of petroleum, comprising using bacterial cellulose-producing microorganisms or bacterial cellulose produced by the microorganisms.
(6) the above (1) Described inBacterial cellulose-producing microorganisms are grown in an oil reservoir to produce bacterial cellulose(Five)The enhanced oil recovery method described.
[0021]
The method for producing bacterial cellulose using the novel microorganism of the present invention eliminates the disadvantages of the conventional method for producing bacterial cellulose using microorganisms, and is simple and inexpensive without complicated operations. This is a useful method in that cellulose can be produced. Further, the enhanced oil recovery method of the present invention utilizes the microorganism of the present invention, which can be cultured in a large amount on an inexpensive medium and actively proliferates under anaerobic conditions to produce insoluble polymers such as biofilms. Based on the nature of the microorganism, the microorganism is preliminarily cultured in an inexpensive medium at an above-ground facility, and then introduced into an anaerobic oil reservoir environment to grow in the oil reservoir environment, thereby efficiently insoluble polymer in the oil reservoir. This is a useful method that can be expected to have an excellent oil recovery effect by selectively plucking the water sweeping channel at a low cost.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
(1) Bacterial cellulose producing microorganism
The bacterial cellulose-producing microorganism of the present invention is a bacterial cellulose-producing bacterium belonging to the genus Enterobacter.
[0023]
The bacterial cellulose-producing microorganism targeted by the present invention is a bacterium belonging to the genus Enterobacter, and further containing the base sequence described in SEQ ID NO: 1 in the sequence listing described later in the base sequence of the 16S-rRNA gene Is included. The base sequence does not prevent the deletion, substitution or addition of one or several bases, as long as the properties of the bacterial cellulose-producing microorganism of the present invention described later are not impaired.
[0024]
The base sequence can be located at any position of the 16S-rRNA gene of Enterobacter bacteria, but is preferably located at positions 435 to 469 from the 5 'end of the 16S-rRNA gene. More preferably, the bacterial cellulose-producing microorganism of the present invention is a bacterium belonging to the genus Enterobacter, wherein the 16S-rRNA gene has the base sequence set forth in SEQ ID NO: 2. Similarly to the above, the base sequence does not prevent the deletion, substitution or addition of one or several bases as long as the properties of the bacterial cellulose-producing microorganism of the present invention are not impaired.
[0025]
Moreover, the bacterial cellulose-producing microorganisms targeted by the present invention include bacteria belonging to the genus Enterobacter and having the following mycological characteristics.
A. Morphological properties (agar medium)
(1) Shape and size of cells: rod-shaped, 0.5-1.0μm × 1.5-3.0μm
(2) Existence of mobility: Existence
B. Culture properties: Growth under various culture conditions
(1) Meat broth liquid culture: (+)
(2) Growth under aerobic conditions (+)
(3) Growth under anaerobic conditions (+).
[0026]
C. Physiological properties:
(1) Gram staining: Negative
(2) O-F test [Hugh leifson method]: (F)
(3) Gas production from glucose: (+)
(4) Indole production: (-)
(5) Production of hydrogen sulfide: (-)
(6) Utilization of organic acids: Citric acid Utilization (+)
(7) Pigment production: (-)
(8) Urease: (-)
(9) Oxidase: (-) [cytochrome c oxidase]
(10) Catalase: (+)
(11) Arginine dihydrolase: (+)
(12) Lysine dicarboxylase: (-)
(13) Ornithine decarboxylase: (+)
(14) Phenylalanine diaminase: (-)
(15) Degradability of gelatin: (-)
(16) Malonic acid degradability: (-)
(17) ONPG: (+)
(18) Growth range: Temperature; 4-45 ° C / pH; 2.2-9.5
(19) Fermentability using L-arabinose: (+)
(20) Fermentability using inositol: (-)
(21) Fermentability using D-sorbitol: (-)
(22) Acid production from D-rhamnose: (+)
(23) Acid production from D-mannitol: (+)
(24) Production of acid from D-adonitol: (-)
(25) Production of acid from D-raffinose: (-)
(26) Acid production from D-sucrose: (+)
D. Other characteristic properties: Degradation of esculin (+).
[0027]
The bacteriological properties of the above bacterial cellulose-producing microorganisms are highly homologous to those of Enterobacter cloacae. Therefore, the microorganisms targeted by the present invention include Enterobacter cloacae having the ability to produce bacterial cellulose.
[0028]
As a specific example of a bacterial cellulose-producing microorganism targeted by the present invention, Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry, which has an address on 1-3-3 Higashi 1-chome, Tsukuba, Ibaraki, Japan, on March 29, 2000 Enterobacter microorganisms deposited at the National Institute of Advanced Industrial Science and Technology Patent Microorganism Depositary as “Microbes” (indication for identification given by the depositor) “Enterobacter sp. CJF-002” and deposit number “FERM P-17799” (Hereinafter, also simply referred to as “CJF-002”).
[0029]
The bacterial cellulose-producing microorganism of the present invention is subjected to mutation treatment by a known method such as chemical treatment using a mutation agent such as NTG (nitrosoguanidine) or various physical treatments such as irradiation. Various mutant strains created by are included. Chemical mutagenesis methods using mutagens are described, for example, in Bio Factors, Vol. 1, p.297-302 (1988) and J. Gen. Microbiol, Vol.135, p.2917-2929 (1989). Can be mentioned. A person skilled in the art can easily obtain a mutant strain of the bacterial cellulose-producing microorganism of the present invention, particularly a mutant strain of CJF-002, based on these known methods.
[0030]
In addition, the bacterial cellulose-producing microorganism of the present invention includes a progeny (for example, subculture of CJF-002 strain) having substantially the above-mentioned mycological properties and bacterial cellulose-producing ability as long as the effects of the present invention are not hindered. Microorganisms) and mutants.
[0031]
Bacterial cellulose to which the present invention is directed includes bacterial cellulose that is conventionally known or can be found in the future, regardless of whether it is water-soluble or water-insoluble, and includes, for example, cellulose and a heteropolysaccharide having cellulose as the main chain, Those containing glucans having α- or β-bonds such as 1,2 bonds, 1,3 bonds and 1,6 bonds are included. In the case of heteropolysaccharides, components other than cellulose are not particularly limited, but usually include hexoses such as mannose, fructose, galactose, and rhamnose, pentoses such as xylose and arabinose, and organic acids such as uronic acid. . These polysaccharides may be a single substance, or two or more kinds of polysaccharides may be mixed by hydrogen bonding or the like.
[0032]
These bacterial celluloses are produced by culturing the aforementioned bacterial cellulose-producing microorganism of the present invention in a medium.
[0033]
(2) Bacterial cellulose production method
Accordingly, the present invention provides a method for producing bacterial cellulose using the aforementioned bacterial cellulose-producing microorganism. The present invention can be carried out by culturing the above microorganism in an appropriate medium and collecting and recovering bacterial cellulose from the resulting culture.
[0034]
The medium used in the method of the present invention may be any medium as long as the microorganism of the present invention has a medium composition capable of producing bacterial cellulose, and any of various synthetic media and natural media can be used. A liquid medium containing saccharides such as monosaccharides and disaccharides is preferred. Examples of such saccharides include monosaccharides such as glucose, fructose and galactose; disaccharides such as sucrose, maltose, sucrose and fructose; polysaccharides such as levan; sugar alcohols such as mannitol, sorbitol and erythritol. More preferably, it is a liquid medium containing any one sugar of monosaccharide or disaccharide. Moreover, it can replace with these saccharides and can also use the composition which has these saccharides, for example, starch hydrolyzate, citrus molasses, beet molasses, cane molasses, beet juice, sugarcane juice, citrus fruit juice components etc. can do.
[0035]
Specifically, glucose, fructose, galactose, sucrose, maltose, sucrose, fructose, mannitol, sorbitol, erythritol, glycerin, ethylene glycol, starch, molasses, corn steep liquor, malt extract, starch hydrolysis Citrus molasses, beet molasses, cane molasses, beet juice, sugarcane juice, citrus fruit juice components, organic acids, etc. alone or in combination; ammonium salts (ammonium sulfate, ammonium chloride, Inorganic nitrogen sources such as ammonium phosphate), nitrates (sodium nitrate, etc.) and organic nitrogen sources such as pharmamedia, peptone, soybean flour, meat extract, yeast extract, casein, urea, and bean concentrate. A mixture of more than seeds It can be mentioned. The organic nitrogen source is preferably an organic nitrogen-containing natural nutrient source such as Bact-Peptone, Bact-Soytone, Yeast-Extract, or Tono.
[0036]
If necessary, the medium may contain amino acids, vitamins, fatty acids, nucleic acids, etc. as organic micronutrients, and phosphates, iron salts, manganese salts, and other metal salts as inorganic salts, either alone or in combination. Can be used.
[0037]
In the present invention, a natural medium and an artificial medium can be used in combination, and the composition ratio of each component in the medium and the inoculation (amount, method) of the bacterial cells to the medium depend on the purpose of use of the microorganism, the culture conditions, and the like. Can be appropriately selected by those skilled in the art.
[0038]
Cultivation of the bacterial cellulose-producing microorganism of the present invention is started by inoculating the microorganism of the present invention into a medium which has been preliminarily steam sterilized or filtered sterilized or has not been treated.
[0039]
The bacterial cellulose-producing microorganism used in the present invention is a facultative anaerobic bacterium. For this reason, the method of the present invention is characterized in that it can significantly reduce the production cost of bacterial cellulose without affecting the cellulose productivity even when cultured under aerobic or anaerobic conditions.
[0040]
Therefore, the culture of the microorganism of the present invention and the production of bacterial cellulose by the culture are not limited by the culture format, and can be carried out widely using known methods used in principle for culturing microorganisms. For example, any culture method such as stationary culture, shaking culture or aeration and agitation culture can be used.Preferably static culture. Stirring culture is a culture method performed while stirring a culture solution. For example, for convenience, stirring tanks such as jar fermenters and tanks; baffled flasks, Sakaguchi flasks, and air lift type stirring tanks; It can be implemented by arbitrarily selecting and using means and devices such as pump-driven circulation of fermentation broth. These can be used alone or in combination of two or more. In addition, the stirring culture can be performed while aeration is performed simultaneously with stirring, as necessary. The aeration can be performed using any of oxygen-containing gas such as air and non-oxygen-containing gas such as argon and nitrogen, and these gases are appropriately selected by those skilled in the art according to the conditions of the culture system. You can choose. In addition, known methods such as batch fermentation, fed-batch fermentation, repeated batch fermentation, and continuous fermentation can also be used in the culture operation method. Further, these culture formats and culture operation methods are appropriately modified or changed. It is also possible to use a method to which is added.
[0041]
The pH of the medium during the cultivation is not particularly limited, and can be carried out in a wide range from an acidic region to an alkaline region, but is usually in the range of pH 2.2 to 9.5, preferably pH 5.5 to 9.3. The culture temperature is preferably a temperature at which the microorganism of the present invention grows well, preferably a temperature at which bacterial cellulose is efficiently produced, and is usually from 4 to 45 ° C, preferably from about 25 to 35 ° C.
[0042]
The various culture conditions described above can be appropriately changed according to the type and characteristics of microorganisms to be used, external conditions, and the like, and optimal conditions can be appropriately selected and prepared from the above ranges according to various situations.
[0043]
Bacterial cellulose produced and accumulated in the medium by culturing may be recovered as it is, or a treatment for removing substances other than bacterial cellulose such as microbial cells in the culture may be performed. The method for removing impurities is not particularly limited, but water washing treatment, pressure dehydration treatment, dilute acid washing treatment, alkali washing treatment, treatment with bleach such as sodium hypochlorite and hydrogen peroxide, lysozyme and other fungi Examples include treatment with a body-dissolving enzyme, treatment with a surfactant such as sodium lauryl sulfate and deoxycholic acid, and heat washing treatment in the range from room temperature to 200 ° C. These can be carried out alone or in combination. .
[0044]
Separation and collection of bacterial cellulose from the culture can be performed according to a general method for collecting and collecting a fermentation product. For example, means such as solvent extraction, gel filtration, distribution, and adsorption chromatography can be used alone or in combination of two or more in any order. For example, when bacterial cellulose is water-soluble, the cellulose is present in the culture solution. Therefore, the solid matter containing the bacterial cells is removed by a method such as filtration or centrifugation according to a conventional method to remove the culture filtrate. Acquire bacterial cellulose from the obtained filtrate according to a conventional method. If bacterial cellulose is water-insoluble, the cellulose is present in the insoluble components (solid content) of the culture, so obtain solids containing bacterial cells by methods such as filtration and centrifugation according to conventional methods. Then, bacterial cellulose is isolated from the obtained solid matter according to a conventional method using differences in specific gravity, molecular weight, solubility and the like. The isolated bacterial cellulose can be purified as necessary, and such purification can be performed according to a conventional method such as a solvent extraction method or column chromatography.
[0045]
The produced bacterial cellulose can be confirmed by analyzing the composition of the sugar component according to a conventional method or analyzing the binding mode of the sugar component, as will be described in detail in the examples described later.
[0046]
(3) Enhanced oil recovery method
The present invention also relates to a method for enhanced oil recovery utilizing the aforementioned bacterial cellulose-producing microorganism and the bacterial cellulose produced by the microorganism.
[0047]
Specifically, the enhanced oil recovery method of the present invention is characterized in that the microorganism of the present invention is press-fitted into an oil layer and grown in the oil layer to produce bacterial cellulose, which is produced in the oil layer. By blocking the highly permeable region with the bacterial cellulose, efficient plaking is possible, improving the water sweeping efficiency and increasing the rate of oil recovery.
[0048]
Therefore, the method used in the present invention may be any method as long as it includes a method of growing the microorganism of the present invention in an oil layer to produce bacterial cellulose in the oil layer.
[0049]
For example, as an enhanced oil recovery method of the present invention, specifically, a culture solution containing the microorganism of the present invention pre-cultured in advance on a ground facility constructed in an oil field, or a medium necessary or useful for the production of bacterial cellulose. Along with the ingredients, it is pressed into the oil reservoir from the production well, and then the operation of the production well is stopped for a certain period of time, in which the microorganisms grow and bacterial cellulose is produced to bring the bacterial cellulose into the highly permeable region in the oil reservoir. One example is a method of recovering oil by letting it out and plugging, and then restarting the production well. As another method, the culture solution of the microorganism of the present invention pre-cultured in advance on the ground facility is continuously injected into the oil reservoir from the injection well together with a medium component necessary or useful for the production of bacterial cellulose. , While the injected fluid passes through the high permeability area in the oil layer and reaches the production well, the microorganisms are grown in the high permeability area and bacterial cellulose is produced. And a method of recovering oil according to a conventional water flooding method.
[0050]
The bacterial cellulose-producing microorganism used in the enhanced petroleum recovery method of the present invention is preferably a microorganism that produces water-insoluble bacterial cellulose among the microorganisms belonging to the various Enterobactors described above. More preferred is CJF-002 strain.
[0051]
In this method, the microorganism of the present invention is used to ensure the amount of bacterial cells capable of efficiently producing bacterial cellulose, and to increase the growth of microorganisms in the production oil layer and the production efficiency of bacterial cellulose. Thus, it is desirable to pre-incubate in the ground facility in advance before entering the oil reservoir. The medium and culture conditions used for such pre-culture are not particularly limited as long as the medium components and culture conditions allow the microorganisms to grow, but in consideration of blockage troubles such as a liquid supply line from the ground facility into the oil reservoir, It is preferable to use a medium or a medium condition in which the production of bacterial cellulose is suppressed, or a medium to which an agent that suppresses the production of bulk bacterial cellulose is added, for example, causing only the growth of the microorganism of the present invention. Examples include a medium containing a carbon source and a medium having a low sugar concentration, a culture under a low temperature condition, or a medium to which an agent that suppresses the agglomeration of bacterial cellulose such as NaCl or cellulase is added.
[0052]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples.
Example 1: Isolation of insoluble polymer-producing microorganisms
Microorganisms used in the microbial attack are preferably those that can reliably grow in the anaerobic oil reservoir, and, in addition, for the purpose of selective plugging by microorganisms, biofilms are used in an oil reservoir environment. It is desirable to produce insoluble polymers such as
[0053]
Therefore, oil layer rocks were collected from the Yuyao Oil Field of Jilin Petroleum Company, Ltd. (76 km of Guo Luo Si Road, Songdo, Jilin Province, China) and used as a sample for bacterial separation. Specifically, the recovered oil layer rock hole core (diameter: about 11 cm, length: about 17 cm) was crushed with a scissors, and immediately after that, with a pick-type hammer that had been flame-sterilized in advance, A small piece was scraped off from the central part of the crushed core, and this was enclosed in a sterilized petri dish as a sample for bacterial separation.
[0054]
Subsequently, the target microorganism was isolated from the obtained sample for bacterial separation. Specifically, about 2 g of the above sample is pulverized using a sterilized mortar until the particle size becomes about 0.5 mm, and 5 ml of sterilized water is added to the obtained oil layer rock powder and suspended in sterilized water. Then, sonication (1 minute irradiation → 1 minute standing) was repeated 10 cycles to suspend the microorganisms. Furthermore, these microorganism suspensions were applied to an agar medium sterilized by autoclaving, and the culture was continued in an anaerobic environment at 30 ° C. until sufficient growth of the bacterial cells could be visually confirmed. The agar medium was prepared by adding agar at a rate of 15 g / l to an aqueous solution containing Chinese beet molasses (composition shown in Table 1) at a rate of 4% (v / v).
[0055]
[Table 1]

[0056]
The anaerobic environment for the culture was constructed according to the anaerobic culture system (Becton Dickinson and Company) and the attached protocol.
[0057]
According to the above separation procedure, about 47 strains of microorganisms were separated from the bacteria-separating sample, and it was confirmed that two of them produced remarkable viscous substances around the colonies. Next, these isolates were inoculated into a liquid medium prepared by autoclaving a 4% (v / v) Chinese beet molasses-containing aqueous solution, and the gas phase was replaced with nitrogen for 3 minutes to cultivate the culture system. After making the anaerobic environment, the culture was allowed to stand at 30 ° C. for 1 day, and the strain forming the insoluble polymer was isolated using the production state of the insoluble polymer in the culture medium as an index (visual observation). From the microscopic observation and the size of the cells, the bacteria were recognized as bacteria.
[0058]
This strain was named Enterobacter sp. CJF-002 on the basis of the mycological characteristics described below, and was located in 1-3-1 Higashi-machi, Tsukuba, Ibaraki Prefecture on March 29, 2000. Deposited with the Institute of Biotechnology, Ministry of International Trade and Industry (Accession Number FERM P-17799). In the following examples, the bacteria are simply referred to as “CJF-002 strain”.
[0059]
Example 2: Mycological properties (morphology, physiological properties)
In order to perform mycological positioning of the CJF-002 strain isolated in Example 1, the morphological properties, growth state in the medium and physiological properties of the CJF-002 strain were analyzed.
A. Morphological properties
The following morphological properties were observed for bacteria grown under culture conditions at 30 ° C. for 24 hours using an agar medium.
[0060]
(1) Cell shape: cocoon
(2) Cell size: width 0.5-1.0μm, length 1.5-3.0μm (observation with optical microscope)
(3) Existence of mobility: Existence
(4) Colony color: white
(5) Colony shape: cocoon-like distorted shape
B. Culture characteristics (growth state in medium)
(1) Meat broth liquid culture: (+)
(2) Growth under aerobic conditions: (+)
(3) Growth under anaerobic conditions: (+).
[0061]
C. Physiological properties
To determine the physiological properties of CJF-002 strain, ID test EB-20 (manufactured by Nissui Pharmaceutical), which is a kit for identifying glucose-fermenting Neisseria gonorrhoeae, was used. Specifically, one platinum loop CJF-002 strain was inoculated into a liquid medium sterilized by autoclaving Nutrient broth (manufactured by Difco), followed by standing culture at 30 ° C. overnight to obtain a preculture solution. This preculture was processed according to the protocol attached to the kit and various measurements on physiological properties were performed. The results are shown below.
[0062]
(1) Gram staining: Negative
(2) O-F test [Hugh leifson method]: (F)
(3) Gas production from glucose: (+)
(4) Indole production: (-)
(5) Production of hydrogen sulfide: (-)
(6) Utilization of organic acids: Citric acid Utilization (+)
(7) Pigment production: (-)
(8) Urease: (-)
(9) Oxidase: (-) [cytochrome c oxidase]
(10) Catalase: (+)
(11) Arginine dihydrolase: (+)
(12) Lysine dicarboxylase: (-)
(13) Ornithine decarboxylase: (+)
(14) Phenylalanine diaminase: (-)
(15) Gelatin degradability: (-)
(16) Malonic acid degradability: (-)
(17) ONPG: (+)
(18) Growth range: Temperature; 4-45 ° C / pH; 2.2-9.53
(19) Fermentability using L-arabinose: (+)
(20) Fermentability using inositol: (-)
(21) Fermentability using D-sorbitol: (-)
(22) Acid production from D-rhamnose: (+)
(23) Acid production from D-mannitol: (+)
(24) Acid production from D-adonitol: (-)
(25) Production of acid from D-raffinose: (-)
(26) Acid production from D-sucrose: (+)
D. Other characteristic properties: Degradation of esculin (+).
[0063]
The physiological properties of CJF-002 strain obtained above are described in Bergey's Manual of Systematic Bacteriology [Vol.1 (1984), Vol.2 (1986), Vol.3 (1989), Vol.4 (1989), Williams & In contrast to the properties of various microorganisms described in Wilkins, Baltimore], the CJF-002 strain of the present invention most closely matches the physiological properties of bacteria belonging to the genus Enterobacter reported so far. It was.
[0064]
Example 3: Mycological properties (chemical properties)
In order to characterize the mycological positioning of the CJF-002 strain obtained in Example 1 from the viewpoint of genetic engineering, the 16S-rRNA gene of the CJF-002 strain was cloned and its base sequence was determined (analyzed). .
(1) Cloning of genes held by CJF-002 strain
For template DNA, CJF- prepared using the InstaGene Matrix method [Lamballerie et al., A one-step microbial DNA extraction method using "Chelex 100" suitable for gene amplification. Res Microbiol 143: 785-790 (1992)] Strain 002 chromosomal DNA was used. Then, 5′-AGAGTTTGATCCTGGCTCAG-3 ′ (SEQ ID NO: 3) is used as the upstream primer, and 5′-AAAGGGGTGATCCAGCC-3 ′ (SEQ ID NO: 4) is used as the downstream primer, and Taq DNA polymerase (Takara Shuzo) is used. PCR amplification). The denaturation reaction, annealing reaction, and extension reaction in PCR were each performed at 94 ° C. for 1 minute, 52 ° C. for 2 minutes, and 72 ° C. for 2 minutes, and this reaction cycle was repeated 30 times. The PCR amplified fragment was cloned into a plasmid vector using pMOS Blue T-vector Kit (Amersham International plc). The recombinant plasmid was prepared in large quantities using QIAquick Gel Extraction Kit (manufactured by QIAGEN). E. coli JM109 [recA1, endA1, gryA96, thi, hsdR17, supE44, relA1, Δ (lac-proAB), F ′ (traD36, proAB +, lac1y, lacZΔM15)] was used as a host fungus. The transformation of the host E. coli was performed according to the method of Sambrook et al. [Molecular Cloning, A Laboratory Manual 2nd ed. (1989)]. The medium was L medium (tryptone 10 g / l, yeast extract 5 g / l, NaCl 5 g / l, glucose 1 g / l, pH 7.2), and 100 μg / ml ampicillin was used as an antibiotic.
(2) Analysis of genes held by CJF-002 strain
Analyze the base sequence of the gene possessed by the CJF-002 strain clone using an automated analyzer ABI DNA Sequencer (Perkin-Elmer Co.) and ABI PRISM Dye Cycle Sequencing Kit (Perkin-Elmer Co.) The base sequence of the 16S-rRNA gene, one of the important indicators of microbial classification, was decoded. The gene sequence is shown in SEQ ID NO: 2. Based on this sequence, the homology of both was observed in comparison with the sequence of 16S-rRNA gene possessed by other microorganisms registered in gene databases such as Gene Bank. The results are shown in Table 2. For the homology analysis, BLAST search program [Altschul et al., Basic local alignment search tool. J. Mol. Biol., 215, 403-410 (1990)] was used.
[0065]
[Table 2]

[0066]
As can be seen from this table, the base sequence of the 16S-rRNA gene of the CJF-002 strain coincided with the base sequence of the 16S-rRNA gene of Enterobacter bacteria at a high rate of 98% or more. In particular, it matched the nucleotide sequence of 16S-rRNA gene of Enterobacter cloacae at a very high rate.
[0067]
From the mycological properties (physiological properties such as physiological properties) shown in Examples 2 and 3 above, the CJF-002 strain of the present invention was judged to be a bacterium closest to the genus Enterobacter.
[0068]
Example 4: Evaluation of microbial-produced bacterial cellulose
For the CJF-002 strain isolated in Example 1, the insoluble polymer material produced by the microorganism was evaluated according to the following procedure.
[0069]
First, Polysaccharide-production medium with Chinese beet molasses liquid medium diluted to 4% (v / v) with water and 2.0% sucrose [Akihiko Shimada, Viva Origino, 23, 1, 52-53, 1995 ] (Hereinafter referred to as “PPM medium”) as the basic medium, 30 ml of medium sterilized by high-pressure steam sterilization was dispensed into 50 ml vials, and CJF-002 strain was inoculated. Static culture was performed at 30 ° C. for 1 day. The CJF-002 strain used was a culture (preculture) that was allowed to stand overnight at 30 ° C. in a liquid culture medium previously sterilized under high pressure steam at Nutrient broth (manufactured by Difco).
[0070]
Subsequently, the insoluble polymers produced by the CJF-002 strain in the above two basic media were analyzed for (1) the composition of the sugar component and (2) the binding mode of the sugar component, respectively. These analyzes were performed using an insoluble polymer freeze-dried product obtained by washing the insoluble polymer obtained by the above-mentioned culturing several times with 500 ml of sterilized distilled water and then heating and washing.
[0071]
(1) Composition of sugar component,
In the composition analysis of the sugar component, the freeze-dried product of the insoluble polymer was hydrolyzed with a commercially available cellulase that had been confirmed in advance to contain no sugar component, and these hydrolysates were then neutralized under the conditions shown below. Carried out by analyzing sugar and uronic acid. The insoluble polymer is hardly hydrolyzed by organic acids or inorganic acids such as trifluoroacetic acid (TFA), hydrochloric acid and sulfuric acid, and the hydrolysis rates are about 12-13%, 5-9% and 20-33%, respectively. Met. On the other hand, the degradation rate of 65-75% was obtained in the enzymatic degradation treatment with cellulase or the like. The degradability of the polymer was not affected by the production conditions, that is, the difference in the medium used for production.
[0072]
(i) Neutral sugar analysis conditions
HPLC system: LC-9A (manufactured by Shimadzu Corporation)
Column: TSK-gel Sugar AXG (φ4.6 mm x 150 mm, manufactured by Tosoh Corporation)
Eluent used: 0.5 mM potassium borate buffer (eluent A)
Flow rate: 0.4 ml / min
Post-column labeling: Using 1% arginine and 3% boric acid, the flow rate was 0.5 ml / min, and the reaction temperature was 150 ° C.
[0073]
(ii) Uronic acid analysis conditions
HPLC system: LC-9A (manufactured by Shimadzu Corporation)
Column: Shimpal ISA-07 (φ4.6 mm x 250 mm, manufactured by Shimadzu Corporation)
Eluent used: 0.5 mM potassium borate buffer (eluent A)
Flow rate: 0.8 ml / min
Post-column labeling: 1% arginine and 3% boric acid were used, the flow rate was 0.8 ml / min, and the reaction temperature was 150 ° C.
[0074]
Table 3 shows the composition ratios of the sugar components obtained under each measurement condition for the insoluble polymer obtained in the 4% (v / v) molasses medium and the insoluble polymer obtained in the PPM medium (containing 2% sucrose).
[0075]
[Table 3]

[0076]
In the analysis of neutral sugars, the retention time of the main peak coincided with the retention time of the commercially available glucose preparation, and the composition ratio of both insoluble polymers produced in the two liquid media was 97% or more. It was. As sugar components other than glucose, mannose, arabinose and galactose were detected at a concentration of about 1% or less. On the other hand, uronic acids such as glucuronic acid and galacturonic acid were not detected.
[0077]
(2) Binding mode of sugar components
The binding mode of the sugar component of the insoluble polymer was estimated from the results obtained by methylation analysis using gas chromatography (GC) and gas chromatography mass spectrometry (GC-MS).
[0078]
Specifically, for the lyophilized product of the insoluble polymer, first, the sugar component is completely methylated by a powder NaOH method, and the resulting methylated polysaccharide is hydrolyzed to a monosaccharide by TFA, followed by an acetic anhydride-pyridine solution. To form a partially methylated sugar alcohol acetyl derivative (partially methylated alditol acetate). This was subjected to GC analysis and GC-MS measurement under the following conditions.
[0079]
(i) GC analysis conditions
GC device: HP5890A (manufactured by Hewlett-Packerd)
Column: SPB-5 (fused silica capillary, 25m x 0.25mm I.D. made by Supergo Japan)
Carrier gas: He
Detection mode: FID.
[0080]
(ii) GC-MS measurement conditions
GC device and mass spectrometry part: JMS DX-303 (manufactured by JEOL Ltd.)
Ionization: EI method.
[0081]
Table 4 shows the results of methylation analysis of insoluble polymers produced in Chinese beet molasses liquid medium.
[0082]
[Table 4]

[0083]
In the gas chromatogram of the sample partially methylated alditol acetate, the mass spectrum of the main peak is the standard mass spectrum of 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylglucitol. In agreement with this, the binding mode of 1,4 bonds was estimated. Further, from the area ratio with each peak obtained in the gas chromatogram, the composition ratio of the main peak was determined as follows: non-reducing terminal glucose (1,5-di-O-acetyl-2,3,4,6-tetra-O— (Methylglucitol) is 29.22, and the composition ratio of other 1,3 bonds and 1,6 bonds is 0.5 or less, so that the main binding mode of the insoluble polymer is 1 Therefore, it was estimated that there were 4 bonds. From the above, it was revealed that the insoluble polymer is mainly composed of 1,4-bonded glucose.
[0084]
Furthermore, as described above, the polymer is difficult to be hydrolyzed by organic acids or inorganic acids, but is relatively easily degraded by cellulase having substrate specificity that selectively degrades β1,4 bonds of glucose. Considering these properties, it was determined that the insoluble polymer is cellulose having β1,4-linked glucose as the main skeleton or a substance very similar thereto.
[0085]
The degradability of the polymer by acid or cellulase, the composition of the sugar component, and the like are not affected by the production conditions of the polymer, that is, the medium used for production. From this, it is presumed that the insoluble polymers produced in the PPM medium and Chinese beet molasses liquid medium are the same or very similar substances. From these facts, it is considered that not only an artificial medium such as a PPM medium but also an inexpensive natural substance such as molasses which is waste from a sugar factory can be used as a medium for the production of the insoluble polymer.
[0086]
Example 5: Proliferation of microorganisms in molasses medium and productivity of bacterial cellulose
The CJF-002 strain isolated in Example 1 was evaluated for the growth in an inexpensive molasses medium and the productivity of bacterial cellulose according to the following procedure.
[0087]
First, as a preculture, the CJF-002 strain was statically cultured overnight at 30 ° C. in 2 ml of 4% (v / v) Chinese beet molasses liquid medium sterilized by autoclaving. Subsequently, as the main culture, 0.1 and 1% (v / v) Chinese beet molasses liquid medium sterilized by autoclaving was used, and the initial concentration of CJF-002 strain was adjusted to 2.2 × 10.^FourAnd 2.2 × 10²CFU / ml was statically cultured at 30 ° C. for 72 hours. During the culture period, the cultures were collected after 4, 6, 8, 10, 12, 14, 16, 20, 24, 28, 32, 36, 40, 44, 48, 56, 64 and 72 hours after the culture. The degree of growth of CJF-002 strain at the collection point was evaluated using the direct counting method shown in (1) below. At the same time, the bacterial cellulose concentration in the medium was measured according to the following procedure (2) to evaluate the productivity of bacterial cellulose.
[0088]
(1) Measurement of the growth degree of CJF-002 strain
The CJF-002 strain produces insoluble bacterial cellulose, and there are many cells in the bacterial cellulose. For this reason, the total number of bacteria of CJF-002 strain in the system was measured by the following analysis method. Specifically, first, 500 mM phosphate buffer (pH 5.5), 0.2% nalidixic acid, and cellulase (sugar component-free) (1% solution) are added to the collected culture medium, and incubated at 30 ° C. for 10 hours. After visually confirming the degradation of the polymer, 8% paraformaldehyde is added to the solution to fix the cells. Next, the immobilized bacterial cell solution was filtered through a black filter, washed with 2 ml of PBS (20 mM phosphate buffer + 130 mM NaCl, pH 7.4), and then 0.01% acridine orange solution (0.01 M Tris-HCl, pH 7.4). ) For 5 minutes and microscopically.
(2) Measurement of bacterial cellulose concentration
Three times the amount of acetone is added to the collected culture solution to precipitate the polymer component (acetone extraction), the polymer component is collected by centrifugation, dried at room temperature overnight, and the conventional phenol-sulfuric acid reaction method Measure sugar concentration at
[0089]
FIG. 1 shows changes with time in the growth of CJF-002 strain (FIG. A) and the productivity of bacterial cellulose (FIG. B). From now on, the initial concentration of CJF-002 will be 10^Four In the case of CFU / ml, the transition from the logarithmic growth phase to the stationary phase was carried out after 10 hours of culture, and it was found that the CJF-002 strain proliferated very early. The growth rate is not affected by the difference in molasses concentration, but the maximum growth concentration depends on the molasses concentration and is about 10 when the concentration of Chinese beet molasses is 1%.⁹ About 10 for CFU / ml, 0.1%⁸ CFU / ml. The initial concentration of CJF-002 is 10² In the case of CFU / ml, the transition from the logarithmic growth phase to the stationary phase after 14 hours of culture, the initial concentration is 10^Four It shifted to about 4 hours later than the case of CFU / ml. The growth rate in this case is not affected by the difference in Chinese beet molasses concentration, and the maximum growth concentration is 10%.^Four The result was the same as in the case of CFU / ml. From these results, it was found that the doubling time in the logarithmic growth phase was approximately 40 minutes when the growth rate of the CJF-002 strain was calculated.
[0090]
For bacterial cellulose productivity, the initial concentration of CJF-002^Four When the concentration of CFU / ml and Chinese beet molasses was set to 1%, 0.2-0.25 g / l of bacterial cellulose was produced by about 14 hours after the start of the culture, and it was found that it became almost constant thereafter. In addition, the initial concentration of CJF-002 strain is 10² In the case of CFU / ml, the same concentration of bacterial cellulose was produced by about 16-20 hours after the start of culture. When the concentration of beet molasses produced in China was 0.1%, the same tendency as the above result was observed, but the production amount of bacterial cellulose was greatly reduced, and the maximum was 0.05 g / l or less.
[0091]
From the comparison of the growth curve of the obtained CJF-002 strain and the production curve of bacterial cellulose, it was found that the production of bacterial cellulose by the CJF-002 strain proceeds with a delay of about 4 hours.
[0092]
Example 6: Effect of molasses concentration on bacterial cellulose productivity
Next, the effect of molasses concentration on the bacterial cellulose productivity of CJF-002 strain was evaluated. First, a pre-culture solution was prepared by stationary culture at 30 ° C. overnight using 2 ml of 4% (v / v) Chinese beet molasses liquid medium obtained by autoclaving CJF-002 strain. Was used for main culture. For main culture, 30 ml of 0.001, 0.01, 0.1, 1, 5 and 10% Chinese beet molasses liquid medium sterilized by autoclaving is used, and the initial concentration of CJF-002 is 1.0 × 10⁶CFU / ml was statically cultured at 30 ° C. for 24 hours. After completion of the culture, the viable cell count of CJF-002 strain was measured by CFU method (colony forming unit) using Nutrient broth (Difco) agar medium, and the bacterial cellulose productivity was extracted from the culture solution. It was evaluated by measuring the dry weight.
[0093]
The results are shown in FIG. As shown in Fig. 2, CJF-002 strain grows when the concentration of Chinese beet molasses is 0.1% (v / v) or higher, but bacterial cellulose is produced using Chinese beet molasses medium of 1% or higher. When a 10% concentration liquid medium was used, 0.4 g / l or more of bacterial cellulose was produced. From these results, it was found that the productivity of bacterial cellulose increases with increasing molasses concentration, and that CJF-002 can produce a large amount of bacterial cellulose in an extremely short time using inexpensive molasses. .
[0094]
Example 7: Biofilm formation ability
The CJF-002 strain isolated in Example 1 was evaluated for the ability to form a biofilm on an oil layer rock.
[0095]
Specifically, after collecting from the Yoyo Oil Field, oil layer rock pieces (thickness 3 mm, diameter 37 mm, permeability [Kair] 607 md) sterilized by autoclaving were fixed vertically in a 1 L beaker and 4% ( v / v) 500 ml of a liquid medium containing Chinese beet molasses and synthetic oil layer water component was poured, and the preculture of CJF-002 strain prepared by the same method as in Example 6 was added at an initial concentration of 1.0 ×. Ten⁶ Inoculated to CFU / ml.
[0096]
The above-mentioned synthetic oil reservoir was determined based on the results of chemical composition analysis of the fluid produced with crude oil collected from the production well located in the Yuyu Oil Field of Jilin Petroleum Co., Ltd. , NaCl; 1210 mg / l, KCl; 23 mg / l, NaHCO_Three; 2820 mg / l, CaCl₂140 mg / l, MgCl₂253 mg / l, FeCl_Three; 2 mg / l, KH₂PO_Four; 10 mg / l, NaHSO_FourThe composition of 3 mg / l [Yonebayashi Eiji, Taguchi Mitsuru, Fujiwara Kazuhiro, Yoshida Shinichiro, Enomoto Yuji, Current Status of Research on Microbial Offenses and Prospects for Field Testing, Journal of Petroleum Technology Association, Vol. 61 (6), 487-493 (1996), Yonebayashi, H., Ono, K., Enomoto, H., Chida, T., Hong, CX, Fujiwara, K., Microbial Enhanced Oil Recovery Field Pilot in a Waterflooded See also Reservoir, Society of Petroleum Engineers, 38070 (1997)].
[0097]
In addition, in order to grasp the biofilm formation ability of CJF-002 strain under conditions closer to the oil reservoir environment, and 1 L beaker containing the liquid medium inoculated with CJF-002 strain was anaerobic jar (Becton Dickinson and Company, An anaerobic environment similar to the inside of the oil layer is established by placing it inside the oil layer, and a stir bar is placed in a 1 L beaker, and the medium is stirred at a rotational flow rate of about 1.7 cm / sec. The fluid flow was reproduced in an accelerated test. Furthermore, the culture temperature was 30 ° C., which is the oil reservoir temperature of the Yoyo Oil Field, and the culture was performed for 36 hours.
[0098]
After completion of the culture, the viable cell count of CJF-002 strain in the culture solution was measured by CFU method using Nutrient broth (Difco) agar medium, and the biofilm formation on the oil layer rock surface was evaluated by visual observation did.
[0099]
FIG. 3B is a drawing showing a state in which a biofilm is formed on the surface of the oil layer rock. As can be seen from this figure, the biofilm completely covered the oil layer rock. The concentration of CJF-002 strain in the culture is 2.2 × 10⁸ Since it is CFU / ml, which is 2 orders higher than the initial concentration, the biofilm formed on the surface of the oil reservoir is estimated to be derived from bacterial cellulose produced by the growth of CJF-002 strain. It was done. From these results, it was found that CJF-002 strain grew under oil reservoir environmental conditions and formed a strong biofilm on the oil reservoir rock surface. In addition, since the time required for biofilm formation was short, it was considered that CJF-002 strain could block the highly permeable region early in the oil reservoir. Therefore, the CJF-002 strain contributes to the short-time treatment or efficiency improvement of the microbial attack, and it can reduce the injection operation cost by using it as a raw material microorganism of the microbial attack. It is a suggestion.
[0100]
Example 8: Increased recovery capability of crude oil
For the CJF-002 strain isolated in Example 1, the enhanced oil recovery capability was evaluated using an oil layer rock saturated with crude oil. First, the oil reservoir core is collected from the Yuyao Oil Field of Jilin Oil Co., Ltd., China Petroleum Natural Oil Co., Ltd., and the plug core (diameter approx. 3.7cm, length approx. 11cm) from the core of 359.2m depth (diameter approx. Pore volume [PV]; 23.7 ml, porosity [φ]; 29.4%, permeability [Kair]; 1051 md). Next, the plug core is sterilized by dry heat at 180 ° C. for 3 days, immersed in the synthetic oil layer water described in Example 7, and degassed overnight using a vacuum pump to saturate the pores in the plug core with the synthetic oil layer water. did. Next, the plug core is attached to the core holder, sterilized at 121 ° C for 240 minutes, the water permeability is measured, and approximately 5.3 PV of crude oil from the Yuyu oil field is fed into the plug core at a flow rate of 60 ml / min. Then, the pore portion of the plug core was saturated with crude oil. Then, the synthetic oil reservoir water was fed again at a flow rate of 40 ml / min, and all the crude oil in the plug core that could be recovered by the water flooding method was recovered to reproduce the oil reservoir at the end of the water flooding (water saturation rate). [% PV]; 56.9%, oil saturation [% PV]; 31.6%, permeability [Kwater]; 208.0md).
[0101]
Next, the CJF-002 strain was injected in succession. First, using a liquid medium in which 0.1% (v / v) Chinese beet molasses, synthetic oil reservoir water component, and 0.005% cellulase (without sugar component) were sterilized by autoclaving CJF-002 strain, The cells were cultured at 30 ° C for 24 hours. Subsequently, the culture solution was mixed with the synthetic oil layer water at a ratio of 1: 100, and pressed into the plug core at a flow rate of 10 ml / min for 1 day. The injection concentration of CJF-002 strain at this time is about 10⁶ CFU / ml. The CJF-002 strain was injected by placing the CJF-002 strain injection solution in a bottle with an appropriate volume and feeding it into the plug core with a feed pump. Before the CJF-002 strain was press-fitted into the plug core, the temperature of the bottle and the liquid feed line was maintained at 15 ° C. in order to prevent the biofilm from being produced in the bottle or the liquid feed line. In addition, 0.1% (v / v) Chinese beet molasses, synthetic oil reservoir water component and 0.01% cellulase (no sugar component) sterilized under high pressure steam sterilization were added to the fresh CJF-002 strain culture solution prepared by the above procedure. As a nutrient solution, a mixture of each of the above was injected into the plug core under the same conditions as the CJF-002 strain, and the synthetic oil layer water was further injected under the same conditions.
[0102]
In this experiment, the total bacterial cell concentration of the eluate collected throughout the experiment period was measured by the CFU method using Nutrient broth (Difco) agar medium, and the survival concentration of CJF-002 strain was determined by the present inventors. Monitored using previously established genetic engineering techniques [Fujiwara, K., Tanaka, S., Ohtsuka, M., Ichimura, N., Yonebayashi, H., Hong, CX, Enomoto, H., Evaluation See also of the Use of Amplified 16S rRNA Gene-Restriction Fragment Length Polymorphism Analysis to Detect Enterobacter cloacae and Bacillus licheniformis for Microbial Enhanced Oil Recovery Field Pilot, Sekiyu Gakkaishi, 42 (5), 342-351 (1999)]. In addition, during the experiment period, the amount of recovered crude oil and the pressure change in the plug core (differential pressure between the injection pressure and elution pressure of various solutions) were evaluated, and the concentration of CJF-002 strain surviving in the plug core after the experiment was completed Was measured using the CFU method and the above genetic engineering technique, and the presence or absence of biofilm formation was evaluated by the method described in Example 5.
[0103]
The results are shown in FIG. As shown in FIG. 4, immediately after the injection of the nutrient solution, the concentration of the CJF-002 strain in the eluate and the differential pressure between the injection pressure and the elution pressure increased. This is considered to be a result suggesting that the CJF-002 strain grew in the plug core due to the injection of the nutrient solution, and a biofilm was formed accordingly. In addition, the crude oil recovery rate increased slightly after the increase in the differential pressure, and a maximum crude oil recovery rate of 25% was observed. This result shows that the highly permeable region of the plug core is blocked by biofilm. This is thought to be because the water / oil replacement efficiency of the press-fit fluid has been improved by changing the flow path of the press-fit fluid. Furthermore, after completion of the press-fitting experiment, the concentration of CJF-002 strain surviving in the plug core was measured and found to be 10 per 1 g (dry weight) of the plug core.⁷ CFU CJF-002 strain was found to be almost uniformly distributed in the plug core, and biofilm formation was also confirmed.
[0104]
From the above, according to the CJF-002 strain, it can grow in an oil layer environment, which is an anaerobic environment, to form a biofilm, and the formed biofilm blocks the highly permeable region of the oil layer. It was suggested that it could have an excellent enhanced oil recovery effect.
[0105]
Example 9: Increased recovery of crude oil in the actual oil reservoir
For the CJF-002 strain isolated in Example 1, the effect of enhanced recovery of crude oil in the actual oil reservoir was evaluated. The experiment was conducted on production well # 22-264 (actual oil reservoir) located in the Yuyu Oil Field of Jilin Petroleum Co., Ltd. A one-cycle huff & puff attack method is used to complete the process. The procedure for the press-in experiment of CJF-002 strain is shown below.
[0106]
First, the CJF-002 strain was statically cultured overnight at 30 ° C. using a liquid medium (1 L) sterilized by autoclaving (containing 1% Chinese beet molasses and 0.01% cellulase). CJF-002 strain used for press-fitting was obtained by inoculating 10 L (eight) of the same medium and further stationary culture at 30 ° C. overnight.
[0107]
Next, prepare four 15kl tank trucks (loading capacity of about 12kL), sterilize the inside of these tanks, and load ground water from the groundwater storage tank of the ground facility into the first tank truck. Eight 10L cultures of CJF-002 strain (average 9.1 × 10⁸CFU / ml = 7.3 × 10^TenCFU / 80L) was mixed and transported to the well site. The road to the well was a rough road, and it took about 30 minutes to transport, and during this time, the injection solution in the tank was being stirred. At the well well, press injection was performed from the annulus portion of the well by a pump car that was heat sterilized in the same procedure as the tank lorry. Subsequently, only the groundwater was used as a spacer for the second unit, and the beet molasses solution made in China diluted with about 5% of the groundwater as the target for the third and subsequent units was sequentially injected in the same procedure as above (total amount of injection: 130 kl). The average press-fitting rate for these press-fittings was approximately 0.5 kl / min (30 kl / h). Then, after injecting a predetermined amount of the injecting solution, about 6 kl of ground water corresponding to a half tank lorry was injected as boosting water in order to inject all of the solution remaining in the annulus portion into the oil layer.
[0108]
After completion of the injection, CJF-002 strain injected into the reservoir was propagated in the reservoir using molasses, and the well was sealed (discontinued) for 10 days in consideration of the time required to form a biofilm. .
[0109]
After the well was sealed for a predetermined period, production was resumed, and the effect of enhanced oil recovery was evaluated by measuring changes in the amount of fluid produced from the production well and the water content (water cut). Furthermore, after the production resumed, after draining the oil reservoir water stagnating in the tubing, the output fluid was sampled over time from the drain part of the wellhead device into the sterilized bottle, and the method described in Example 8 was used. The concentration of CJF-002 strain surviving in the production fluid was monitored.
[0110]
FIG. 5 shows the production behavior of the output fluid and crude oil in the press-fitting experiment. As a result of the injection of CJF-002 strain, the yield of the output fluid was reduced by approximately 35%, and an increase in crude oil recovery by 4-5 times was observed. Further, in the output fluid collected over time, 10%^Four-Ten^FiveCJF-002 strain of about CFU / ml was detected frequently. From these results, the high permeability area existing in the oil reservoir near production well # 22-264 was blocked by the biofilm produced by CJF-002 strain, resulting in a decrease in the production fluid output. At the same time, it is considered that the unrecovered crude oil was swept away due to the change in the fluid flow path in the oil reservoir, and the amount of recovered crude oil increased.
[0111]
From the results of crude oil scavenging attack experiments and Huff & Puff tests using CJF-002 strains in Examples 8 and 9 above, it was confirmed that CJF-002 strains had an enhanced oil recovery effect, and the bacteria were used in microbial attack. It turned out to be a very effective bacterium.
[0112]
【The invention's effect】
As described above, according to the present invention, a novel microorganism that produces bacterial cellulose, a bacterial cellulose produced by the microorganism, a method for producing the bacterial cellulose, and an enhanced recovery method for the microorganism and the petroleum of the bacterial cellulose, which were the initial objectives. How to use is provided.
[0113]
The microorganism of the present invention is a microorganism that can be mass-cultured using an inexpensive medium with a simple device and can actively grow under anaerobic conditions to produce an insoluble polymer such as a biofilm. It is useful as a microorganism for efficiently carrying out oil recovery by microbial attack at low cost.
[0114]
According to the present invention, it is expected that bacterial cellulose, which is attracting attention as a natural material, can be supplied at a low cost in recent years when development of environmentally compatible materials is urgently required for global environmental problems. In addition, in the present invention, the microorganism of the present invention is effectively used for selective plugging of a highly permeable region by a microorganism-produced insoluble polymer, which is one of the effective utilization methods of microorganisms in the microbial attack. It turns out that this is possible, and the enhanced recovery of oil in various oil fields is highly expected.
[0115]
[Sequence Listing]

[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing bacterial growth (FIG. A) and cellulose productivity (FIG. B) when the bacterial cellulose-producing microorganism of the present invention (CJF-002 strain) is cultured in beet molasses medium (FIG. 1). Example 5).
FIG. 2 is a graph showing the influence of beet molasses concentration on the growth of bacterial cellulose-producing microorganisms (CJF-002 strain) of the present invention and the productivity of bacterial cellulose (Example 6).
FIG. 3 is a view showing a state in which a biofilm is formed on the surface of an oil layer rock (Example 7). Fig. A is a diagram showing the state of oil layer rock fragments before use in the experiment, and Fig. B is a diagram showing the state of oil layer rock fragments after inoculating CJF-002 strain into the oil layer rock fragments and culturing. Yes (Example 7).
FIG. 4 is a diagram showing the result of examining the enhanced recovery capability of crude oil using an oil layer rock saturated with crude oil in Example 8.
5 is a diagram showing the results of a Hough & Puff test performed in Example 9. FIG.

Claims (6)

A bacterial cellulose-producing microorganism that is an Enterobacter sp. CJF-002 ( FERM BP-8227 ) strain , or a progeny or variant thereof having the bacteriological properties and bacterial cellulose-producing ability of the strain . A method for producing bacterial cellulose, comprising culturing the bacterial cellulose-producing microorganism according to claim 1 and collecting and recovering bacterial cellulose from the culture. Method for producing bacterial cellulose according to claim 2, wherein the culturing bacterial cellulose producing microorganisms according to claim 1 in a liquid medium containing at least one monosaccharide or disaccharide. The method for producing bacterial cellulose according to claim 2 or 3 , wherein the cultivation is performed by a stationary culture method. A method for enhanced recovery of petroleum, wherein the bacterial cellulose-producing microorganism according to claim 1 or the bacterial cellulose produced by the microorganism is used. 6. The method for enhanced recovery of petroleum according to claim 5 , wherein the bacterial cellulose-producing microorganism according to claim 1 is grown in an oil layer to produce bacterial cellulose.

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