ڟ֎׮বǿȕǼǒțߞߞҍޙإƝॻਙࠏҍডȉǧțবঢۉޙإƣ ӴǺDZȕǚDz➚Ǥಓہ

ڟ֎׮বǿȕǼǒțߞߞҍޙإƝॻਙࠏҍডȉǧțবঢۉޙإƣ ӴǺDZȕǚDz➚Ǥಓہ

ଥѼ֥ࢫҚԆગԆ➙ԆবӁЋ➙ÿ૴੬ผ๓

঩ӁЋ➙➙ϺଚԾఽ➛ગ׾ࣵ໡➛ںஎࢂࡵ

1.ƤƍƶƠ

➙݄ਫ਼౗ॻࣂື֥ࢫƜŷǀ݄҉ UASB ǿȕǣǡƤ౗ॻࣂືұ୤ſŹƛǿȕǼǒțߞƣ૭৅Ɓবƍ➛໡܇Ɵ౗ॻࣂືƁ඼

ƶƟƄƟǀƝŹŻเચƁƌƥƌƥಓবƎǀ➜1985వƠWiegant ƾƁඓݒƌƛƀƾƎƜƠ18 వƁٶұƌƛŹǀƠƷƀƀDŽ ƾƏƈƣเચƣ໡܇ƟӃೖඝඟƤ฀ƕഖผƜŷǀ➜ƈƣเચӂڎƣࠃ܁Ƥ➛݄҉ڟ֎पڕҌƜǿȕǼǒțߞƣൌӂƠտ

๹ƌƛŹǀ฀ƕඓݒƁƟƊǁƛŹƟŹೝবൊ٘ƠŷǀƝܯŽƾǁǀ➜ƈƣƽŻƟৎƿ܁ƽƿ݄҉ڟ֎ডपڕҌƠſŹƛ ǿȕǼǒțߞƣൌӂƠտ๹ƌƛŹǀೝবൊƁ Imachi ƾƠƽƿൌຽƊǁ्੠्࡫ƝƌƛবືԆ୴௫ଓƁඓݒƊǁƔ➜ඓݒ ƊǁƔೝবൊƤિ௰ƜƣǿȕǼǒțߞƣൌӂƁܶŽƟƀƘƔ➜ƈƣƈƝƤ➛ǿȕǼǒțߞൌӂಣѰƣ೺ࢺࠧ๭ǐǵȓǖ

➚൱ҍ(∆G˚’)ƀƾƷൌƀǀƽŻƠ➛׷ŹׅǐȓǜțಣѰƜŷǀƔƶƜŷǀ(Table 1-(1))➜ǿȕǼǒțߞƣൌӂƠಠŹॻਙ

ƁবঢƊǁ➛ൌӂտ๹ೝবൊƁॻਙൌωƣझࣲƠƽƿবϽƁݤఙƠƟǀƔƶǿȕǼǒțߞƣൌӂƁॠܶƌƟŹƣƜŷǀ➜

ƙƳƿ➛ॻਙLJࣖטƎǁƥǿȕǼǒțߞƣൌӂƁॠƵƣƜŷǀ(Table 1-(3))➜ƽƘƛ➛ǿȕǼǒțߞൌӂޙإƤ➛ǿȕǼ ǒțߞƣൌӂƠƽƿবঢƊǁƔॻਙLJາ຋ƎǀॻਙࠏҍডƣೝবൊƝ׷ۋƟ׮বٵLJܟ૩ƌƟƆǁƥǿȕǼǒțߞƣൌ

ӂƁܶŽƟŹ׮বޙإƜŷǀ➜ƈƣǿȕǼǒțߞൌӂࠜƠſƆǀ׮বդٞƤ➛ॻਙƁ࢟ຑƟǽljǗǧ➚ƝƟƘƛſƿ➛

ॻਙ஋઱׮বդٞƁঢƿແƘƛŹǀƝŹŽǀ➜ƈƣॻਙ஋઱LJૂƘƛŹǀƷƣƁǺDZȕǚDz➚ǤƝۊƥǁǀܼਙƜŷǀ➜

ܼਙƤ➛DNAČmRNAČܼਙƣࣁ಺ƜবঢƊǁ➛ܼਙবঢ໤Ƥ mRNA ಓہ໤Ơೊ໻ƌƛŹǀƝۄDŽǁƛŹǀ➜ƒƈƜ➛

ƈƣܼਙLJإ੾ఊƜ݉ঢƎǀƔƶƣ৓ٽॴƝƌƛಓہƌƛŹǀ mRNA ϸ஋߰LJଠƮǀƈƝƜڟ֎ড౗ॻࣂືѪ୯ఊƜƣ ॻਙ஋઱׮বդٞೝবൊƣবઆొςƁƜƂǀƝܯŽƾǁǀ➜ƒƈƜ➛ේڪאƤǿȕǼǒțߞߞҍޙإƝॻਙࠏҍডȉǧ țবঢۉޙإƣ݄҉ڟ֎׮বपڕҌƠſŹƛǺDZȕǚDz➚ǤLJǛ➚DZƌƛŹǀ mRNA ƣϸ஋߰ౡ་ƣڎୖׄƨϸ஋߰ୖ

໤ҍLJࠌƴƔ➜

2.࠾ڷඝඟ

➙2.1 ǿȕǼǒțߞߞҍޙإ(Pelotomaculum thermopropionicum strain SI)ƣǺDZȕǚDz➚Ǥϸ஋߰ౡ་

➙݄҉ǿȕǼǒțߞߞҍޙإƜŷǀ P.thermopropionicum strain SI(DSM 13744)ƣǺDZȕǚDz➚Ǥϸ஋߰ౡ་ƤඓݒƊǁƛ ŹƟŹ➜ہޟඓݒƊǁƛŹǀঊ➼ƣǺDZȕǚDz➚Ǥ-NJȇǶߞౡ་LJ຋Źൽ੨ডƣ݄Ź໦ϼƽƿǿȑnjȆ➚LJޮঢƌ➛SI Ԣ ƽƿ૸ࢪƌƔǚǶȈDNA Ơ઀ƌPCR➛Ǘȕ➚dzțǘLJӀƌѨռౡ་ƣ௙ୖLJࠌƴƔ➜DNA૸ࢪƤ SDS+ǻ➚Ǣǻ➚

Ǩ➚LJ຋ŹƔ➜

➙2.2ॻਙࠏҍডȉǧțবঢۉޙإ(Methanothermobacter thermautotrophicus strain ∆H)ƣǺDZȕǚDz➚Ǥϸ஋߰ౡ་

➙M.thermautotrophicus strain ∆H(DSM 1053)ƤਆѨռౡ་ƁڎୖƊǁƛŹǀ➜ේڪאƜǧ➚ǚǫǰƝƎǀǺDZȕǚDz➚Ǥ Ƥmv-ǺDZȕǚDz➚Ǥ(mvhDGAB)ƝƌƔ➜

➙2.3 ǺDZȕǚDz➚Ǥ-mRNAಓہƣଠݺ

Ǖ➚ȗ➚DZ➠׮বޙإ➛ॻਙ஋઱׮বդٞ➛ǺDZȕǚDz➚Ǥ➛mRNA➛RT-PCR➛Real-Time PCR

༘ວড়➠ċ940-2188➙्ԑڭଥѼ߲झഛѼଚ1603-1➙ଥѼ֥ࢫҚԆગԆ➙Ր׵ǟǡǮȈ܋Ԇ Tel.0258-47-1611 (ext.6646)➙Fax.0258-47-9630➙E-mail: [email protected] Table 1 Standard Gibbs free-energy changes (∆G˚’) at 55˚C.

(1) CH3CH2COO^-(aq)+3H2O(liq)ŤCH3COO^-(aq)+HCO3

-(aq)+H⁺(aq)+3H2(g) ∆G˚'=+62.2kJ/reaction (2) HCO3

-(aq)+4H2(g)+H⁺(g)ŤCH4(g)+3H2O(liq) ∆G˚'=-122.5kJ/reaction (3) CH3CH2COO^-(aq)+3/4H2O(liq)ŤCH3COO^-(aq)+1/4HCO3

-(aq)+1/4H⁺(aq)+3/4CH4(g) ∆G˚'=-60.2kJ/reaction 土木学会第58回年次学術講演会（平成15年9月）

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Table 3 Results of quantitative analysis by R-PCR.

16S rDNA 16S rRNA hydrogenase-mRNA (copies/ng) (copies/ng) (copies/ng) strain SI 8.0 x 10⁶ 2.8 x 10⁶ 7.5 x 10¹ strain ∆H 2.8 x 10⁶ 3.9 x 10⁴ 2.1 x 10⁰ strain SI 6.4 x 10⁴ 1.5 x 10⁴ 2.5 x 10¹ strain ∆H 4.0 x 10⁶ 5.9 x 10⁴ 4.4 x 10² pure culture

co-culture

sample name Target cell

Table 2 PCR primers used in this study

Sequence position*

primer 1F ACICGIATYGARGG 73-90 primer 4R CAYTAYYTISARGCICT 613-630 primer 9R-1 AYCCITGYITSGCIT 1720-1737

*: Escherichia coli hydrogenase-1 large subunit primer Nucleotide sequence (5'-3')

➙mRNAಓہƣଠݺƠƤRT-PCR (Reverse Transcription-PCR)LJ຋ŹƔ➜ࢽॾإԢƽƿ RNA LJ૸ࢪƌ➛Ҍໆ௫ϯஊౡ་ǿ ȑnjȆ➚LJ຋ŹӴϸ஋߰ƣֿஈࡈLJܶƘƔ➜ֿஈࡈ۩ cDNA Ơ઀ƌǺDZȕǚDz➚ǤƠ௫ϯ୴ƟǿȑnjȆ➚LJ຋Ź PCR ಣ ѰLJܶƘƔ➜RNA૸ࢪƠƤISOGEN(Nippongene)+ȅȒǰȕțȃȊǠDznjǞ➚LJ຋ŹƔ➜

➙2.4 Real-Time PCRƠƽǀϸ஋߰ୖ໤

➙ϸ஋߰ୖ໤ƠƤ Light Cycler (Roche)LJߦ຋ƌ➛ӴǺDZȕǚDz➚Ǥ-mRNA ϸ஋߰ƣୖ໤ҍLJࠌƴƔ➜Ɵſ➛njțǧ➚Ǔ Ȕ➚ǟȏțǨnjƝƌƛSYBR Green ILJ຋ŹƔ➜

3.࠾ڷڑҜ

➙SI ԢƣǺDZȕǚDz➚Ǥϸ஋߰ƣ௙ୖƠޝƌƛ➛ہޟඓݒƊǁƛŹǀঊ➼ƣǺDZȕǚDz➚Ǥ-NJȇǶߞౡ་LJ຋Źൽ੨ড ƣ݄Ź໦ϼƽƿझໆശƠ2࡫➛ҌໆശƠ1࡫ƣٽ3࡫ƣǿȑnjȆ➚LJޮঢƌƔ(Table 2)➜ǿȑnjȆ➚ƤDNAౡ་झƜƤ ԼਆЀ૥ƎǀശϠƁࣧƟƄݮ݉ѨռLJ੯ঊ๧ƎƷƣƝƟƘƛƌƳƘƔ➜

ƒƣƔƶnjǶǟț(I)LJ຋ŹǀƈƝƜݮ݉ѨռঊLJھƾƌƔ➜SI Ԣ ƽ ƿ૸ࢪƌƔǚǶȈ DNA Ơ઀ƌझໆശ 2 ࡫ƣǿȑnjȆ➚1F,4R LJ຋Ź PCRಣѰLJܶƘƔ➜ಣѰবঢൊLJNJǔȕ➚ǡǚȓƠƽƿǝnjǢൌҸƌ➛

ǚȓƀƾ DNA LJӃࡹ➟ম঱ƌƛ௨Ɣ੏ിߘൊƣѨռౡ་LJǗȕ➚dz țǘLJӀƌƛڎୖƌƔ➜ڎୖƌƔѨռౡ་૴Ơ௫ϯ୴ƟǿȑnjȆ➚LJ

ޮঢƌ➛ҌໆശƠޮঢƌƔǿȑnjȆ➚9R-1 LJ຋Źƛ௙ງƣޮؐLJܶŹ Ѩռౡ་LJڎୖƌƔ➜ƈƣਲޮƠƽƿ௨Ɣϸ஋߰ౡ་LJ BLASTX Ơƽ

ǀNJȇǶߞƝƣ਼௙ডڥ޷LJܶŹ֋ృƣ௙ୖLJܶƘƔ➜Desulfovibrio ੠ƣǺDZȕǚDz➚ǤƁ

਼௙ড 49%ƝއƷ݄Ƅ➛ƒƣ੮਼௙ডƣŷǀƷƣƎƮƛƁǺDZȕǚDz➚ǤƜŷƘƔ➜NJȇ ǶߞƝƣ਼௙ডƤЀರƠ 30%Ϟझŷǁƥ਼௙ডƁŷǀƝŹDŽǁƛŹǀࠔƽƿ➛ݣӃ௨Ɣϸ

஋߰ƤǺDZȕǚDz➚Ǥƣ֋ృLJ๧ƌƛŹǀƝܯŽƾǁƔ➜ࠝƠ➛௨ƾǁƔϸ஋߰Ɓ࠾ޝƠಓ ہƌƛŹǀƀLJRT-PCRƠƽƿǩǏǫǗƌƔ➜ȅǠǮNjǾǛțǰȕ➚ȓƝƌƛ16S rRNA LJ

຋ŹƔRT-PCRƷ௙ࠜƠܶƘƔ➜ƒƣڑҜ➛ݣӃ௙ୖƌƔ SIԢƣǺDZȕǚDz➚Ǥ-mRNA ϸ

஋߰ƣಓہƁӼభƊǁƔ(Fig.1)➜ಓہƁӼభƜƂƔƔƶ➛mRNA ಓہ໤ƣୖ໤LJܶƘƔ➜ש ࠌǝțǿȓƝƌƛ➛ǼȓǻțߞƜౣຖƌƔ SI Ԣࢽإ➛ॻਙƜౣຖƌƔ∆H Ԣࢽإſƽƨ➛SI ԢƝ∆H ԢƣǿȕǼǒțߞƠƽǀ׮বౣຖٵLJ຋ŹƔ➜૸ࢪƌƔ Total RNA Ơ઀ƌҌໆ௫ϯ ஊౡ་ǿȑnjȆ➚LJ຋ŹֿஈࡈಣѰLJܶ

Ź➛ಣѰবঢൊƜŷǀ cDNA ƣୖ໤LJܶ

ƘƔ➜௙ࠜƠ16S rDNAϸ஋߰LJǧ➚ǚǫ ǰƝƌƔ DNA ƣୖ໤ƷܶƘƔ➜ୖ໤ڑҜ LJTable 3ƠࠤƎ➜

4.ƳƝƶ

➙ේڪאƠƽƿ௙ୖƊǁƛŹƟŹ֋ృϸ஋߰LJ௙ୖƌƔ➜Real-Time RT-PCR LJ຋ŹƛǺDZȕǚDz➚Ǥ-mRNA ƣୖ໤Ɓܶ

ŽƔ➜ǺDZȕǚDz➚Ǥ-mRNA ϸ஋߰ಓہ໤Ƥ 16S rRNA ϸ஋߰ಓہ໤Ơ઀ƌ 2➴5 ǒ➚Ǩ➚ƣಓہ໤ƣϷŹƁӼభƊǁ Ɣ➜धࠜಓہƌƛŹǀƝۄDŽǁƛŹǀ 16S rRNA ϸ஋߰Ɲƣಓہ໤ƣϷŹƁગƂŹࠔƽƿ➛ǺDZȕǚDz➚Ǥϸ஋߰Ƥध

ࠜಓہLJƌƛŹƟŹࠔƁܯŽƾǁǀ➜ݣ۩Ƥ➛ॻਙ஋઱׮বٵೝবൊƣবઆӂৃLJॠƶǀƮƄ➛ງ➼ƟౣຖपڕҌƠſ ƆǀǺDZȕǚDz➚Ǥϸ஋߰ಓہLJଠݺƎǀ➜

ࡑࠩ➙ේڪאƣЀശƤ➛्ǐǵȓǖ➚➟ߘ֥ؐࢫਿ݉әಓ֋ܟ(NEDO)ƣÚবൌӂ➟ࣂືȉǓdzǢȈƣӂৃƝঝ۪֥ࢫ әಓÛƣࠔؐƝƌƛ࠾ߺƌƔ➜

土木学会第58回年次学術講演会（平成15年9月）

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