Identification of Indigenous Acidophilic Bacteria by Polymerase Chain Reaction and 16S rRNA Sequences

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Vol. 49, No. 4 O2012PG pp. 507-520

ண෍ตী઴৐ࢱଦր4S3/"સ׆ছવଡଲ૳෉

ഠచ෹ॺনࢮഓࠤੰଭܛ୨

׌ࣲச

Â ଍۩૽

Â ࢽֽਐ

Â ন౿ْ

Â ًౖశ

Â ࢮవઽ

Identification of Indigenous Acidophilic Bacteria by Polymerase Chain Reaction and 16S rRNA Sequences

Bong-Ju Kim, Dae-Woong Wi, Keun-Sik Baik, Chi-Nam Seong, Nag-Chol Choi and Cheon-Young Park

Abstract : Polymerase chain reaction and 16S rRNA sequence were investigated to identify the indigenous acidophilic bacteria from the Goseong and Yeonhwa in Korea, and the Japanese bacteria in Japan. In comparison sequences with the type strain of Acithiobacillus ferrooxidans(ATCC 23270), the similarity of Goseong and Yeonhwa bacteria were obtained the range of 96.99-97.79% and 97.59-97.90%, respectively. By comparison sequences with type strain both A. ferrooxidans and A. caldus(ATCC 51756) the similarity were founded range of 97.26-97.97% and 93.84% in the Japanese bacteria, respectively. The similarity of these bacteria were found 98.5% with the type strain of Acithiobacillus ferrooxidans by using DGGE in the cluster analysis. When these bacteria were incubated in growth-medium with and without sulfur, the pH value were rapidly decreased more containing ferrous iron medium than medium with sulfur.

Key words : Indigenous acidophilic bacteria, Polymerase chain reaction, 16S rRNA sequence, Type strain, Acidithiobacillus ferrooxidans

څ أ ČՁ, ٍজфێ҆ࢹ޳঒ԓՁчࢬν؉εʴ܁ॠşڦॠيܼ०মՙٍթъڿę16S rRNA ّşԴَ

қԵںսॱॠٕɰ. 3Òݓً֨ΒقԴصرݕ16S rRNA ّşԴَںशܵŒܳۍAcidithiobacillus ferrooxidans (ATCC 23270)ٮpairwise Ҽİ܁͵(alignment sequence) ѓѪڷͿڮԐʪεҼİॠٕɰ. शܵŒܳٮڮԐʪε

ҼİॢĀę, ČՁфٍজчࢬν؉ۆّşԴَڹڮԐʪÀÁÁ96.99-97.79%ٮ97.59-97.90%ͿǣࢍǮɰ.

ێ҆чࢬν؉əशܵŒܳٮۆڮԐʪÀ97.26-97.97%ͿǣࢍǮČA. caldus शܵŒܳٮə93.84%ۆڮԐʪε

ٕ҃ɰ. ѺՁĵѕõۻşٖʴڷͿ3Òݓًчࢬν؉˞قʂॢķݚқԵںսॱॢĀęČՁ, ٍজфێ҆

чࢬν؉əϿ˃əAcidithiobacillus ferrooxidansٮ98.5%ۆڮԐʪεٕ҃ɰ. ۋ˞чࢬν؉˞ںۋڌॠيՁۤ

-֬ॹںսॱॢĀęڙՙডںप॥ॠəѕتؚقԴ҃ɰferrous ironںप॥ʽѕتؚقԴʌӇβópHÀ

Çՙॠٕɰ.

ܳڅر ࢹ޳঒ԓՁчࢬν؉, ܼ०মՙٍթъڿ, 16S rRNA ّşԴَ, शܵŒܳ, Acidithiobacillus ferrooxidans

2012ț5ښ7ێۿս, 2012ț8ښ20ێ֮ԐٰΒ 2012ț8ښ23ێóۦঝ܁

1) ܓԸʂॡİقȃݓۙڙėॡę

2) տߎʂॡİԦНॡę

3) ۻǫʂॡİėغşցٍĵՙ

*Corresponding Author(чߎٖ) E-mail; [email protected]

Address; Department of Energy and Resource Engineering, Chosun University, 375 Seouk-dong, Dong-gu, Gwangju 501-759, Korea

Դ΁

঒ԓՁ чࢬν؉ۍ Acidithiobacillus ferrooxidans (Thiobacillus ferrooxidans)əTemple and Colmer(1951) قۆ३ߌڼڷͿѕتقՁėॢۋ͒ψڹܛΪۆڮڌ Ś՚ڌ߻قۋڌʼČەɰ. A. ferrooxidansəজॡИş

ۙÀٖتߕۋş˺Лق, ݌ИşٖتՁқ, ʂşܼۆИ ş࢏ՙŔνČferrous ironęঞڙ঍ࢗۆডںԓজ֨

ࡈটڌॠəԦνॡۺ࣢ՁںÍČەş˺Лقي͠ܛ

ٍĵȦЛ

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ΪۆডজġНͿҙࢢڮڌŚ՚ۋ٣ںڌ߻֨ࢅəɠͳ ںÀݓČەɰ(Leduc and Ferroni, 1994). ۋ˞чࢬν

؉˞ڹܼŚ՚ۋ٣ۆʫՁقǴՁںÍČەş˺Лق (Tuovinen et al., 1971; Das et al., 1997), ডʴԵ, Խ؉

ٍԵ, ѓٍԵ, ডߏԵ, ڮҼߏԵ, ۙΪߏԵ, ৅ʴԵˣڷ ͿҙࢢڮڌŚ՚ںڌ߻֨ࢅəʚۋڌॠČەںӼχ؉

ɦ͆ডߏԵۋǣڮҼߏԵق॥ڮʼرەəŚںধսॠ əʚʪۋڌʼČەɰ(Bhakta and Arthur, 2002). ۋ͠

ॢчࢬν؉ۆɠͳںডজġНقۺڌॠϸ҃ɰ۹Ͷॢ

ҼڌڷͿڮڌŚ՚ںধսॣսەČঞąۺڷͿЛ܃À

äۆьԦॠݓ؍əɰ. ˰͆Դইۦə۹ुڦġԵقԴ ҙࢢČुڦ܁ġقūݓŔνČ֬ॹ֬սܵقԴҙࢢ

آٽইۤۆheap leachingقۋβşūݓйԦНڌ߻ق

টڌʼČەɰ(Petersen and Dixon, 2002; Sampson et al., 2005). A. ferrooxidansəۋ͠ॢɠͳʍқقġԓ ѕս(AMD), ʴڌ߻ݓً̚əڍ͆ɔġԓݓًڷͿҙ ࢢ Čߕѕݓ(solid medium)ε ۋڌॠي տսқν(pure culture)ॠٕڷǣ(Manning, 1975; Mishra et al., 1983;

Johnson et al., 1987; Visca et al., 1989; Johnson and McGinness, 1991), জॡИşۙÀٖتߕۆ࣢ՁڷͿۍ ॠيڮşНۋ॥ڮʽČߕѕݓق۞Ձۤॠݓ؍əɰ (Bryner and Jameson, 1958; Lobos et al., 1986; Rawlings et al., 1999; Rojas-Chapana and Tributsch, 2004). Čߕ ѕݓقԴտսѕتʽчࢬν؉قʂॠيԦজॡۺ࣢ݜ

˞ںٍĵॠČқΪॠٕɰ. Ŕ͠ǣۋ͠ॢशই঍(phenotype) ۆқΪѓѪقəي͠ÀݓॢćÀەؽɰ. ࣢০ڍνǣ

͆قԴA. ferrooxidansǣA. thiooxidansεۋڌॠيй ԦНڌ߻ۋǣܼŚ՚܃äقěॢٍĵ˞ۋݕॱʼؽڷ ǣʂҙқԜغۺڷͿқتыڹчࢬν؉˞ۋٕɰ(чߎ

ٖˣ1997; ۋʴݕˣ, 2003; Čϼսˣ, 2009; ťڰս

ˣ, 2011). чߎٖ ˣ(2010, 2011), чߎٖę ܓÌৠ (2010) ŔνČчߎٖęťҋܳ(2010)قۆॠيࢹ޳঒

ԓՁчࢬν؉ε ۋڌॠي ডজġНͿҙࢢ йԦНڌ߻

֬ॹ˞ںսॱॠٕڷǣϿ˃чࢬν؉ܛ(species)ۋő ϼʼݓ؍ڹٍĵ˞ۋٕɰ. ڍνǣ͆قԴࢹ޳঒ԓՁч ࢬν؉ق ʂॠي ܼ०মՙٍթъڿę 16S rRNAε ۋ ڌॠيܛқΪε֨ʪॢٍĵəۻИॢ֬܁ۋɰ. ࢹ޳

঒ԓՁчࢬν؉˞ڹŔݓًঞąقۋйԸ࢘(selection) ʼČ ۺڿ(adaptation)ۋ ٰՁʽ ێܛۆ ԦНۙڙۋɰ.

ġԓѕսۆজॡۺ࣢ՁڹŔݓًقԴԓ߻ʼəġԵġ Нۆ ݓĵজॡۺ ࣢Ձۋ ŔʂͿ ъٖʼČ(Lottermoser, 2007), ̚ॢۋġԓѕսقԴ֩ॠəࢹ޳঒ԓՁчࢬν

؉˞ڹŔݓًġԵġНͿҙࢢڌ߻ʽܼŚ՚ۋ٣ۆ

ʫՁقۋйǴՁ(tolerance)ۋ঍Ձʽìۋɰ(Das et al., 1998). ˰͆ԴġԵġНͿҙࢢڮڌܼŚ՚ۋ٣ںڌ߻

ॠČۙॠəąڍ, Ŕݓًġԓѕսǣࢹتقۺڿʽࢹ

޳чࢬν؉εۋڌॠϸڮڌŚ՚ۋ٣ںমęۺڷͿڌ

߻֨࢈սەɰ(Shi et al., 2006; Astudillo and Acevedo, 2008). Ŕ͠дͿ ڍνǣ͆قԴ ԓ߻ʼə ġԵġНͿҙ ࢢڮڌŚ՚ۋ٣ںйԦНںۋڌॠيڌ߻֨ࡈǴşڦ ३Դəওڹ١ّʽࢹتڷͿҙࢢܼŚ՚ۋ٣ں܃äॠ ş ڦ३Դə, Ŕ ঞąق ۺڿʽ ࢹ޳঒ԓՁчࢬν؉ۆ

܁ঝॢ ܛ(species) қΪÀ ज़څॠɰ.

ٍ҆ĵۆЀۺڹČՁʴġԓݓًۆࢹتقԴ, ٍজ ġԓۆġԓѕսقԴŔνČێ҆ԓՁ٣ߎսقԴ֩ॠ əࢹ޳঒ԓՁчࢬν؉ε޽ࠄॠٕɰ. ۋ˞ࢹ޳঒ԓՁ чࢬν؉ق ʂॠي ܼ०মՙٍթъڿę 16S rRNA ّ şԴَںۋڌॠيćࣀьԦॡۺڷͿڍ۾ܛۆܛқΪ ε őϼॠČۙॠٕɰ.

ࢹ޳чࢬν؉֨Β޽ࠄ ćʂѕتфՁۤ֬ॹۆ

ѕتؚܓՁ %/"қνфқԵѓѪ

ࢹ޳чࢬν؉֨Β޽ࠄ

ČՁࢹ޳чࢬν؉əąԜǫʪČՁķԘԓϸйΗν قՙۦॠəԘԓ܃ێġԓदġԵۺ࠘ۤۆࢹتقԴ޽

ࠄॠٕɰ(ۋॠČՁчࢬν؉, Goseong bacteria; G). ۋ

ࢹتڹʴġԵقۆ३֮ॠó١ّʼرۺÄԟڷͿѺ ԟʼرەؽɰ. ࢹت100 gεݒΪս100 mlقঔ०ॠČ

30қۋԜݕ࢖ॠČ1֨ÂۋԜѓ࠘ॠٕɰ. ࢹتԜˣ ս(pH=3.5) 10 mlε޽ࠄॠيՁۤ-ѕتؚ150 mlقۿ ܛॠČ२٣ѕتşقԴ32Ϳڮݓॠٕɰ. ٍজࢹ޳

чࢬν؉əÌڙʪԘߍ֨Àčϸॄčνٍজ܃2ġԓ

ġԓѕս(pH=2.96)قԴ޽ࠄॠٕČ(ۋॠٍজчࢬν؉, Yeonhwa bacteria; Y), ێ҆ࢹ޳чࢬν؉əێ҆१߶

цΘݓَьۻՙݓًۆԓՁ٣ߎս(pH 3.36)قԴ޽ࠄ ॠٕɰ(ۋॠێ҆чࢬν؉, Japanese bacteria; I). ġԓѕ սٮԓՁ٣ߎս10 mlεÁÁՁۤ-ѕتؚقۿܛॠČ

२٣ѕتşقԴ 32Ϳ ڮݓॠٕɰ.

ćʂѕتфՁۤ-֬ॹۆѕتؚܓՁ

чࢬν؉ۆ ćʂѕتڹ ɰڼę Ïۋ ИşٖتՁқę

ИşԓজقȃݓڙڷͿĵՁʽѕتؚ(ATCC)قԴսॱ ॠٕɰ. ИşٖتՁқڹ^Þ§¡_Ñß_Ï¬¨_Ñ 0.2 g/l, ¦ƅ¬¨_Ñ읆Ô¡_Ï¨ 0.5 g/l, ſƊ_Ï 0.25 g/l ф¤¡_Ï©¨_Ñ 3.0 g/lں3޲ݒΪս

1.0 νࢢ(liter)قڌ३֨ࡎɰ. Иşԓজقȃݓڙڹڙՙ

ডқϊ1.0 g/lęƃ¬¨_Ñ읆Ô¡_Ï¨ 5.0 mg/lں ИşՁқق

ߐÀॠٕɰ. ࢹ޳чࢬν؉˞ۆćʂѕتڹ30ێܳşͿ

սॱॠٕɰ. ČՁ, ٍজфێ҆чࢬν؉قʂॢćʂѕ تۙΒεTable 1ق܁νॠٕɰ. ۋ˞чࢬν؉˞قʂ

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Table 1. The data of periodic subcultures for the acidophilic indigenous bacteria in the growth-medium for 30 days at 32

Subculturing times(months)

Goseng bacteria (2010.2.)â Yeonhwa bacteria (2010.8.)â Japanese bacteria (2009.7.)â

pH Eh (mV) pH Eh (mV) pH Eh (mV)

0 - - - - 2.64 436

1 - - - - 2.54 445

2 - - - - 2.45 460

3 - - - - 2.41 468

4 - - - - 2.38 488

5 - - - 2.02 490

6 - - - - 1.96 496

7 3.52 383 - - 2.71 422

8 3.48 391 - - 2.54 430

9 3.45 385 - - 2.42 436

10 3.13 399 - - 2.4 441

11 3.74 481 - - 3.54 493

12 4.1 457 - - 4.06 476

13 3.4 481 3.52 436 3.39 495

14 2.3 478 2.76 469 2.42 481

15 2.52 415 2.5 474 2.24 489

16 2.34 437 2.09 474 2.14 431

17 2.14 430 2.11 408 3.01 360

18^b 2.1 441 2.08 432 2.86 363

19 2.08 451 2.06 451 2.74 369

20 2.06 458 2.01 455 2.54 371

21 2.01 468 1.99 462 2.5 378

22^c 1.99 482 1.95 468 2.48 381

23 2.01 480 1.94 469 2.49 382

a; The dates of the sample sites

b; The beginning date of DNA extraction experiment c; The beginning date for growth-experiment

ॢDNAқԵڹ8ѥݫćʂѕت֨Βεۋڌॠٕɰ. ̚

ॢۋ˞чࢬν؉˞قʂॢՁۤ-֬ॹڹ22ѥݫćʂѕ ت֨Βεۋڌॠٕɰ. Ձۤ-֬ॹڹćʂѕتڷͿۋڌ ʼؽʏ ѕتؚ(ATCC)ę Tuovinen and Kelly(1973)ۆ

ѕتؚقԴÁÁսॱॠٕɰ. Tuovinen and Kelly(1973) ۆ ѕتؚڹ K2HPO4 0.4 g/l, MgSO4·7H2O 0.4 g/l, (NH4)2SO4 0.4 g/lقFeSO4·7H2O 33.3 g/lںقȃݓڙڷ ͿԐڌॠٕɰ.

DNA қνѓѪ

ࢼઑઁଞߦऀഉ DNAଭ ंࠤ ѕتؚڷͿҙࢢgenomic DNA қν

чࢬν؉ ѕتؚں ڙ֮қνşε ۋڌॠي ࠞۻ֨ࢇ

঳ чࢬν؉ۆ genomic DNAεқνॠٕɰ. чࢬν؉

ࠞۻНق SET(20% Sucrose, 50mM EDTA, 50 mM Tris-HCl [pH 7.6]) 1.8 ຸٮlysozyme solution(5 mg/

ຸ in TE) 62 ືںߐÀॠČ, 37°CقԴ30қÂrotating ovenقԴъڿ֨ࢇ঳25% SDS 16 ືںߐÀॠيʴ ێॢ٣ʪقԴ30қÂrotating ovenقԴʌъڿ֨ࡎɰ.

ъڿНقproteinase K(20 mg/ຸ in D.W) 50 ືںȏČ

55°C قԴ2֨Ârotating ovenقԴʌъڿ֨ࡎɰ. ۋ

ъڿНق ʴ͟ۆphenol : chloroform : isoamylalcohol (25:24:1) ڌؚںߐÀॢ঳ڙ֮қνॠيԜˣؚںʴ

͟ۆchloroform : isoamylalcohol (24:1) ڌؚۋ˞رە ə ԞͿڏ tubeق ٦Ąɰ. Ԝˣؚق 1/10 ҙक़ۆ 8M قԴovernight ֨ࢇ঳ڙ֮қνॠيԜˣؚں܃äॠ

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Č, 70% ethanolͿwashing ॠٕɰ. æܓʽDNAںTE buffer(10mM Tris-HCl, 1mM EDTA)ق ڌ३ ֨ࢇ ঳

֬ॹق Ԑڌॣ˺ūݓ -20قԴ Ǽʴ ҃ěॠٕɰ.

DNA տʪ ࠑ܁

Genomic DNAۆshearing фϼʪεঝۍॠşڦ३

1% agaroseقԴۻşٖʴॠيঝۍॠČ, DNA տսՁ ڹ spectrophometer(Ultrospec 2100 pro, Amersham Biosciences)Ϳ ܁͟ॠٕɰ.

16S rRNA କୢୀ ஹඒଡ ଍෉ PCR ࢱଦ

16S rRNA ڮۻۙεݒफॠşڦ३ՃŒق࣢ۋۺڷ εԐڌॠٕɰ(Lane, 1991). PCR ݒफںڦॢъڿؚ

ĵՁڹ ܳ঍ DNA 10 ng, 200 uM dNTP, 10 mM Tris-HCl(pH 9.0), 40 mM KCl, 0.15 mM MgCl2, 3mM MgSO4,20G BSA, 1U TaqpolymeraseŔνČ forword ٮreverse primer ÁÁ0.5 uMͿߪvolumeں50 ulͿ

ॠٕɰ.

PCRڹGenAmp^TMPCR System9700(AppliedBiosystem) ں ۋڌॠٕڷ϶, PCR ъڿܓæڹpre-denaturation ę

܁ڷͿ94قԴ3қÂսॱॠٕČ, denature(94, 30 ߣ), annealing(50, 30ߣ), elongation(72, 5қ) ъڿ ںߪ30ধъ҄ॠČpost-elongation (72, 10қ)ںս ॱॠٕɰ. PCR ԓНڹ1% agarose gelقۻşٖʴॢ

঳ݒफԓНںঝۍॠٕɰ.

Cloning ࢫ DGGE ंজଡ ധ෉ ࢠ঍ࢄ ֞ு ंজ

Cloning

ݒफʽ double-strand DNAə Wizard PCR Preps DNA Purification System(Promega, USA)ں ۋڌॠي

܁܃ॠٕɰ. ܁܃ʽPCR ԓНںɳێّşԴَںÀݕ

cloneڷͿқνॠşڦ३pGEM-T easy vector system (Promega, USA)ںۋڌॠٕɰ. PCR ݒफԓНęvector ۆȬʪε3:1 ҼڱͿߐÀॠČ2× ligation buffer, T4 DNA ligaseεঔ०фъڿ঳E. col DH5쩀Gcompetent cellق঍ݗۻঞ֨ࡈX-Gal (5-bromo-4-chloro-3-indoly- 쩁-D-galactopyranoside; Promega, USA), IPTG (isopropyl- 쩁-D-thiogalacto-pyranoside; Promega, USA), ampicillin (50 mg/ຸ)ۋ प॥ʽ LB agar ѕݓقԴ blue/white colony ԸѻѓѪقۆ३঍ݗۻঞʽwhite cloneںԸ ѻॠٕɰ. ےۆͿԸ܁ॢwhite colonyεdirect reamplified PCR ѓѪڷͿݒफॠٕɰ. ۋ˺Ԑڌॢprimerəvector

1999), PCR ܓæڹ16S rRNA ڮۻۙٮʴێॢѓѪ ڷͿ ॠٕɰ.

DGGE(denaturing gradient gel electrophoresis) DGGE PCRə 1޲ PCR ԓНں ܳ঍(template)ڷͿ

Ԑڌॠي, 2޲PCR ॠٕɰ. 2޲PCRڹ16S rRNA ڮۻ

ۙۆV3 ҙқق३ɾॠəprimerقGC-clampεҙ޳ॢ

341F (5'-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG-3')ٮ 518 (5'-ATT ACC GCG GCK GCT G-3')ε܃ۚॠيԐڌॠٕɰ. PCR ъڿؚڹ1޲PCR ٮʴێॠ϶, PCR ܓæڹ94G5қÂߣşDNA ѺՁ

঳, 94G30ߣ, 65ҙࢢ56ūݓ2cycle υɰ1؂

٣ʪεǴͲÀ϶30ߣ, 72G30 ߣ঳ŔνČ94G30 ߣ, 55G30ߣ, 72G30ߣ؂10ধъ҄ॠ϶υݓφڷͿ

72قԴ 7қÂ ъڿॠٕɰ.

DGGE ۻşٖʴ սॱڹ D-code system(Bio-Rad)ں

ۋڌॠٕɰ. ۋ˺Ԑڌॢpolyacrylamide gel Ȭʪə8%

ۋ϶gel ࡾşə20 × 16 cm(W × H)Ϳ܃ۚॠٕɰ. Gel ڹ˃ƍ1 mm ͿॠČ, 40% polyacrylamide bis-solution 29:1(3.3% C) 40% ٮ60% ԐۋͿȬʪĵѕÀսݔڷ Ϳێ܁ॠó঍Ձॠʪ΀܃ۚॠٕɰ. ۋ˺ԐڌॢѺՁ

܃Ϳə7 M ureaٮ40%(w/v) formamideۋɰ. Running bufferͿəTAE buffer(20mM Tris, 10mM acetic acid, 0.5mM EDTA, pH 8.0)ۋ϶, loading dye(0.05% bromophenol blue, 0.05% xylene cyanol, 70% glycerol)ٮPCR ԓН ں 1:1Ϳ ঔ०ॢ ঳ 60 VͿ 12֨Â ۻşٖʴ ॠٕɰ.

ّşԴَқԵںڦ३ݒफʽї˚ε١ͲϹŒʽ2޲

ݒΪս30 ືقॠΘʴ؋Ǽۤ҃ěॠٕɰ. 16S rRNA ڮۻۙۆ V3 ҙқۆ GC-Clampε ܃äॠş ڦ३Դ

GC-ClampÀ ػə primerε ۋڌॠي ۦ ݒफॠٕɰ.

ݒफʽ ԓНں ܁܃ ę܁ں äࠚ ঳ pGEM-T easy vectorقԙۓॠيEscherichia coli DH5쩀ق঍ݗۻঞ ॠٕɰ. ঍ݗۻঞʽࢁ΁ںԸѻॠيّşԴَқԵॠ

ٕɰ.

ۻşٖʴԜPCR ԓНۆ֨ΒÂɳठۆڦ࠘қԵڹ

Gelcompar II program(Applied Maths, Belgium)ںۋ ڌॠٕڷ϶, similarity matriceۆcluster қԵڹUPGMA ε ۋڌॠٕɰ.

16S rRNA କୢୀଭ સ׆ছવ ծധंজ

ّşԴَÂۆ ڮԐʪε ঝۍॠş ڦॠي EzTaxon

(5)

Fig. 1. SEM image of indigenous acidophilic bacteria, a;

Goseong bacteria, b; Yeonhwa bacteria, c; Japanese bacteria. The scale bar is 5.0 G (a, b and c) in length.

Fig. 2. The photograph of electrophoresis for the genomic DNA from the separated samples(a) and for the amplifi- cation of 16S rRNA genes(b). 1; Goseong bacteria, 2;

Yeonhwa bacteria, 3; Japanese bacteria.

server(http://www.eztaxon.org/)ںۋڌॠٕɰ. Cloneۆ

Ā܁ʽ ّşԴَę databaseق صرݕ ّşԴَڹ

(http://plaza.snu.ac. kr/~jchun/phydit/)ںۋڌॠيClustal X multiple alignmentͿ ܁͵ॠي ćࣀս(phylogenetic tree) ܃ۚق ۋڌॠٕɰ. ّşԴَÂ ڮۻۺ äνə

Jukes & Cantor distance model(Jukes & Cantor, 1969) ͿԐڌॠي߸܁ॠٕڷ϶, Neighbor-joining methodͿ

ćࣀսεۚՁॠٕɰ. Neighbor-joining dataۆbootstrap қԵ֨1,000ধۆresamplingںۺڌॠيtree topology ۆथÀşܵڷͿԐڌॠČ(Felsenstein, 1985) ۋεࢹ ʂͿ ćࣀսεۚՁॠٕɰ(Maidak et al., 1994).

DNA қԵѓѪ ਓ෠ էր

Ճप ѕتؚ Ǵ Œܳ ঝۍ

Ճपѕتؚںڙ֮қνॠيȬ߹ʽѕتؚ10 ືε

slide glassقʪϊॠيġॡইйąںࣀ३ ঝۍॠٕɰ.

ČՁ, ٍজфێ҆чࢬν؉ѕتؚǴՃपۆ঍ࢗə

ݥڹφʂ঍(short rod) ওڹφʂ঍(rod) ՃŒ˞ۋSEM قԴ ě޶ ʼؽɰ(Fig. 1).

ՃŒDNAۆ қν

G Genomic DNA ߸߻ ф 16S rRNA ڮۻۙݒफ ѕتؚ50mlεȬ߹ॠيgenomic DNAε߸߻ॢĀ ę, ČՁ, ٍজŔνČێ҆чࢬν؉֨ΒۆDNA Ȭʪ əÁÁ1.5, 1.1 ŔνČ1.2 Ϳঝۍʼؽɰ(Fig. 2a).

߸߻ʽgenomic DNAεܳ঍ڷͿ16S rRNA ڮۻۙε

PCR ॠي3Ò֨ΒϿ˃أ1.5kbق३ɾॠəݒफԓ Нں ঝۍॠٕɰ(Fig. 2b).

Cloning фDGGE қԵں ࣀॢ йԦН ķݚ қԵ

G 16S rRNA cloning ںۋڌॢ ՃŒ˞ۆćࣀқԵ

ࢹ޳঒ԓՁчࢬν؉ǴՃŒۆķݚںқԵॠşڦ ३ ՃŒق ࣢ۋۺۍ 27F forwardٮ 1492R reverse primerεԐڌॠيأ1,500bp ࡾşق३ɾॠəPCR ԓ Нںঝۍॠٕɰ. ݒफʽ16S rRNA ڮۻۙεpGEM-T vectorقԙۓॢ঳E. coli DH5쩀Gcompetent cellق঍ݗ

ʪۓॠيصرݕcloneں1Ò֨Βɾ10Òࢁ΁ںИۚ

ڦۺڷͿ Ըѻॠي pGETf forwardٮ pGETr reverse primerͿۦݒफॠيԙۓʽ16S rRNA ڮۻۙۆࡾş εۻşٖʴĀęϿ˃1.5 kbۆࡾşےںঝۍॠٕɰ.

(6)

Fig. 3. The photograph of electrophoresis for the cones.

G1-G10; Goseong bacteria, Y1-Y10; Yeonhwa bacteria, I1-I10; Japanese bacteria.

Fig. 4. Alignment of the 16S rRNA sequences isolated from Goseong bacteria with those of Acidithiobacillus ferrooxidans (ATCC 23270 type strain).

Fig. 5. Phylogenetic tree for strains of acidophilic indigenous bacteria. (G1-G10); Goseong bacteria, (Y1-Y10); Yeonhwa bacteria; (I1-I10); Japanese bacteria.

3ÒۆԢ॔قԴصرݕߪ30ÒcloneۋۻşٖʴقԴ

16S rRNA ّşԴَۋқԵʼؽɰ(Fig. 3). 30Òۆclone

ܼ, ČՁ чࢬν؉ G1ں ʂशۺڷͿ Ը࢘ॠي ATCC 23270 शܵŒܳۍAcidithiobacillus ferrooxidansۆّş Դَںpairwise Ҽİ܁͵(alignment sequence) êԐε

Fig. 4ق܁νॠٕɰ. қԵʽّşԴَڹEzTaxon server εۋڌॠيʴ܁ॠٕڷ϶ŔĀęق˰δڮԐՁĀę ə Table 2ق ǣࢍǴؽɰ.

ČՁчࢬν؉قܕۦॠə10Òࢁ΁˞ڹϿ˃Acidithio- bacillus ferrooxidansशܵŒܳ(ATCC 23270)ٮ96.99- 97.79%ۆڮԐʪݓսεٕ҃ɰ. ̚ॢԴͿÂۆّşԴ

َڮԐʪݓսə98.36-99.93% ѩڦͿϔڍȭóқԵ ʼؽɰ. ˰͆Դ10Òࢁ΁Ͽ˃əɳێܛڷͿԐΒʼ϶, ݕজۺڮٍěćεǣࢍǴəćࣀʪقԴʪۋεঝۍॣ

ս ەɰ(Fig. 5).

ٍজ чࢬν؉ق ܕۦॠə 10Ò ࢁ΁˞ڹ Ͽ˃

Acidithiobacillus ferrooxidansशܵŒܳٮ97.59-97.90%

ۆڮԐʪݓսεٕ҃ɰ. ̚ॢԴͿÂۆّşԴَڮԐ ʪəݓսə99.57-100% ѩڦͿϔڍȭóқԵʼؽɰ.

˰͆Դ10Òࢁ΁Ͽ˃əɳێܛڷͿԐΒʼ϶, ݕজۺ

ڮٍěćεǣࢍǴəćࣀʪقԴʪۋεঝۍॣսە

(7)

Table 2. The similarity of 16S rRNA sequence for cloning from the indigenous acidophilic bacteria Sample

Name

Clone

No. Nearest type strain Strain No.

(ATCC) NCBI No. Pairwise

Similarity Diff/Total nt*

G-bacteria G1 Acidithiobacillus ferrooxidans 23270(T) CP001219 97.32 40/1494

G2 A. ferrooxidans 23270(T) CP001219 96.99 45/1494

Y-bacteria Y1 A. ferrooxidans 3270(T) CP001219 97.59 36/1493

Y2 A. ferrooxidans 23270(T) CP001219 97.66 35/1494

I-bacteria I1 A. ferrooxidans 23270(T) CP001219 97.26 41/1494

I2 A. ferrooxidans 23270(T) CP001219 97.59 36/1494

I4 Acidithiobacillus caldus 51756(T) ACVD01000050 93.84 91/1478

ATCC : American Type Culture Collection

NCBI : National Center for Biotechnology Information Diff/Total nt* ; difference/total nucleotide

G-bacteria; Goseong, Y-bacteria; Yeonhwa, I-bacteria; Japanese bacteria.

ɰ(Fig. 5).

ێ҆чࢬν؉قܕۦॠə8Òࢁ΁˞ڹϿ˃Acidithio- bacillus ferrooxidans शܵŒܳٮ97.26-97.97%ۆڮԐ ʪݓսεٕ҃ɰ. ̚ॢԴͿÂۆّşԴَڮԐʪəݓ սə98.86-99.93% ѩڦͿϔڍȭóқԵʼؽɰ. ˰͆

Դ8Òࢁ΁Ͽ˃əɳێܛڷͿԐΒʼ϶, ݕজۺڮٍ

ěćε ǣࢍǴə ćࣀʪقԴʪ ۋε ঝۍ ॣ ս ەɰ

(Fig. 5). Ŕ͠ǣ˃Òۆࢁ΁ܼI4 ࢁ΁ڹAcidithiobacillus caldus शܵŒܳٮ 93.84%ۆ ڮԐʪ ݓսε ٕ҃ɰ.

Stackebrandt et al.(1994)ۋ܃֨ॢ16S rRNA ڮۻۙ

ڮԐʪݓս97%ۆşܵڷͿܛĵқѪق˰βϸԞͿ ڏŒܳͿқԵʾսەɰ. ̚ॢI5 ࢁ΁ڹAcidithio- bacillus ferrooxidansशܵŒܳٮÀۤÀŲóқԵʼؽ ݓχ, 92.82%ͿϔڍǰڹڮԐʪݓսε҃ۋ϶, ćࣀ ʪقԴʪAcidithiobacillus ՚ęɰδŔΝڷͿ঍Ձʼ رۋ ŒܳʪԞͿڏ ՚ওڹ ܛۋʾ ս ەɰ.

G DGGE (denaturing gradient gel electrophoresis)ε

ۋڌॢ ՃŒķݚқԵ

DGGE ѓѪںࣀ३16S rRNA ڮۻۙۆV3 ҙқق

(8)

Table 3. The distribution of dominants for acidophilic bacteria using denaturing gradient gel electrophoresis (DGGE) Band

No. Sources Nearest type strain Accession

No. Similarity Diff/Total nt 1 G-bacteria Thermoflavimicrobium dichotomicum KCTC 3667(T) AF138733 93.6 11/172 2 I-bacteria Amphibacillus tropicus Z-7792(T) AF418602 65.4 37/107 3 G-bacteria Acidithiobacillus ferrooxidansATCC 23270(T) CP001219 98.5 3/194 4 Y-bacteria Acidithiobacillus ferrooxidansATCC 23270(T) CP001219 98.5 3/194 5 I-bacteria Acidithiobacillus ferrooxidansATCC 23270(T) CP001219 98.5 3/194 6 G-bacteria Luteibacter anthropi CCUG 25036(T) FM212561 91.8 16/194 7 I-bacteria Luteibacter anthropi CCUG 25036(T) FM212561 91.8 16/194 8 G-bacteria Deinococcus aquatilis CCUG 53370(T) AM940971 98.3 3/181 9 Y-bacteria Acidiphilium organovorum ATCC 43141(T) D30775 99.4 1/168

10 I-bacteria Iamia majanohamensisF12(T) AB360448 93.5 11/169

G-bacteria; Goseng bacteria, Y-bacteria: Yeonhwa bacteria, I-bacteria; Japanese bacteria Fig. 6. The photograph of denaturing gradient gel electro-

phoresis (DGGE) for acidophilic indigenous bacteria of 16S rRNA. A; Goseong bacteria, B; Yeonhwa bacteria;

C; Japanese bacteria, M; marker. Arrows indicate the cloning and analyzed sequence bands.

ʂॢःࢤڹFig. 6ęÏɰ. ߪ10Òۆband (ČՁчࢬ ν؉, 4 band; ٍজчࢬν؉, 2 band; ێ҆чࢬν؉, 4

band)εԸѻॠٕɰ(Fig. 6). Ըѻşܵڹbandۆݕॠş ٮÁsampleںҼİॠيԴͿɰδڦ࠘قǣࢍǦband εelutionॠٕɰ. Áї˚ۆّşԴَқԵ঳EzTaxon serverε ࣀ३ қԵॢ Āęə Table 3ق ǣࢍǴؽɰ.

DGGE ї˚ԜقԴݒफԓНۋψںս΀ŔঞąǴ࣢

܁ŒܳÀڍ۾ॢɰČॣսەɰ. ČՁчࢬν؉, ٍজ

чࢬν؉, ێ҆чࢬν؉֨ΒϿ˃قԴÀۤݕॠóݒ फڦ࠘ε҃ۋəҙқڹÁÁ3, 4, 5ѥї˚ڦٕ࠘ɰ.

ۋՃї˚Ͽ˃əʴێॢڦ࠘ͿࣺϼʼؽČ, ّşԴَ

қԵĀęAcidithiobacillus ferrroxidans शܵŒܳٮÀ

ۤÀūڏڮԐʪݓսͿǣࢍǮɰ. ̚ॢ3Òۆї˚ə

ّşԴَۋ100% ێ࠘ॠٕɰ. ۋĀęədirect cloning ڷͿ қԵقԴ 2Òۆ ࢁ΁ں ܃ٽॢ 28Òۆ ࢁ΁ۋ

Acidithiobacillus ferrroxidansٮÀۤÀŲóқԵʽì ę ێ࠘ॢɰ.

ČՁчࢬν؉֨ΒقԴə1, 8=6, 3 ѥտڷͿї˚ۆ

ȬʪÀȭڹìںঝۍॣսەؽɰ. ّşԴَқԵĀę

ÁÁAcidithiobacillus ferrooxidans(98.5%), Luteibacter- anthropi(91.8%), Deinococcusaquatilis(98.3%), Thermo- flavimicrobiumdichotomicum(93.6%)ͿÀۤÀūڏڮ Ԑʪ ݓսε ٕ҃ɰ.

ٍজчࢬν؉֨ΒقԴə4, 9ѥۋȬʪÀȭڹìں

ঝۍॠٕČ, ّşԴَқԵĀęÁÁAcidithiobacillus ferrooxidans(98.5%)ٮLuteibacter anthropi(91.8%) Œ

ܳٮ Àۤ Àūڏ ڮԐʪ ݓսε ٕ҃ɰ.

ێ҆чࢬν؉֨ΒقԴə5, 10, 2, 7 ѥտڷͿȭڹ

Ȭʪεٕ҃Č, ّşԴَқԵĀęÁÁAcidithiobacillus ferrooxidans(98.5%), Iamiamajanohamensis(93.5%),

(9)

Fig. 7. The similarity analysis(UPGMA) of denaturing gradient gel electrophoresis(DGGE) for acidophilic indigenous bacteria of 16S rRNA sequence. G-bacteria; Goseong bacteria, I-bacteria; Japanese bacteria, Y-bacteria; Yeonhwa bacteria, C; M1 and M2; marker.

Amphibacillustropicus(65.4%), Luteibacteranthropi(91.8%) ŒܳٮÀۤÀūڏڮԐʪݓսεٕ҃ɰ. ࣢০2ѥї

˚À ڮԐʪ ݓսÀ ǰó ǣࢍǦ ìڹ ݒफ ࡾşÀ

107bp(ࣀԜ160-200 bp)ڷͿPCR ݒफ֨࣢܁ҙڦÀ

deletion ওڹchimeraÀ঍ՁʽìڷͿࣺɳʽɰ. ˰͆

Դ ۋ ї˚ə ܃ٽÀ ʼرآ ॣ ìڷͿ ԐΒʽɰ.

3ÒۆԢ॔ۆDGGE ї˚ःࢤÂԜ঒ٍěěćε

ࣷ؊ॠČۙUPGMA ѓѪڷͿқԵॠٕɰ(Fig. 7). қԵ

ĀęČՁчࢬν؉ٮێ҆чࢬν؉Àٍজчࢬν؉ق

Ҽ३ Ԝ঒ ڮԐʪÀ ȭڼں ঝۍ ॣ ս ەؽɰ.

ڍ۾ ՃŒۆ ࣢Ձ

ČՁчࢬν؉, ٍজчࢬν؉фێ҆чࢬν؉֨Β قܕۦॠəՃŒϿ˃əAcidithiobacillus ՚(genus)ق

՚ॠəܛ(species)ڷͿқԵʼؽɰ. Acidithiobacillus՚

ڹқΪॡۺڦ࠘əProteobacteria Л(phylum), Gamma- proteobacteria Ì(class), Acidithiobacillales Ѐ(order), Acidithiobacillaceae ę(family)ق ՚ॠ϶, Acidithio- bacillaceae ęقəڮێॠóAcidithiobacillus ՚χܕ ۦॢɰ. Acidithiobacillus՚ڹ Kellyٮ Wood(2000)ق

ۆ३ Thiobacillus ՚ۆ Thiobacillus thiooxidans, Thiobacillus ferroxidans ф Thiobacillus caldus ܛں

ইۦۆAcidithiobacillus ՚ڷͿۦқΪॠٕɰ. Acidithio- bacillus՚ۆशܵŒܳəAcidithiobacillus thiooxidans ۋ϶, ইۦ5ܛۋܕۦॢɰ(http://www.bacterio.cict.fr/a/

acidithiobacillus.html).

Ձۤ֬ॹ

ČՁ, ٍজ ф ێ҆ чࢬν؉˞ۆ Ձۤ-֬ॹ(growth- experiment)ں ѕتؚں ۋڌॠي սॱॠٕɰ. 22Òښ

ćʂѕتʽ(Table 1) ۋ˞чࢬν؉˞ۋप॥ʽѕتؚ

10 mlںÁÁćʂѕتقԐڌॠٕʏѕتؚ(ATCC)ę

Tuovinen and Kelly(1973)ۆ ѕتؚق ۿܛॠČ 32

Ϳڮݓॠٕɰ. ČՁфٍজчࢬν؉εՁۤ-֬ॹقԴ

pH ÇՙٮEh ݒÀεҼİॢĀęۋ˞чࢬν؉˞ڹ

Ͽ˃ ćʂѕتۆ ѕتؚ(ATCC)قԴ ҃ɰ Tuovinen and Kelly(1973)ۆѕتؚقԴঽ؃۞ՁۤॠəìڷͿ

ǣࢍǮɰ(Fig. 8, Fig. 9, Fig. 10). Tuovinen and Kelly (1973)ۆѕتؚقԴpH ѺজəČՁ҃ɰٍজчࢬν

؉قԴʌǰóŔνČEh ѺজəʌȭóǣࢍǮɰ. ێ

҆чࢬν؉ə32, 42G ф52قԴÁÁՁۤ-֬ॹ ںսॱॠيpH фEh ѺজεҼİॠٕɰ(Fig. 10). ً

֨ێ҆чࢬν؉˞ۆՁۤʪćʂѕتۆѕتؚقԴ҃

ɰTuovinen and Kelly(1973)ۆѕتؚقԴʌǰڹpH Ѻজεٕ҃ČEhəʌȭóǣࢍǮɰ. ࣢০Tuovinen and Kelly(1973)ۆѕتؚܼ32ۆѕتؚۋÀۤǰ ڹ pHٮ Àۤ ȭڹ Eh Éںٕ҃ɰ.

ࢹۆ

ܼ०মՙٍթъڿę16S rRNA ّşԴَқԵşցڹ

ԓՁġԓѕսقԴ֩ॠəчࢬν؉قʂॠيݔۿۺڷ Ϳܛ(species) ĵқںॣսەəÌͳॢşցۋɰ. ࣢০

ۋşց˞ڹঞą֨ΒقԴԐۻقտսѕتںä࠘ş؍

ČйԦН˞ۆܛ(species)ںÇ܁ॣսەɰ. ߌڼڷͿ

ঞą֨ΒقԴ 5S rRNA ّşԴَں ۋڌॠي Stahl et al.(1984)ۋ ३۹َսĵق Դ֩ॠə ডԓজчࢬν؉ٮ

ۋ˞ėԦԦН˞ںÇ܁ॠٕČ, ً֨Stahl et al.(1985) ڹ 5S rRNA ّşԴَқԵڷͿ Yellowstoneۆ ٣ߎս قə3ܛΪۆڍ۾ܛйԦН, ݌ডںԓজॠəČՃŒ (archaebacteria)Ϊ, Thermus spp.ٮڮٍěćقەə2 ܛΪۆйԦНߕ˞ںőϼॢцەɰ. 5S rRNA ّşԴ

َқԵڹLane et al.(1985)قۆॠيʴġԓڌ߻սق ʪۺڌॠيThiobacillus neapolitanus, T. ferrooxidans, T. thiooxidans, T. intermedius, T. perometabolis, T. thioparus, T. versutus, T. novellus, T. acidophilus, Thiomicrospira pelophila, Acidiphilium crptumˣۋܕۦॠəìںő ϼॠٕɰ. ŔνČBond et al.(2000)ڹ16S rRNA ّşԴ

َқԵں࠭νपɦ؉ۆRichmond mineۆԓՁġԓѕ սقۺڌॠيLeptospirillum sp.ęSulfobacillus spp.ں

Ç܁ॢцەڷ϶, Lopez-Archilla et al.(2001)ڹ֟गۍ ۆTinto ԓՁġԓѕսقProteobacteraۆ쩂GgroupۍT.

ferrooxidans, T. thiooxidansٮ쩀GgroupۍAcidiphilium sp.

L.eptospirillum ferrooxidansŔνČGram positive чࢬ ν؉ۍ Bacillus megaterium, B. subtilis, B. amylolique- faciens, B. stearothermophius, B, cerus ˣںÇ܁ॢц

(10)

Fig. 8. The variation of pH and Eh in growth-medium with Goseong bacteria at 32. ; Tuovinen and Kelly(1973) medium, ; ATCC medium.

Fig. 9. The variation of pH and Eh in growth-medium with Yeonhwa bacteria at 32. ; Tuovinen and Kelly(1973) medium, ; ATCC medium

ەɰ. ̚ॢYin et al.(2008)ڹܼĶʴġԓۆġԓѕս قԴ Proteobacteria, Acidobacteria, Actinobacteria, Nitrospira, Firmicute, Chlorella ˣں Ç܁ॠٕɰ.

ČՁ, ٍজфێ҆чࢬν؉֨ΒقܕۦॠəՃŒڹ

Ͽ˃ Acidithiobacillus ՚(genus)ق ՚ॠə ܛ(species) ڷͿқԵʼؽɰ. ۋٮÏۋԴ֩ঞąۋɰδݓًقԴ

޽ࠄʽчࢬν؉ےقʪҝĵॠČϿ˃Acidithiobacillus

՚(genus)ۋڍ۾ܛڷͿǣࢍǦۋڮəড(¬^ƍ)ęߏՁқ (ƃ^{Ï â})ۋॄҙॢѕتؚقԴćʂѕتں١͒ʴ؋ݓ՚

ʼر ٵş ˺ЛڷͿ ԐΒʽɰ. ֬ॹ֬قԴ чࢬν؉ε

ѕتॢɰəۙߕəۋйٍۙঞąۆܓæں܃ʂͿъٖ

ॠݓ Їॠə ìۋɰ(Head et al., 1998; Amann et al., 1995). ܼ०মՙٍթъڿ şցę 16S rRNA ّşԴَ

қԵۋѕتॠşūɰͿڏওڹѕتॣսػəчࢬν

؉قʂॠيÇ܁(identification) ॣսەəÌͳॢʪĵ Ϳۋڌʼؽݓχ, Ԑ֬ڹي͠ÀݓࠑϸقԴ١३ǣठ þۋьԦॣսەɰ. чࢬν؉ۆnucleic acidεঞą֨

ΒقԴ߸߻ॠəߣşɳćقԴ֮Áॢठþ(bias)ۋڮ ьʾսەɰ. ঞą֨Βقप॥ʼرەəчࢬν؉˞ۆ

nucleic acidε߸߻ॠəɳćقԴНνۺওڹজॡۺ

ѓѪںۺڌॢɰ. Ŕ͠ǣۋѓѪڷͿঞą֨Βقप॥

ʼرەəϿ˜чࢬν؉ۆnucleic acidÀŒˣॠóڌ ν(lysis)ʼݓ؍əɰəìۋɰ. ٚε˞ϸ, Gram positive чࢬν؉ ҃ɰ Gram negative чࢬν؉À ʌ ֖ó

nucleic acidÀڌνʼČ, ݔąۋۚڹчࢬν؉҃ɰݔ ąۋԜʂۺڷͿࢀчࢬν؉˞ۋʌ֖ónucleic acid Àڌνʽɰ. ۋͩó࣢܁чࢬν؉ۆnucleic acidÀԸ

࢘ۺڷͿڌνʽ߸߻НںPCRͿݒफॠϸ֮Áॢ١ ΪÀьԦॢɰ(Amann et al., 1995; Head et al., 1998).

ঞą֨Βۆ١ّНݗۋ॥ƍ߸߻ʼرPCR ݒफںѓ ३ॠəąڍ, PCR ݒफۆठ޲, PCR ݒफقԴࢅϭ͆

(chimera)ٮÏڹНݗۋԦՁˣقۆॠي֮Áॢ١Ϊ ۆ ॥܁ق Ӈݗ սەɰ(Wintzingerode et al., 1997).

ČՁ, ٍজфێ҆чࢬν؉قʂॢՁۤںҼİॢĀ ęATCC ѕتؚقԴ҃ɰTuovinen and Kelly(1973) ۆѕتؚقԴʌǰڹpHٮʌȭڹEh Ѻজεٕ҃ɰ (Fig. 8, Fig. 9, Fig. 10). ČՁ҃ɰٍজчࢬν؉Àʌ

ǰڹpHٮʌȭڹEh Ѻজεٕ҃ɰ. ێ҆чࢬν؉ə

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Fig. 10. The variation of pH and Eh in growth-medium with Japanese bacteria at 32, 42G and 52. ; Tuovinen and Kelly(1973) medium, ; ATCC medium.

32GѕتؚقԴÀۤǰڹpHٮÀۤȭڹEh Ѻজε

ٕ҃ɰ. ATCC ѕتقԴ҃ɰTuovinen and Kelly(1973) ۆѕتؚۋʌǰڹpHٮʌȭڹEh Ѻজε҃ۋə

ڙۍڹ16S rRNA ّşԴَқԵقԴőϼॠٕˢۋۋ˞

঒ԓՁчࢬν؉˞ۆڍ۾ܛۋAcithiobacillus ferrooxidans ق३ɾʼş˺ЛۍìڷͿԐΒʽɰ. A. ferrooxidansə

ferrous ironęڙՙড(¬^×) ̚əঞڙ঍ࢗۆডںԓজ ॠəɠͳںÍČەəчࢬν؉ۋɰ(Olson, 1991; Das and Mishra, 1996). Ձۤ-֬ॹقԴ, ڙՙডۋप॥ʽć ʂѕتڌѕتؚ҃ɰTuovinen and Kelly(1973) ѕت

ؚقԴʌǰڹpH ѺজٮʌȭڹEh ѺজÀǣࢍǣə

ڙۍڹڍ۾ܛۍA. ferrooxidansÀڙՙড҃ɰferrous ironںʌ۞ԓজ֨ࢅəɠͳںÍČەş˺ЛڷͿԐ Βʽɰ.

څأфĀ΁

ČՁ, ٍজ ф ێ҆ ࢹ޳঒ԓՁчࢬν؉ε Ç܁ॠş

ڦॠي ܼ०মՙٍթъڿę 16S rRNA ّşԴَ қԵ ں սॱॠٕɰ. 16S rRNA ڮۻۙε ݒफॠş ڦॠي

27F forward ф1492R reverse primerεԐڌॠٕČ, أ

1,500bp ࡾşق३ɾॠəPCR ԓНںঝۍॠٕɰ. 3Ò

ݓًࢹ޳чࢬν؉֨ΒقԴصرݕ16S rRNA ّşԴ

َں शܵŒܳۍ Acidithiobacillus ferrooxidans(ATCC 23270)ٮpairwise Ҽİ܁͵(alignment sequence) ѓѪ ڷͿڮԐʪεҼİॠٕɰ. शܵŒܳٮڮԐʪεҼİॢ

Āę, ČՁ ф ٍজ чࢬν؉ə ڮԐʪÀ ÁÁ 96.99- 97.79%ٮ 97.59-97.90%Ϳ ǣࢍǮɰ. ێ҆ чࢬν؉ə

शܵŒܳٮۆ ڮԐʪÀ 97.26-97.97%Ϳ ǣࢍǮČ A.

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caldus शܵŒܳٮə93.84%ۆڮԐʪεٕ҃ɰ. ѺՁ ĵѕõۻşٖʴڷͿ3Òݓًчࢬν؉˞قʂॢķݚ қԵںսॱॢĀęČՁ, ٍজфێ҆чࢬν؉əϿ˃

əAcidithiobacillus ferrooxidansٮ98.5%ۆڮԐʪε

ٕ҃ɰ. Ҽ΀ČՁ, ٍজфێ҆ࢹ޳঒ԓՁчࢬν؉˞

ۋAcidithiobacillus ferrooxidansٮϔڍȭڹڮԐՁں

ٕ҃ݓχտսѕتںࣀ३ۋεঝۍ३آॣìڷͿԐ Βʽɰ. ۋ˞чࢬν؉˞ںۋڌॠيՁۤ-֬ॹںսॱ

ॢ Āę ڙՙ ডں प॥ॠə ѕتؚقԴ ҃ɰ ferrous ironںप॥ʽѕتؚقԴʌӇβópHÀÇՙॠٕɰ.

˰͆Դferrous ironۋप॥ʽডʴԵęڮҼߏԵęÏڹ

ডজġНقڍ۾ܛۍ҆A. ferrooxidansεۿܛ֨ࡈй ԦНڌ߻ںսॱॢɰϸমęۺڷͿڮڌŚ՚ۋ٣ۋڌ

߻ʾ ìڷͿ ԦÁॢɰ.

ԐԐ

҆ ٍĵə 2011țʪ ܓԸʂॡİ ॡցٍĵҼ ݓڙں

ы؉ٍĵʼؽڷ϶, ٍĵҼεݓڙ३ܵܓԸʂॡİقÇ Ԑॢɰ.

޷ČЛॶ

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૳ౢ,”෉֝஺֜ਏਆഗվ෈ฎ஺,୪46֫2෹, pp. 239-251.

׌ଗ৤, ୢต೿, ଲஂ૶, 2011, “ࢠ঍ࢄ෈ୡจॺฃ઩ଭ෉

ૈસഠઑٛணׁুࢫणীଭ૳ౢ,”෉֝஺֜ਏਆഗվ

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ࢮవઽ, ׌ࣲச, 2010, “ഠచࢮഓࠤੰ઩ଭ෉จܛজଭ݊ୢ

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11-15.

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׌ࣲச

ইۦ ܓԸʂॡİ ėęʂॡ قȃݓۙڙėॡę ԵԐ (欧G 彳櫾躇G 缧48嘳G 缧1埲G 垾畢)

ࢽֽਐ

1996ț տߎʂॡİ ԦНॡę ۋॡԐ 1999țտߎʂॡİԦНॡęۋॡԵԐ 2005țտߎʂॡİԦНॡęۋॡчԐ

ইۦ տߎʂॡİ ԦНॡę ٍĵڙ ф ֨ÂÌԐ (E-mail; [email protected])

ًౖశ

1999țܓԸʂॡİۙڙėॡęėॡԐ 2001țܓԸʂॡİۙڙėॡęėॡԵԐ 2007țČͲʂॡİݓĵঞąęॡęۋ

ॡчԐ

ইۦ ۻǫʂॡİ ėغşցٍĵՙ ٍĵİս (E-mail; [email protected])

଍۩૽

2011ț ܓԸʂॡİ قȃݓۙڙėॡę

ėॡԐ

ইۦ ܓԸʂॡİ قȃݓۙڙėॡę ʂॡڙԦ (E-mail; [email protected])

ন౿ْ

1983țԴڐʂॡİйԦНॡęۋॡԐ 1985țԴڐʂॡİйԦНॡęۋॡԵԐ 1992țԴڐʂॡİйԦНॡęۋॡчԐ

ইۦ տߎʂॡİ ԦНॡę İս (E-mail; [email protected])

ࢮవઽ

ইۦ ܓԸʂॡİ ėęʂॡ قȃݓۙڙėॡę İս (欧G 彳櫾躇G 缧48嘳G 缧1埲G 垾畢)

Identification of Indigenous Acidophilic Bacteria by Polymerase Chain Reaction and 16S rRNA Sequences

ண෍ตী઴৐ࢱଦր4S3/"સ׆ছવଡଲ૳෉

ഠచ෹ॺনࢮഓࠤੰଭܛ୨

׌ࣲச

Â ଍۩૽

Â ࢽֽਐ

Â ন౿ْ

Â ًౖశ

Â ࢮవઽ

Identification of Indigenous Acidophilic Bacteria by Polymerase Chain Reaction and 16S rRNA Sequences

Bong-Ju Kim, Dae-Woong Wi, Keun-Sik Baik, Chi-Nam Seong, Nag-Chol Choi and Cheon-Young Park

Դ΁

ࢹ޳чࢬν؉֨Β޽ࠄ ćʂѕتфՁۤ֬ॹۆ

ѕتؚܓՁ %/"қνфқԵѓѪ

Ձۤ֬ॹ

ࢹۆ

څأфĀ΁

ԐԐ

޷ČЛॶ

׌ࣲச

ࢽֽਐ

ًౖశ

଍۩૽

ন౿ْ

ࢮవઽ

ண෍ตী઴৐ࢱଦր4S3/"સ׆ছવଡଲ૳෉

ࢹ޳чࢬν؉֨Β޽ࠄ ćʂѕتфՁۤ֬ॹۆ

Ձۤ֬ॹ