Bioremediation of coastal areas 5 years after the Nakhodka oil spill in the Sea of Japan

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Bioremediation of coastal areas 5 years after the Nakhodka oil spill in the Sea of Japan

著者 Siti Khodijah

journal or

publication title

博士学位論文要旨論文内容の要旨および論文審査結果の要旨／金沢大学大学院自然科学研究科

volume 平成18年1月

page range 95‑99

year 2006‑01‑01

URL http://hdl.handle.net/2297/16712

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氏，名学位の種類学位記番号学位授与の日付学位授与の要件学位授与の題目

SITIKHODIJAH 博士（理学）

博甲第690号平成１６年９月３０日

課程博士（学位規則第４条第１項）

Bioremediationofcoastalareas5yearsafterthe陥肋ｏｏＩＡａｏｉｌｓｐｉｌｌｉｎ

ｔｈｅＳｅａｏｆＪａｐａｎ

（"ナホトカ号"によるオイル流出事故後５年間にわたる日本海沿岸のバイオ

レメディエーション）

田崎和江（自然科学研究科・教授）

奥野正幸（自然科学研究科・教授），加藤道雄（自然科学研究科・教授），

古本宗充（自然科学研究科・教授），中村省吾（富山大学鉾教授）

論文審査委員(主査）

論文審査委員(副査）

学位論文要旨

Abstract

Thisstudydealsprimarilywiththeisolationandcharacterizationofhydrocarbonbacteriaindigenous ｔｏｔｈｅＭｚ肋o肱aoilspillafterfiveyearsofbioremediation，ａｓwellaswhatenvironmentalfactors influencingthebioremediationoftheMz肋odkaspilLSevenbacterialstrains,whichwereidentifiedusing l6SrDNAsequenceanalysisasBacjﾉﾉzJsspp.，児Demomo"αｓｓｐ.，andPZzracocczJsspp.，showedtheir abilitytogrowwellonaliphatichydrocarbons,butnotonaromatichydrocarbons,ａｎｄａｌｌｏｆｔｈｅｍｗｅｒｅａｂｌｅｔｏｇｒｏｗｗｅｌｌｏｎｈｅａｖｙｏｉＬＴｈｅｂｅｎevolentinteractionofbacteriawithfUngioccurredduringthe 429-daybioremediationoftheMzkﾉio(Zkaoilspill,eventhoughtheenvironmentalparameter(i､e､,ｐＨ）

wasnotmaintainedHydrocarbonbacteriahadtendencytoplaygreatestｒｏｌｅａｓｔｈｅｓｏｌｕｔｉｏｎｐＨｗａｓ

､eutral-alkalineconditionOnthecontraryｳｗｈｅｎthepHsolutionchangedtobeacidic，ｔｈｅｆＵｎｇｉｔｏｏｋ overtodegradeheavyoiLFungalsuccessionwaspredominatedbyRhj"ocﾉα`iejZzJsp.，whileallfungi whichsurvivedindegradingheavyoilwereidentifiedasR/､j"ocJadjeJJasp.,ASpeγgi"zJssp.,acre"zo"、腕 sp.，ａｎｄＰｅ"jcjJljzJ腕ｓｐ・Duringthatperiod，aliphatichydrocarbonswerereducedsignificantly，whereaｓ aromatichydrocarbonsremainedrelativelyconstant，andtherewastheproductionofmetabolitesafter 429daysofbioremediationOfallmetalscontainedintheheavyoil，seawater，andbeachsands，ＣＯ appearedtoinhibithydrocarbonbacterialgrowth，whileSitendedtoenhancehydrocarbonbacterial growthlnaddition，theclaymineralspresentinthemarｉｎｃａｎｄ､coastalsitestendedtostimulate

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、C L

hydrocarbonbacteriaIgrowth・Theseresultsprovideasignificantinsightintohowbioremediationoｆｔｈｅ MukﾉMkaoilspilltakeplaceandwhatenvironmentalfactors(i､e､,Sicompoundandclayminerals）

influencethatbioremediation,therebygivingmorefruitfulinfOrmationfOrthebioremediationofthｅｏｉｌ spill-contaminatedmarineandcoastalenvironments．

KeyWOrds:MzA/tozZlEZzoilspill,bioremediation,heavyoil,heavymetals,clayminerals,montmorillonite,kaolinite，

BacjJJⅢｓｓｐｐ.,片ezJdo"zo"αｓｓｐｐ､Pzzracocc"ｓｓｐｐ.,RAi"ocladielﾉﾋzsp.,乱SpeZgiノルｓｓｐ.,ＡＣ”"、"z""dsp.,Ｐｅ"iciJ肋、^●

sp．

StudiesonbacterialstrainsdegradingaliphaticandaromatichydrocarbonsattributabletooU spillsreceivedmuchattcntion・However,onlyfewstudieshavebeenconducteｄｏｎｔｈｅｌ９９７ＮＺｚＡﾉiMltnoilspillmrelationtothelong-termbioremediationalstudy,aswellashydrocarbon degraders､Hence,acarefUlbioremediationalrcsearｃｈｏｆｔｈｅＭｚ肱oc1kaoilspillalongseashores ｉｎＳｅａｏｆＪａｐａｎａｓＷｅｌｌａｓｔｈｃｉｎｖｅｓｔｉgationofwhatenvironmentalfactorsinfluencingthat

biorcmediationwerecarriedoutsincel997．

Theprimaryobjectiveofthisstudywastoisolatcanddescribehydrocarbon-degrading

、

bacteriafiFomtheMz肋ｏＣｊｌ(zzoilspill-contaminatedseashoresintheSeaofJapanafterfiveycars ofbioremediationaswellastoinvestigatewhatenvironmentalfactorsmfluencingthe bioremediationofthelVZzModﾉhzoilspilL

SevenbacterialstrainscapableofutilizingtheheavyoilspilledfroｍｔｈｅＭＪ肋ocZkzzRussian oiltankerwereisolatedfromthreecoastalareas(namelyKatanoSeashoreofFukuiPrefecture，

OsawaandAtakeseashoresoflshikawaPrefecture)andtheMz肋ocZAzJRussianoiltankerafter fiveyearsofbioremediationA11bacterialstrainsshowedtheirabilitytogrowwellonaliphatic hydrocarbons，butnotonaromatichydrocarbons，ａｎｄａｌｌｏｆｔｈｅｍｗｅｒｅａｂｌｅｔｏｇｒｏｗｗｅｌｌｏｎ heavyoiLTheywcreidentifiedusinｇｌ６ＳｒＤＮＡｓｅｑｕｅｎｃｅａｎａｌｙｓｉｓａｓＢａｃiJJ"ｓｓｐｐ，

RsezJdomo"αｓｓｐ,ａｎｄP〃αcocczJsspp．

ThebenevolentinteractionofbacteriawithfUngioccurredduringthe429-day bioremediationofthe」ＶＭﾉiMAzzoilspillatlaboratoryscale，eventhoughtheenvironmental parameter(i､e､,ｐＨ)wasnotmaintainedoruncontrolled・Hydrocarbonbacteriahadtendencytｏ

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PlaygreatcstroleasthesolutionpHwasneutral-alkalmecondition(7~7.8）Onthecontrary，

whenthepHsolutionchangedtobeacidic(pＨ２~4),thefimgitookovcrtodegradeheavyoiL Fungalsuccessionduringthe429-daybioremediationwaspredoｍｉｎａｔｅｄｂｙＲｈｊ"oc/ααieJJbzsp.，

whileallfUngiwhichsurvivedindegradingheavyoUwereidentifiedasＲ肱"ocJα`ie"αｓｐ.，

ASpelgiﾉJzJssp.,Acremo"i"ｍｓｐシａｎｄＰｅ"ici"izJ腕ｓｐ､Duringthatperiod,aliphatichydrocarbons wcrereducedsignificantly，whcreasaromatichydrocarbonsremaincdrelativelyconstant，and

therewastheproductionofmetabolitesafter429daysofbioremediation・

E1ementallevelsinheavyoilshowedwiderangesinalltheheavyoilsamplesconsistingof Si,Ｓ,Ｔｉ,Ｃｒ,Ｍ､,Ｆｅ,ＣＯ,Ｎｉ,Ｃｕ,Ｚｎ,ａｎｄＰｈＣｏｍｐｏｕｎｄｓｏｆＳｉ,Ｓ,andCrwcreobservedathigh levels,whilethoseofnMn,Fe,CO,Ni,Cu,Zn,andPbwercobscrvedatlowlevels,Ofall heavymetals，Ｃｏａｐｐｅａｒｅｄｔｏｂｅｔｏｘｉｃｆｂｒａｌｌｂａｃｔｅrialgrowthatconccntrationsof＞１ｐｐｍ，

whilcthepresenceofTi,ＣＬａｎｄＣｕａｔＯ・O１ｔｏｌＯｐｐｍｗｃｒｅfOundnottoinhibitgrowthofaU bacterialstrains・Itissuspectedthatthepresenceofheavymetalsmayhaveasignificanteffect onthccompositionofthebactcrialconⅡnunity,(i､6.,alkane-degradingbacterialisolates),as wellasonthebiodegradativeproｃｅｓｓｅｓｏｆｔｈｅＭＷｉＭﾉIzzoilspmduringthe5-year

biorcmediation．

C1ayminerals，whichareusuallypresentinthemarineorcoastalenvironments contaminatedwithhighconcentrationofoilspills,werecapableofpromotingmicrobialgrowth

andallowcdmicroorganismstoprolifCratefavorablyabovelevclsofnon-clay-contaming

controlatanextremelyhighconcentratioｎｏｆｈｅａｖｙｏｉｌ（１５０９/1）（Fig.1).C1aysatlowto moderateconcentrations(Ｏ５ｔｏｌＯｇ/1)tendedtoenhanccbacterialgrowth・Onthecontrary，

claysathighconcentrations(〉１０９/1)appearedtoinhibitgrowthofhydrocarbondegrading

bactcria,resultingfromthelowredoxpotentialvaluesoftheliquidphascandnotfromthe toxicityofelements(ｓｕｃｈａｓＳｉａｎｄＡ１)containedmclayMincedissolvedSiandA1were observedatlowconcentrationsintheaqueousphasc、Ｔｈｅｐｒｅｓｅｎｃｅｏｆｃｌａｙｓａｔｈｉｇｈ Concentrationsgenerallyyieldsthelowdissolvedoxygenlevelsinthebulksolution,thusbeing consideredaspollutants，hversely，ｉｆｃｌａｙｍineralsarepresentatlowconcentrationsthat

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rcmainsustainableoxygenlevelsfOrbacterialgrowth,thcybecomestimulatorfOrhydrocarbon

bactcrialgrowthbyfOrnngtheC-O-N4-Sicomplexesonthcsurfaceofhydrocarbonbacterial

cells・However,theuptakeOfSielementbybacterialcellsappearedtobemorepredominant thanA1clcment・Theseresultsindicatcthatthecnhancementofhydrocarbonbactcrialgrowth maybethcrcsultsofclayslikelyservingastraccelementsrequiredfOrbacterialgrowthaswcll asgrowthsupportingcarrierlnaddition，theseresultsshowedthatA1clcmentcontamcdin claysdidnotresultinthctoxiceffectonhydrocarbonbactcrialgrowthThetoxicityofA1were notobservedatlevelsofclays(0.5ｔｏ３０ｇ/1)usedinthisstudyHydrocarbonbactcriausedin thisstudytendedtobefavorablctoSielemcntofclays・ＩｔｉｓｓｕｓｐｅｃｔｃｄｔｈａｔＳｉｉｓａｎｅｌｅｍｃｎｔ facilitatingthehighhydrocarbonbacterialgrowth,andthcC-O-Na-SicomplcxesontheSurface ofbacterialccllwallsarethestimulatorfbrhydrocarbonbacterialgrowth．

Thisstudyprovidesevidencethatafterfiveyearsofbioremediation，thebacterial communityoftheMWiod）hzonspill-contaminatedcoastalsitesisprcdominatedby^o alkane-degradingbacteriaamongwhichHsezJ血加o"αsspeciesisadominantorganism・

Howeverlnotonlydohydrocarbonbacteriaplaysignificantrole，buthydrocarbonfimgi predominateｄｂｙＲｈｉ"ocJaaieJJaspalsoparticipateinthedegradationofheavyoil,wherethcir roleisdependentuponthepHoftheaqueousphase・Inaddition,theelcments(inparticularSi）

andtheclaymineralsareofenvironmentalfactOrsenhancmgthehydrocarbonbacterialgrowth inthebioremediationoftheMzAﾉｶo`！RzJoUspill．

７７７７７７０鄙鄙副鄙馳副鄙６５４３２１０（ヨミロ隅〉）⑭ヨ⑩：一合唇

0２０４０６０８０１００１２０Ｔｉｍｅ(days）

－●－R11He日ｖｙｏｊｌ＋Mic｢ootg因nbms＋ⅢonhnoriUonibe eR2kHeaVyoil＋MicrooIFan七ｍs＋Xao1mite -A-R5:HefH7yojl＋Mic｢omgan鱈、臼

Fig.１.】M[icrolMgr･wll,(bllcM同ａｌｌＭｍｇｉ）ａ＄ＶＭ)lccclMn coIonyfbrmmgHM(CFU)pcrmlinUhccxpcrimcntalsyl;t亡ms cont同iningcInysamdinUlccoxMrolcXpcHim巳xutal5y5tcmcontniniJ3g noncl８５/旨.Ｅ８ｍｒｂ洞r5rcprc5cntst⑧ndnrddwi3tionS．

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;≦二三鑿

領

iIl

^{ｉ凹ｕｍａｌＫｚＩｒ}

llllmZI

uＶ

9曇ＩＩＰ

Fig.２．Transmissionelectronmicrographoftheattractivenetwork formationofbacterialceUsconsistingofshortaridlongrods-shaped bacteriaandsphericalbacteriaattachedtotheedgesofhexagonal kaolinite,Thecellsappeartoconnectwithoneanotherandareattachedざ

totheedgesofhexagonalkaolinitebyanadhesiveagent,assulnedtobe extracellularpolymericsubstances(EPS)．

学位論文審査結果の要旨

平成１６年７月１５日に第１回学位論文審査委員会を開催し，７月２８曰に口頭発表を行い，同日最終の審査委員会を開催した．協議の結果以下のとおり判定した．本学位論文は，ナホトカ号重油流出後５年間の重油の分解に寄与した炭化水素分解細菌の単離と特性評価を行い，バイオレメディエーションに影響する要因について研究した。ナホトカ号から抜き取った重油や海岸（片野，大沢，アタケ）の漂着重油・砂・海水の試料から３９菌株が単離され，７菌株が炭化水素（n-アルカン）を炭素源として培養できることを明らか

にした．

この７菌株についてl6SrDNA塩基配列解析を行い，比cZZ/hzsspp.’んeudbmmassp.，HRI1acDcaLsspp・

を同定した．４２９曰間のＣ重油分解実験では，炭化水素分解細菌は共存する菌類とｐＨ範囲に応じて炭化水

素を分解する働きがあることを明らかにした．すなわち，ｐＨ7.0～７．８の領域では細菌が主に炭化水素分解を担い,pH2.0～４．０では菌類が分解する．さらに,重油と粘土鉱物が共存する系での培養実験を行った結果，

粘土鉱物の存在は分解細菌の成長を助長する効果があることも明らかになった．本研究結果はナホトカ号重油流出事故後に自然環境下で土着の微生物による重油の分解過程やバイオレメデイエーションに影響する環境要因ついて重要な知見を提供し、バイオレメディエーションの実用化へ向けた有用な情報を与えた．参考論文は国際誌２編、国内誌１編が既に受理されている。

５名の審査員はいずれも博士（理学）に相当する質の高い論文であると評価した．

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