Table Of ContentSpringer J apan KK
K. Horikoshi, K. Tsujii
(Eds.)
Extremophiles in Deep-Sea
Environments
With 122 Figures
Springer
Koki Horikoshi
Director General
The DEEPSTAR Group
Japan Marine Science and Technology Center (JAMSTEC)
2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan
Kaoru Tsujii
TeamLeader
The DEEPSTAR Group
Japan Marine Science and Technology Center (JAMSTEC)
2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan
ISBN 978-4-431-68008-6
Library of Congress Cataloging-in-Publication Data
Extremophiles in deep-sea environments I K. Horikoshi, K. Tsujii
(eds.).
p. cm.
Includes bibliographical references.
ISBN 978-4-431-68008-6 ISBN 978-4-431-67925-7 (eBook)
DOI 10.1007/978-4-431-67925-7
1. Extreme environments-Microbiology. 2. Benthos-Microbiology.
3. Submarine trenches. I. Horikoshi, Koki. II. Tsujii, Kaoru.
QRlOO.9 .E96 1999
579'.1777-dc21
99-36537
Printed on acid-free paper
© Springer Japan 1999
Originally published by Springer Verlag Tokyo Berlin Heidelberg New York in 1999
Softcover reprint of the hardcover 1s t edition 1999
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Preface
OnFebruary 28, 1996,Japanese researchers working on the DEEPSTARprogram
at the Japan Marine Science and Technology Center (JAMSTEC) broke their own
world record by sending the unmanned submersible Kaiko (the Japanese word for
"trench")to 10898metersbeneaththePacificOcean. Kaikosentbackvideoimages
oflifein thedepths oftheMarianaTrench, thedeepest pointin the world'soceans.
AbriefbutexcitedfaxfromthemothershipdescribedthescenecapturedonKaiko's
camera:'ThebedoftheMarianaTrenchwasfilledwithafinemudofreddishbrownish
particles. There were norocks orcracksatall and itresembledadesert. However,
very unusual organisms were observed here and there." In the following fax, they
describedtypesofseaurchin,jellyfishmovingfairly fast, and theexcrementofsea
organisms. My colleagues also saw a fast-moving shrimp about three centimeters
long. Therewasalsoakind ofseacucumber,which was thesamesizeas thejelly
fish. Kaikoscoopedoutsamplesofmud to bringtothesurface. Now we would be
able to isolatelivingcreaturesfrom thedeepestpointoftheMarianaTrench.
Notsomanyyearsago,almostall biologistsbelievedthatlifecouldsurviveonly
within so-called moderate environments; so when microbiologists looked around
for interesting bacteria and other life-forms, they attempted to isolate microorgan
isms only from moderateenvironments.
About30yearsago, Iwas visiting Florence, Italy. Iwas lookingat the Renais
sance buildings, which were very differentfrom Japanese architecture. About500
yearsago,noJapanesecouldpossiblyhaveimaginedthisRenaissanceculture. "There
mightbeawhole new worldofmicroorganisms indifferent unexploredcultures," I
thought."Couldtherebeanentirelyunknowndomainofmicroorganismsatalkaline
pH?" The acidic environment was being studied, probably because most food is
acidic. However, hardlyany work had been done in the alkalineregion.
Science,justas much as the arts, relies upon asense ofromance and intuition.
Upon my return to Japan, I prepared an alkaline medium containing I% sodium
v
VI Preface
carbonate,placedasmallamountofsoilinit,andincubateditovernightat37°C. To
mysurprise,variousmicroorganismsflourished inall 30testtubes. Herewasanew
alkaline world which was utterly different from the neutral world discovered by
Pasteur. I named these microorganisms which thrive in alkalineenvironments"al
kaliphiles," and conducted systematic microbial physiological studies ofthem. At
the same time I focused my intereston theenzymes produced by thesealkaliphilic
microorganisms as well as on their molecular genetics. The results showed that
thesemicroorganisms,whicharecompletelydifferentfromanypreviouslyreported,
werewidelydistributedontheglobeandthey producednewproducts.This wasmy
first encounter with a kind ofextremophile and led to my subsequent work with
alkaliphiles.
Thefive-yearSuperbugProjectoftheJapaneseERATOprogramwaslaunched
in 1984tosearchforextremophilesthatgrowinextremeenvironmentsand totry to
usetheiruniquepropertiestoestablisha"NewBiotechnology." Thisresearchproject
resulted inmanysignificantscientificdiscoveries. TheSuperbugProjectwasarea
sonablysuccessfulERAIDproject,in thatsomenew, youngscientistsweretrained
and several successful patentshavecomeoutofthe project. Forexample, onedis
coverywasavery strongenzymethatcan function in highalkalinity (pH 12)while
degrading modified cellulose. This has already been incorporated in a Japanese
detergentcalled"Attack." Anotherdiscovery hasexceededallconceptionsofwhat
constitutes a viable environment, an unusual, feisty life-form that can thrive in a
50% solution ofvery toxic toluene, in sharp contrast to ordinary organisms which
perishinasolutionofjust0.1%. WewereabletoisolatetheDNAfromthetoluene
resistant bacterium and introduce it into toluene-sensitive mutants. It should ulti
mately bepossibletoisolatethespecial genesand transfersolvent-tolerance.
Inmorerecenttimes,manyorganismshavebeenfoundinextremeenvironments.
Moreover, some ofthem cannot survive in so-called moderate environments. For
example,thermophilicbacteriagrowinenvironmentswithextremelyhightempera
tures,butwillnotgrowat20°Cto40°C. Somealkali-lovingbacteriacannotgrowin
anutrientbrothat pH7.0,butflourish atpH 10.5. Barophilicmicroorganisms iso
lated from the MarianaTrench absolutely require higherpressures (higher than 80
MPa) for growth. Ifa moderate environment for conventional organisms such as
Escherichiacoliweresuperimposedonthatforextremophiles,the"moderate"envi
ronment would be anextremeenvironment. Thus, the ideaofan extreme environ
mentisrelative,notabsolute. Clearlywehavebeentooanthropocentricinourthink
ing. Weshouldthereforeextendourconsiderationtootherenvironmentsinorderto
isolateandcultivatenewmicroorganisms.
A new l5-year research program called International DEEP STAR (Qeep-sea
EnvironmentExplorationfrogram,ScienceandTechnologyforAdvancedResearch)
was launchedinOctober 1990inYokosukathroughJAMSTEC. Ihavebeenplaced
in chargeofthis program and wouldlike to expand thesources ofmicroorganisms
for study from thesurfaceofthe globetothedeepsea. JAMSTEChastwosubma
rines (Shinkni 2000and Shinkai 6500), two unmanned submersibles (Dolphin 3K
and Kaiko), and can collectsamples from the deepest parts ofthe oceans. The re-
Preface VII
motely controlled Kaiko was used tocollect samples from the deepest pointofthe
Mariana Trench as described above. When I accepted the DEEP STAR post, my
classmate,fonnerPresidentofJAMSTECIsaoUchidatoldme: "JAMSTECcannot
study fish, because they belongto the Ministry ofAgriculture and Fisheries, and it
can'tstudythestructureoftheseaorports, sincethis is undertheMinistry ofCon
struction." JAMSTEC decided to tum to microorganisms "because they belong to
nobody." SoIhadgoodscientificandeconomicreasonsfordirectingamajorproject
tostudymicrobiallifeatthebottomofthesea. Onepotentialgoal istocollectnew
infonnationonthe originsoflife.
The program atYokosuka turns out to be rather unusual in that5 to 20million
dollars a year has been spent since 1991. At JAMSTEC there are 30 researchers
workingondeep-searesearch,andfrom 1992to 1998another 45 researchers from
tencountriescametoworkwithus. Engineersandscientistsfrom thisprojectspent
twoyearsconstructingan incrediblefacility thatallowsthemtocollectmicroorgan
ismsunderdeep-seahydrostaticpressureand togrowthemcontinuously inculture,
transferring them constantly under high pressure.These scientists also havedevel
oped the capability of working at very high temperatures, to better preserve and
studydeep-seahydrothennal-ventbacteria.
Thegroups in DEEPSTARarefocusing on thefollowing areas:
1. Organicsolvent-tolerantmicroorganisms. Ittooktwoyearstoisolateanorgan
icsolvent-tolerantbacteriumin 1989. However,in 1991 ourgroupdevelopeda
sophisticatedmethodtoisolateorganicsolvent-tolerantbacteriafrom deep-sea
samples. Using thismethod, theisolation frequency ofsolvent-tolerantmicro
organisms was about 100times higherthan that ofthe isolation previously re
ported. Someofthese microorganisms can completely digest crudeoil in sea
water. This finding has paved the way to removing oil spills. These organic
solvent-tolerantmicroorganisms haveconsiderablepotential for application in
bioreactorsassolvent-tolerantmicrobial catalysts.
2. Hyperthermophiles. Morethan50strainsofhyperthennophileshavebeen iso
latedfromhydrothennalventsintheOkinawaarea. Oneofthesehyperthenno
philes is Pyrococcus horikoshii, isolated in collaboration with The Center of
MarineBiotechnologyoftheUniversityofMaryland.The wholechromosomal
DNAsequenceofthisstrainwasdetenninedbytheNational InstituteofTech
nology and Evaluation in 1998. Several enzymes in hyperthennophiles have
been investigatedfor industrialapplications.
3. Psychrophiles,bacteriathatgrowatvery lowtemperatures. Thesebacteriaare
agoodresource toisolatenovelenzymesthatareactiveat lowertemperatures.
4. Barophiles, bacteria that grow only under hydrostatic pressure. DEEP STAR
researchershavesucceededinisolatingmanyobligatelybarophilicbacteriathat
function only under high pressure and have isolated the genes responsible for
barophily. Phylogeneticstudiesoftheisolatessuggestedthattheisolatedstrains
belongtoanewsub-branchofthegammagroup. Itwasdiscoveredthatthelac
promoter was activated under high pressure in the absence of inducers. AI-
VIII Preface
though the enzymes produced by these barophiles have not been studied, it is
highlylikelythatitwillbepossibletoclonenovelgenesfrombarophilesandto
expressthem inconventionalmicroorganisms for industrial applications. Fur
thermore,thephysiologyofSaccharomycescerevisiaehasbeenextensivelystud
ied underelevatedhydrostatic pressurestoestablish barophysiology.
5. WholegenomeanalysisofalkaliphilicBacillushaloduransC-125,abacterium
producing alkaline protease, alkaline cellulase, and alkaline xylanase. Alka
liphily andthe environmental adaptation mechanism will beclarified from the
whole DNAsequencedeterminedby "shotgunsequencing."
6. Work is proceeding on preservation and culture collection, and research has
begunon thegeneticsofthesemarine bacteria.
DEEPSTARresearchershavesuccessfulIymovedtothesecondphase(1998-2005)
with thefollowingresults.
I. Bytheendof1998about250articleshadbeenpublishedinrespectedjournals.
2. InJanuary 1998, DEEPSTARorganizedthe International CongressonExtre
mophiles atYokohama. About 350participants, including 150scientistsfrom
abroad, discussedextremophiles.
3. ThesciencejournalExtremophileswaslaunchedin 1997bySpringer-Verlag.
wasappointedthefirstChiefEditor.
This book is written to commemorate oureight-year studies ofdeep-sea microor
ganisms. Someoftheworkhasnotbeenpublishedinscientificjournalsforvarious
reasons: somebecauseitisnotyetready forsubmission,someforpatentandother
reasons. However, thesepersonalreportsshouldbeuseful in thestudyofdeep-sea
microorganisms. All the individuals involved have worked very hard to develop
deep-seamicrobiology,includingbarophysiology. Iamextremelygratefulfortheir
cooperationinthisprojectduringthefirst phase.
I would also like to pay special thanks to former Presidents I. Uchida, andW.
IshizukaandPresidentT.HiranoofJAMSTEC,whokindlygavemetheopportunity
toexpandthe new worldofextremophiles.
K. Horikoshi
Contents
Preface V
Part 1 Biodiversity in Deep-Sea Environments
I Isolation and Characterization ofMicroorganisms from
Deep-SeaMud
HidetoTakami 3
2 MolecularAnalyses ofthe Sediment and Isolation of
Extreme Barophilesfrom the DeepestMarianaTrench
Chiaki Kato 27
3 Taxonomy and BiotransformationActivities ofDeep-Sea
Actinomycetes
AlanT. Bull, Joy A. Colquhoun, and Stephen C. Heald 39
4 Microbial Diversity in the Sediments Collected from
Cold-SeepAreas and from Different Depths ofthe Deep-Sea
LinaLi andChiaki Kato 55
IX
X Contents
Part 2 Adaptation and Response of Extremophiles
Toward Extreme Deep-Sea Conditions
5 Barophiles (Piezophiles)
Chiaki Kato 91
6 Thermophiles
Juan M. Gonzalez 113
7 Deep-Sea Psychrophiles
TetsuoHamamoto 155
8 Organic Solvent-Tolerant Microorganisms
AkiraInoue................................................................................... 175
9 Microbial ProcessesAssociated with aMethane Seep
Tubeworm
Takeshi Naganuma 211
Part 3 NewApproaches and Future Scope for Deep
Sea Microbiology
10 Barophysiology (Piezophysiology)
FumiyoshiAbe andChiaki Kato 227
11 Genome Analysis ofFacultativelyAlkaliphilic Bacillus
halodurans C-125
HidetoTakami 249
12 Future Scope
Kaoru Tsujii 285
Appendix: International Congress on Extremophiles '98 at
Yokohama> '" 293
Appendix: List ofPublications ofthe First DEEPSTAR Project:
October 1, 1990 to September 30, 1998 301
Subject Index 311
Contributors
Koki Horikoshi
TheDEEPSTARGroup, Japan MarineScienceandTechnology Center,
2-15 Natsushima-cho,Yokosuka, Kanagawa 237-0061, Japan
HidetoTakami
TheDEEPSTARGroup, JapanMarineScienceandTechnologyCenter,
2-15 Natsushima-cho,Yokosuka, Kanagawa237-0061, Japan
ChiakiKato
TheDEEPSTARGroup, Japan MarineScienceandTechnology Center,
2-15 Natsushima-cho,Yokosuka, Kanagawa237-0061, Japan
AlanT.Bull
DepartmentofBiosciences,UniversityofKent,
Canterbury, Kent,CT27NJ,UK
LinaLi
TheDEEPSTARGroup, JapanMarineScienceandTechnologyCenter,
2-15 Natsushima-cho,Yokosuka, Kanagawa237-0061, Japan
JuanM.Gonzalez
CenterofMarineBiotechnology, University ofMarylandBiotechnology Institute,
701 E. PrattSt.,Baltimore,MD21202,USA
TetsuoHamamoto
BiotechnologyTransferServices,
4-14-9Kamiogi, Suginami-ku,Tokyo 167-0043,Japan
AkiraInoue
TheDEEPSTARGroup, Japan Marine ScienceandTechnology Center,
2-15 Natsushima-cho,Yokosuka, Kanagawa237-0061, Japan
XI
