MICROBIAL ECOSYSTEM IN MARINE HYDROTHERMAL SYSTEM IN THE EARLY
AND PRESENT EARTH
Shingo Kato1 and Akihiko Yamagishi1
1
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432-1
Horinouchi, Hachioji, Tokyo 192-0392, Japan
Fax: +81-426-76-7145
yamagish@toyaku.ac.jp(Received September 14, 2010; Accepted October 28, 2010)

geological, geochemical and microbiological data forvarious hydrothermal fields, the diversity of marinehydrothermal
systemsanddiversityofmicroorganisms living there has been evaluated.
Knowledge of the microbial ecosystem in the present
diverse hydrothermal systems will provide
information about the early microbial ecosystem inancient hydrothermal systems before the evolution ofphotosynthesis. This paper reviews the geological,
geochemical and microbial diversities of the present
hydrothermal systems, and possible metabolic
functions of the microorganisms in the ancienthydrothermal system in the early Earth.

(Abstract)
Marine hydrothermal fields are thought to be anenvironment where microorganisms thrived in early
Earth. However, the physiological characteristics ofthese microorganisms are unknown. The study of
microorganisms in the present hydrothermal fields
will provide clues to elucidate the ancient ecosystem.
For a better understanding of the ancient ecosystem,
it is important to know the relationship between
geological, geochemical and microbiologicaldiversities of the present marine hydrothermal
systems. Microbiological breakthroughs for both
culture-dependent and -independent methods areneeded to reveal the physiology of the
microorganisms living in the present marinehydrothermal fields.
(Keywords)
Ancient life, marine hydrothermal
hyperthermophile, Archaea

2. Marine hydrothermal fields as an analog of
habitats in the early Earth
In the late 1970’s, a deep-sea hydrothermal field
discharging warm fluids was discovered in the
Galapagos Rift 8. Unusual animal communities were
found in the hydrothermal field. As soon as this
discovery was made, other hydrothermal fields
venting high temperature fluids (300ºC or higher)
were found in the East Pacific Rise (EPR) 9. To date,a number of marine hydrothermal fields have beenfound in various areas of the world (Fig.1). A
database of active submarine hydrothermal vent
fields (the “InterRidge Vents Database”) is available(http://www.Interridge.org/irvents) 10.
In marine hydrothermal systems, cold
oxygenated seawater penetrates into the deep
sub-seafloor. The penetrating seawater passes
through sub-seafloor oceanic crusts, reacts withheated rocks and dissolves ionized metals (such andFe2+, Cu2+ and Zn2+), and volatiles (such as H2, SO2,
CH4 and CO2) degassed from magma (Fig. 2A). The
hot fluids, which have dramatically changed
physicochemistry compared to the original seawater,
rise toward the seafloor and discharge while mixing
with seawater under and above the seafloor. Redox
reactions between reductants (such as H2, H2S, CH4
and Fe2+) in the hydrothermal fluids and oxidants(such as O2 and SO42-) in seawater generate chemicalenergy that could support the growth of various
chemolithoautotrophic microorganisms in marinehydrothermal fields 11.
A marine hydrothermal field is thought to be
one of the environments where ancient life thrived 12.
There are geological records indicating that marinehydrothermal systems were present in the early Earth.
All of the compounds required for the growth of
microorganisms, i.e., energy and carbon sources andtrace elements, are provided in the present
hydrothermal system 13. An ecosystem in marinehydrothermal fields does not require solar energy.
However, it is noted that the ecosystem of present

system,

1. Introduction
The ancient ecosystem on early Earth is one of
the most attractive topics for not only scientists but
also the general public. The present ecosystem ismostly fueled by solar energy, and thus is called aphotosynthetic ecosystem. In this ecosystem, plants
and cyanobacteria play the roles of primary producers
that fix inorganic carbon and transform it into organiccarbon using solar energy. These organic carbon
compounds sustain the growth of various organisms
by providing energy and carbon sources. In contrast,in the early Earth before the evolution ofphotosynthesis, the ancient ecosystem was probably
sustained by energy supplied from compounds
produced from the Earth. Microorganisms are able touse inorganic- and organic-compounds such as H2,
H2S, Fe2+, CO2, CH4, amino acids and peptides,
which were generated or synthesized by abiotic
reactions within the Earth’s crust, as energy and/or
carbon sources. Evidence supporting the presence ofancient life in the early Archaean, at least 3.8-3.2
billion-years-ago (Gya), has been reported 1-7.
However, the characteristics of the ancient ecosystem
are still unclear: What microorganisms were there?
What did they do? From when, where and how did
they thrive in the early Earth? Many researchers haveaccumulated data in various scientific fields, such as
geology, mineralogy, geochemistry, microbiology
and molecular biology, and tried to answer thesequestions. It is necessary to integrate these data tobuild a comprehensive view of the ancient ecosystem.
A marine hydrothermal field is one of the mostprobable environments where the ancient ecosystem
was present. Along with the accumulation ofViva Origino 38 (2010) 46 - 55
© 2010 by SSOEL Japan- 46 -

Viva Origino 38 (2010) 46 - 55

Fig. 1. The location of marine hydrothermal fields. The map was modified from Tivey (2007). The abbreviations areshown in the box at the bottom of the map.

microorganisms
were
also
found
in
3.5-billion-year-old hydrothermal deposits 2,4.
Notably, deep-branching Archaea in the phylogenetic
tree
containanaerobic
hyperthermophilic
methanogens and sulfate reducing microorganisms 16.
This fact suggests that these microorganisms retain
the characteristics of ancient life on the early Earth 16.
This notion is partially supported by experimental
data demonstrating that ancestral enzymes showed
high thermal stability 17-19. Regardless of thephysicochemical conditions of the ancient ocean (i.e.,
oxic or anoxic, hot or cold), which are controversial
20,21, the venting fluid in the ancient hydrothermal
fields must have been anoxic and hot. These datasuggest that anaerobic (hyper)thermophiles might
have thrived in marine hydrothermal fields in theearly Earth at 3.2-3.5 Gya. The fluids would havecontained H2 and Fe2+ that were generated byrock-water reactions, SO2 and CO2 degassed frommagma, and carbon compounds (such as CH4,
methanol, formic acid, amino acids and peptides) thatwould be abiotically produced in the hydrothermal
system 22-27. These compounds can be used as carbon
and energy sources by anaerobic (hyper)thermophiles.
Thus, anoxic and hot environments in the present
hydrothermal fields may be analogous to those in theancient hydrothermal fields.

Fig. 2. Illustration of the generation process ofhydrothermal fluids. (A) Magmatic and (B)non-magmatic hydrothermal systems are shown,respectively.

marine hydrothermal systems is not completely
independent of photosynthetic ecosystems because
the main electron acceptor, O2, is produced byphotosynthesis. Geological evidence indicates thatmicroorganisms lived in ancient hydrothermal fields
at 3.5-3.2 Gya 1-6. Filamentous microfossils were
found in a massive ancient volcanogenic sulfide
deposit 3 and in rocks 6. The morphologies of the
microfossils resemble some present-day filamentous
thermophilic microorganisms detected in bothterrestrial and marine hydrothermal fields 14,15.
Massive sulfide deposits are commonly observed in
the present hydrothermal fields. Methane with lightercarbon isotopic composition recognized as a productofmethanogenesis
was
detectedin
3.5-billion-year-old hydrothermal precipitates 1.
Sulfides with lighter sulfur isotopic composition
recognized as products of sulfate reducing

3. Diversity of geochemical characteristics andgeological settings of hydrothermal systems
To understand the ancient ecosystem in themarine hydrothermal fields of the early Earth, it isimportant to investigate extant marine hydrothermal
systems. After the discovery of marine hydrothermal
fields, studies of these hydrothermal systems haverevealed an unexpected diversity of geochemical
characteristics of venting fluids and geological- 47 -

Viva Origino 38 (2010) 46 - 55

settings of the fields 28,29. A variety ofphysicochemical characteristics of venting fluids arefound in the present hydrothermal fields. This
physicochemical diversity is mainly generated by thefollowing three processes: (1) water-rock reactions,(2) magmatic input and (3) phase separation.
The type and amount of minerals contained in
the host rock influence qualitatively andquantitatively the chemical species contained inhydrothermal fluids that are generated byseawater-rock reactions. In particular, pH and H2,
H2S and Fe2+ concentrations are controlled by phaseequilibrium of diverse minerals in the rocks 30,31. The
presence/absenceofmagmatic
input
also
dramatically controls pH and chemical compositionof hydrothermal fluids. The magmatic gases mainly
consist of CO2 and SO2 with H2, CH4 and CO as
minor components 32,33. The SO2 reacts with H2, O2
and minerals during the upwelling process. This
reaction generates H2S or H2SO4, the latter lowering
the pH of hydrothermal fluids. In sub-seafloor
environments, hydrothermal fluids are separated into
a vapor phase with a relatively low Cl-concentration
and a liquid (or brine) phase with a relatively highCl-concentration. The process is called phaseseparation 34. For example, higher concentrations ofvolatiles (such as H2, H2S and CH4) are found in thefluids of the vapor phase; in contrast, higher
concentrations of Ca2+ and Ba2+ are found in thefluids of the liquid phase 35,36. These three processes
are strongly influenced by differences in thegeological settings of the fields described below.
Marine hydrothermal fields have been found on
mid-ocean ridges (e.g., East Pacific Rise,
Mid-Atlantic Ridge, Central Indian Ridge),island-arcs (e.g., Izu-bonin Arc, Kermadec Arc,Mariana Arc) and back-arc basin spreading centers(e.g., Lau Basin, Manus Basin, Mariana Trough) (Fig.
1). In contrast to mid-ocean ridges, island-arcs andback-arc basins exist in the subduction zone (Fig. 3).
Magmatic input of island-arcs and back-arc basins isdifferent from that of mid-ocean ridges, whichconstrains the geochemistry of hydrothermal fluid
venting, thus affecting the pH and concentration ofvolatiles in the hydrothermal fluid 37,38. The fluids ofthe subduction zone are frequently lower in pH thanthose of mid-ocean ridges. Another magmatichydrothermal system called a hotspot has been found,such as the Loihi seamount near Hawaii 39. The

Fig. 3. A variety of geological settings of marinehydrothermal fields.
- 48 -

mechanism of magma generation at the hotspot is still
controversial 40. The features of the end-member (i.e.,
pure hydrothermal fluid, not mixed with seawater) of
these venting fluids in these magmatic hydrothermal
fields are very high temperature (~350-400ºC) andlow pH (~5). The basement of mid-ocean ridges
consists of basalts (mafic rock); however, in someareas (e.g., Rainbow site in the Mid-Atlantic Ridge),
the basement consists of peridotites (utramafic rock).
The basement of island-arcs and back-arc basinsconsists of andesite or dacite (intermediate- orintermediate-felsic rock). The variety of basement
rocks is one of the factors responsible for thegeochemical diversity of venting fluids 28,29.
Recently, a non-magmatic hydrothermal field,
called the Lost City hydrothermal field, wasdiscovered in the western Mid-Atlantic Ridge 41. In
contrast to the venting fluids in magmatichydrothermal fields, the fluids of the Lost City haveunique geochemistry. The fluid temperature canreach approximately 100ºC and the pH is very high(~ 11). In the Lost City, the basement rock is mantle
peridotite rifted up from deeper regions of oceanic
crust. The warm, alkaline fluid is likely to begenerated only by seawater-peridotite reactions (Fig.
2B). These are exothermic serpentinization reactions:peridotite containing olivine and pyroxenes (amineral group high in magnesium and iron) reacts
with seawater producing serpentinite, brucite[Mg(OH)2], magnetite (Fe3O4) and H2 42,43. Highconcentrations of hydrocarbons (such as methane,
ethane, propane and butane) were detected in thefluids of the Lost City 22 as well as the Rainbow siteof a magmatic hydrothermal field of the Mid-Atlantic
Ridge 23, which are likely to be produced abioticallyby Fischer-Tropsch type reactions: hydrocarbons areproduced from H2 and CO2 with metal catalysts suchas Co, Fe, and Ni, which are rich in peridotites.
Although non-magmatic hydrothermal fields have notbeen found except for the Lost City, other suchhydrothermal fields may currently exist, and also may
have existed in the early Earth. The pH gradient, i.e.,
proton gradient, between the seawater and alkalinehydrothermal fluids could have supported ATP
synthesis of the first cell 44.
Mid-ocean ridges have a variety of spreading
rates: fast (>80 mm/yr), mid, (60-80 mm/yr), slow
(20-60 mm/yr) and ultra-slow (<20 mm/yr) 45.
Studies of mid-ocean ridges showed that the
spreading rate quantitatively reflects the type ofbasement rocks present. Areas with fast- andmid-spreading rates consist of basalts (mafics). In
contrast, in regions of mid-ocean ridges with slowand ultraslow-spreading rates, mantle peridotites(ultramafics) are exposed on the seafloor or close to
the seafloor (e.g., the Rainbow site of a segment of
the Mid-Atlantic Ridge 23). The peridotites found in
the Lost City, an off-ridge field, may be derived froma part of the Mid-Atlantic Ridge with ultra-slow
spreading rate (12 mm/yr) 46. The difference in thetype of basement rocks, mafic or ultramafic, is related
to the venting fluid geochemistry at hydrothermal
systems as described above. Although there areViva Origino 38 (2010) 46 - 55

principal electron donor, such as methanogens (e.g.,
Methanopyrus kandleri 56), hydrogen-oxidizing
sulfur-reducers (e.g., Pyrodictium occultum 57),
hydrogen-oxidizing
sulfate-reducers(e.g.,
Archaeoglobus veneficus 58), and hydrogen-oxidizing
iron-reducers (e.g., Geoglobus ahangari 59), havebeen detected in various hydrothermal environments
including mid-ocean ridges, back-arc basins andisland-arcs 60. Thus, it seems that the presence orabsence of chemolithoautotrophic hyperthermophilic
Archaea is not associated with differences in global
geological settings but in more local environmental
factors.
Fermenting microorganisms are also living inmarine hydrothermal environments. Fermentation
generates ATP from carbon compounds including
amino acids and peptides by substrate-level
phosphorylation, reactions that do not require
exogenous electron acceptors (e.g., O2) as opposed torespiration. Members of the Thermococci, ahyperthermophilic archaeal group, are often detectedin hydrothermal environments 48,50,61-63. Thistaxonomic group includes fermenting species 64. In
addition, a carbon monoxide (CO) utilizing species
belonging to the Thermococci, which can grow using
CO as sole carbon and energy sources, was isolated
from a marine hydrothermal field 65. Recently, thewhole genome sequence of a member of theKorarchaeota, “Korarchaeum cryptofilum”, wasdetermined by metagenomic analysis of anenrichment culture 14. This group had been detected
in marine and terrestrial hydrothermal fields by
culture-independent methods 50,52,66-68 but not cultured
for over 20 years. The genome sequence includes
gene sets that include peptide and amino acidfermentation pathways, suggesting that K.
cryptofilum is likely to be a peptide- and aminoacid-fermenting archaeon 14. Some members of the
Thermoprotei can also grow by peptide- and/or
amino acid-fermentation, and their 16S rRNA genesequences have been detected in marine hydrothermal
environments 69. It should be noted that amino acids
and peptides could be abiotically synthesized inmarine hydrothermal systems 24,25 and CO is provided
by magma degassing 32. This means that peptide- andaminoacid-fermenting
andCO-utilizing
microorganisms
could
grow
as
well
as
chemolithoautotrophs in these environments, and in
the absence of any other organisms.

hydrothermal fields on back-arc basin spreading
centers, the relationship between spreading rate andbasement rock is not clear.
Thus, the geochemical diversity of fluids is
associated with the geological diversity ofhydrothermal fields. The diversity of various
physicochemical conditions (qualitative and/or
quantitative differences in temperature, pH andchemical species) must constrain the composition of
microbial communities.
4. Diversity of microorganisms living in marinehydrothermal fields
Diverse microorganisms of both Bacteria andArchaea are living in marine hydrothermal fields asshown by culture-dependent and -independent
methods.
Novel
extremophiles,
includingthermophiles (heat-loving), acidophiles (acid-loving)and piezophiles (pressure-loving; also calledbarophiles), have been cultured and isolated fromvarious hydrothermal fields. Unsurpassed physiologyof these extremophiles has expanded the known
limits of life. Culture-independent moleculartechniques based on 16S rRNA gene sequences haverevealedthat
diversenot-yet-cultivatedmicroorganisms exist in various habitats ofhydrothermal fields, such as fluids, sulfide chimneys
and microbial mats 47-52. Furthermore, using therecently developed and more powerful sequencing
method “pyrosequencing”, the presence of asurprisingly high diversity of microorganisms(especially of Bacteria) in hydrothermal fluids was
revealed 53. Although the physiological functions of
microorganisms cannot be directly determined fromtheir sequence information such as the 16S rRNA
gene and even the whole genome sequence, highphylogenetic diversity of microorganisms in thehydrothermal environments suggests that a variety ofphysiological functions exist there.
Microorganisms living in marine hydrothermal
fields include diverse chemolithoautotrophs that cangrow using inorganic materials as energy sources andCO2 as a sole carbon source 54, which are likely toplay important roles as primary producers in the
microbial ecosystem in these environments. They arecapable of using diverse redox reactions between H2,
H2S, Fe2+, CH4, NH4+, Mn2+, S0 and/or S2O32- aselectron donors and O2, NO3-, SO42-, Sº, S2O32-, CO2,
Fe3+ and/or Mn4+ as electron acceptors.
Chemolithoautotrophs synthesize ATP (adenosine
triphosphate,the"molecularcurrency"of
intracellular energy transfer) using chemical energy
generated by the redox reactions. The redox reactions
occur in thermodynamic nonequilibrium conditions
created upon mixing reduced hydrothermal fluidswith oxygenated seawater. The energy obtained by
the redox reactions can be calculated as Gibbs freeenergy 55. The amount of energy, as well as reaction
rate of the metabolism, constrain which
chemolithoautotrophs thrive and what kind of the
microbial ecosystem is constructed in thehydrothermal environment. Chemolithoautotrophic
hyperthermophilic Archaea that use hydrogen as a

5. Possible metabolic reactions in the ancient
ecosystem
Most of the deep-branching microorganisms in
the universal phylogenetic tree constructed from 16S
rRNA
gene
sequencesareanaerobic
hyperthermophiles, implying that anaerobicity andthermophilicity may be the ancestral characteristics
of life 16. Many anaerobic hyperthermophiles havebeen isolated from hydrothermal fields, especiallymembers of the Archaea as shown in Fig. 4. The
order of emergence of these archaeal lineages is still
controversial.
Some of these deep-branching anaerobic- 49 -

Viva Origino 38 (2010) 46 - 55

ancient ecosystems in the hydrothermal fields in theearly Earth. In terrestrial environments, Stevens and
McKinley 74 first suggested and Chapelle andcoworkers 75 reconfirmed that a hydrogen-based
subsurface lithoautotrophic microbial ecosystem(SLiME) dominated by methanogens existed in deep
continental basaltic crust. Hydrogen, the energy
source of methanogens, could be supplied fromwater-basalt reactions 76 or tectonic activity 77. The
SLiME could sustain itself completely independent ofa photosynthetic ecosystem. In marine environments,
Takai
and
coworkers
suggestedthatahydrogen-based
hyperthermophilic
SLiME(hyperSLiME) may be present below a deep-sea
hydrothermal field, an idea supported by geochemical(the presence of methane with lighter carbon isotopic
composition and high H2 concentration in thehydrothermal fluids) and microbiological (thepredominance of hyperthermophilic methanogens)evidence 63. Ueno and his colleagues exhibited
geological evidence for the presence of methanogens
in an ancient hydrothermal environment 1. It is highly
likely that H2 and CO2 were present in the ancienthydrothermal system. The phylogenetic tree of all life
based on small subunit rRNA gene sequencessuggests that one of the deepest-branching membersof the Archaea is a hyperthermophilic methanogen,
Methanopyrus (Fig. 4). This genus contains the most
hyperthermophilic microorganism identified to date,
M. kandleri strain 116, which can grow at 122ºC 78.
These
facts
supportthepresenceof
hyperthermophilic methanogens in the ancient
ecosystem.

hyperthermophilic Archaea gain energy bymethanogenesis,
fermentation,andhydrogen-oxidizing and sulfate-, sulfur- oriron-reducing reactions. These members could live inmarine hydrothermal environments without any othermicroorganisms because all essential matter for their
growth is abiotically supplied from the marinehydrothermal system. Furthermore, these anaerobic
hyperthermophilic Archaea have been detected in all
types of magmatic hydrothermal fields 60, except the
Hotspot (i.e., Loihi seamount) and the non-magmatichydrothermal field (i.e., the Lost City). It should be
noted that quantitative data regarding the abundanceof each archaeal group in these environments and theefficiency of each energy-yielding reaction does notdirectly indicate the order of emergence of theselineages. If the Gibbs free energy of theenergy-yielding reactions is less than zero 70 (lessthan -20 kJ mol-1 is favorable 71), the microorganism
could grow using any of these energy-yielding
reactions. As long as H2, CO2, CO, Fe3+ (potentiallygenerated by photooxidation of Fe2+)72, SO42- and S0(potentially generated by photolysis of SO2)73, aminoacids 24 and peptides 25 are abiotically supplied fromthe ancient marine hydrothermal system and ocean
surface,anymicroorganisms
using
these
energy-yielding reactions (both respiration andfermentation) could have existed in the ancienthydrothermal environment as primary producers in
the ancient ecosystem before the evolution ofphotosynthesis.
It is the most likely scenario that
hyperthermophilic methanogens dominated in theFig. 4. A phylogenetic tree of the domain Archaea constructed based on the 16S rRNA gene sequences. The tree wasconstructed using Phyml 91 by maximum-likelihood analysis of 1081 homologous positions in the alignment dataset
generated using ClustalW 92, using Thermus thermophilus (NC_006461), Aquifex aeolicus (NC_000918) and Thermotoga
maritima (NC_000853) as the out-group (not shown). All 16S rRNA gene sequences (total 62 taxa) used for the tree werecollected from the whole genome sequences of each archaeon or from fosmid library sequences. Bold branches indicate
groups including hyperthermophiles. MHVG-1: Marine Hydrothermal vent group 1. The Nanoarchaeota was excluded
from the tree to avoid long-branch attraction. Bootstrap values (higher than 50%) based on 100 replicates are shown atbranch points. The scale bar represents the expected number of changes per nucleotide position.
- 50 -

Viva Origino 38 (2010) 46 - 55

Hyperthermophilic hydrogen-oxidizing andsulfate-reducing microorganisms also potentiallyexisted in the ancient hydrothermal fields. It has beenfound that sulfide minerals in Archaean rocks were
isotopically fractionated 2,4,79. Sulfide precipitates
produced by sulfate reducing microorganisms havelower values of δ34S80. The Archaean sulfide minerals
with relatively lower δ34S values than seawater
sulfate indicate that microbial sulfate reduction
occurred before 3.4 Gya 80. Hyperthermophilichydrogen-oxidizing and sulfate-reducing Archaea
and Bacteria have been detected in the present
marine hydrothermal fields. The Archaeogolobi is thesole archaeal group containing hydrogen-oxidizing
and sulfate-reducers, e.g., Archaeoglobus veneficus,
which was isolated from a deep-sea hydrothermal
field 58. Sulfate must have been present in the earlyocean, although the concentration and origin are still
79,81
controversial.
Thus,hyperthermophilichydrogen-oxidizing
andsulfate-reducing
microorganisms could have grown in the ancienthydrothermal fields.
Hyperthermophilic hydrogen-oxidizing andsulfur- or iron-reducing microorganisms, such as
members of the Thermoprotei, could also have beenpresent in the ancient hydrothermal fields, if Fe3+ andS0 had been present in the Archaean ocean72,73.
However, the iron or sulfur isotopic fractionations
resulting from metabolic reactions catalyzed byhyperthermophilic
sulfuroriron-reducing
microorganisms are inconclusive. Further geologicalevidence and analysis of microbial isotopic
fractionation are required to verify the presence ofhyperthermophilic
sulfuroriron-reducing
microorganisms in the ancient hydrothermal fields.
Amino acid- (or peptide-) fermentation isthought to be an earlier energy-yielding metabolic
reactionthanmethanogenesisor
otherhydrogen-based respirations because of its metabolic
simplicity: amino acid-fermentation requires fewer
enzymes than respiration 82. In addition, thedeep-branchingarchaealgroups,such
asThermococci, Archaeoglobi, Thermoprotei andKorarchaeota, include amino acid-fermenting species.
The ubiquity of amino acid-fermentation among the
deep-branching Archaea also supports the idea
proposed by Clarke and Elsden 82. If all matter,
including amino acids and trace elements that areneeded for the growth of fermenting microorganisms
had been continuously and abiotically supplied by theancient hydrothermal system of the early Earth 13,24,
these organisms could have functioned as primary
producers in the microbial ecosystem as well as
methanogens. However, this hypothesis has aproblem: the H2-producing fermentation could notrelease energy from the reaction for the growth of
microorganisms at high H2 concentration, whereas
the H2 concentration at the ancient hydrothermal
system would be high and abiotic production ofamino acids would occur easier at higher H2concentration 24. If there was an anaerobic
environment around the ancient hydrothermal field
where amino acids accumulated at low H2- 51 -

concentration, fermenting microorganisms could
have thrived there: it is possible that amino acids
adsorbed onto minerals (for example, Si or mineraloxides) in hydrothermal fluids 83, were precipitated,pooled and concentrated on the seafloor where theamino acid fermentation reactions could proceed. It
goes without saying that this hypothesis needs
additional experimental evidence.
6. Detection, cultivation and characterization ofdeeper and deeper lineages of life
What can we do to characterize the ancient lifein the early Earth? The simplest approach for a
microbiologist is to find and describe all prokaryotes
existing in the present Earth, construct the correct
phylogenetic tree of them and determine thedeepest-branching species. The physiologicalcharacteristics of the deepest-branching species may
be most similar to those of the ancient life in the early
Earth. However, this is not enough to unveil theancient life. It is important to propose hypotheses
based on the theoretical, experimental, andobservational approaches based not only on
microbiological but also on other related scientificfields.
Unrecognized,
deeper-branching
microorganisms may be living in the present
hydrothermal fields. Although the characteristics ofthe deeper-branching species do not directly describe
ancient life in the early Earth, it will provide
information on early evolution. There may be no end
to this approach as a game of hide-and-seek.
However, we know that this strategy could help us
not only to understand ancient life but also tounderstand what life is: the limit of life, habitabilityof life and unexpected novel function of life.
Culture-independent molecular analysis (mainly
based on PCR) has suggested that only 1% or less ofthe microorganisms living on Earth have beencultivated 84. In fact, while diverse 16S rRNA genesequences of both Archaea and Bacteria have been
detected in marine hydrothermal fields 47-52, most of
this diversity is as yet uncultured. Furthermore,
technical
limitationsof
PCR-based
culture-independent molecular methods must be
considered 83. The PCR primers used formetagenomic analysis have been targeted to common
regions within genes, e.g., 16S rRNA, based on the“known”
sequences.
If
certainuncultured
microorganisms have sequence differences in theseregions, they may never be detected by traditional
PCR analysis even if they are abundant in particularenvironments. This problem can be solved by using
PCR-independent metagenomic analysis, e.g., wholegenome shotgun sequencing (WGS). Actually, WGS
analysis has revealed the presence of uncultured
Archaea in an acid mine drainage 85, which had neverbeen recognized because they have unique 16S rRNA
gene sequences. To date, there are no reports of
microbial communities at deep-sea hydrothermal
fields using WGS analysis of environmental DNA.
These facts suggest that not-yet-recognized, much
more diverse microorganisms are likely to be present
in marine hydrothermal fields.

Viva Origino 38 (2010) 46 - 55

Fig. 5. A phylogenetic tree of the MHVG-1 based on 16S rRNA gene sequences. The tree was constructed using Phyml 91
by maximum-likelihood analysis of 710 homologous positions in the alignment dataset generated using ClustalW 92, using
sequences of the Korarchaeota as the out-group (not shown). Bootstrap values (higher than 50%) based on 100 replicatesare shown at branch points. The scale bar represents the expected number of changes per nucleotide position.

found. Microbiology of hydrothermal systems is also
still developing. The present image of the ancient
ecosystem described in this paper and other previous
reports is based on limited knowledge. To propose
the most reliable hypothesis of the primary producer
in the ancient ecosystem, it is important to expand
our knowledge of the geology, geochemistry andmicrobiology of present hydrothermal systems. Moreexploration of the seafloor, additional understandingof hydrothermal systems and more microbiologicalbreakthroughs for both culture-independent and-dependent methods are needed.

The Marine Hydrothermal Vent Group-1(MHVG-1), which is one of the deepest-branchingarchaeal lineages 52 (Fig. 4), was detected in various
marine hydrothermal fields including mid-ocean
ridges, an island-arc and back-arc basins (Fig. 5). The
MHVG-1 Archaea may represent hyperthermophiles
based on the estimated optimum growth temperature.
The optimum growth temperature of 88ºC was
calculated based on the GC content of the 16S rRNA
gene sequences of MHVG-1 using a correlation
between the optimum growth temperature and GC
content 86. The physiological function (e.g., theenergy-yielding metabolism) of this deep-branching
MHVG-1 Archaea would provide useful information
about early evolution of life. However, no isolate
species of MHVG-1 have been reported. The
sequence information alone does not tell us thephysiology, e.g., optimal growth temperature and pH,
growth rate, morphology, or the complete metabolic
function of the microorganism. Classical cultivation
methods (e.g., batch culture) might be not suitable forthe cultivation, isolation and characterization of
MHVG-1. High-pressure cultivation is a provenmethod for cultivation of novel thermophiles living inmarine hydrothermal fields 87. Another useful method
is a flow-type cultivation system called continuous
cultivation or chemostat 88. In fact, several novel
thermophiles have been cultured from marinehydrothermal samples using a flow-type cultivation
system 89,90. Such contrivances of the cultivation
systemmayberequiredtocultureas-yet-not-cultivated microorganisms living in marinehydrothermal fields.

9. Acknowledgments
We thank the editors of this journal for giving
us the opportunity to publish this review. This
research was funded by the Ministry of Education,
Culture, Science and Technology (MEXT), Japan,
through a special coordination fund (Project TAIGA:
Trans-crustal Advection and In-situ biogeochemical
processes of Global sub-seafloor Aquifer).
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