RESEARCH LETTER
Molecular characterization of the microbial community in
hydrogenetic ferromanganese crusts of the Takuyo-Daigo
Seamount, northwest Paci¢c
Shota Nitahara1, Shingo Kato1, Tetsuro Urabe2, Akira Usui3 & Akihiko Yamagishi1
1
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, Japan; 2Department of Earth and Planetary Science,
University of Tokyo, Bunkyo-ku, Tokyo, Japan; and 3Department of Natural Environmental Science, Faculty of Science, Kochi University, Kochi-shi,
Kochi, Japan
Correspondence: Akihiko Yamagishi,
Department of Molecular Biology, Tokyo
University of Pharmacy and Life Science,
1432-1 Horinouchi, Hachioji, Tokyo 1920392, Japan. Tel.: 181 426 76 7139; fax: 181
426 76 7145; e-mail: [email protected]
Received 14 February 2011; revised 9 May
2011; accepted 24 May 2011.
Final version published online 23 June 2011.
DOI:10.1111/j.1574-6968.2011.02323.x
MICROBIOLOGY LETTERS
Editor: Aharon Oren
Keywords
16S rRNA gene; Bacteria; Archaea;
ferromanganese crust; microbial ecosystem.
Abstract
The abundance and phylogenetic diversity of the microbial community in the
hydrogenetic ferromanganese crust, sandy sediment and overlying seawater were
investigated using a culture-independent molecular analysis based on the 16S
rRNA gene. These samples were carefully collected from the Takuyo-Daigo
Seamount, located in the northwest Pacific Ocean, by a remotely operated vehicle.
Based on quantitative PCR analysis, Archaea occupy a significant portion of the
prokaryotic communities in the ferromanganese crust and the sediment samples,
while Bacteria dominated in the seawater samples. Phylotypes belonging to
Gammaproteobacteria and to Marine group I (MGI) Crenarchaeota were abundant
in clone libraries constructed from the ferromanganese crust and sediment
samples, while those belonging to Alphaproteobacteria were abundant in that from
the seawater sample. Comparative analysis indicates that over 80% of the total
phylotype richness estimates for the crust community were unique as compared
with the sediment and seawater communities. Phylotypes related to Nitrosospira
belonging to the Betaproteobacteria and those related to Nitrosopumilus belonging
to MGI Crenarchaeota were detected in the ferromanganese crust, suggesting that
these ammonia-oxidizing chemolithoautotrophs play a role as primary producers
in the microbial ecosystem of hydrogenetic ferromanganese crusts that was formed
as precipitates from seawater.
Introduction
Ferromanganese deposits are often found at the boundary
between the hydrosphere and the lithosphere in natural
environments. Rocks coated with ferromanganese oxides
are found on modern seafloors as ferromanganese nodules
and crusts (hereafter, Mn nodules and Mn crusts) depending on their mode of occurrence (e.g. Usui & Someya, 1997;
Glasby, 2006; Wang & Müller, 2009). Mn nodules and crusts
mainly consist of Mn and Fe oxides, more than 30% of the
total mass (Mero, 1962), and contain other economic
metals, for example, Co, Ni, Cu, Zn, rare earth elements
and Pt (Hein et al., 2000). Oceanic ferromanganese deposits
grow extremely slowly at rates of about 1–10 mm Myr1 as
determined by radioisotope dating (Hein et al., 2000; Usui
et al., 2007). Although hydrothermal ferromanganese deFEMS Microbiol Lett 321 (2011) 121–129
posits occur in areas associated with volcanic activity,
hydrogenetic ferromanganese deposits are distributed
widely on the deep seafloor (Rona, 2003). Considering the
wide distribution of Mn nodules and crusts on the seafloor
and their potential for future mineral resources (Rona,
2003), the study of microorganisms attached to the Mn
nodules and crusts is important to understand the significance of the role of microorganisms in the elemental cycle
between the ocean and the hydrogenetic oxides. This knowledge is likely to help us develop deep-sea mining techniques
utilizing microorganisms in future (Ehrlich, 2001).
Despite the early discovery of Mn nodules and crusts on
the seafloor, little is known about the microbial communities and their role in Mn nodule formation. In terrestrial
environments, microbial communities on ferromanganese
oxides have been reported from caves (Northup et al., 2003),
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122
soils (He et al., 2008) and freshwater sediments (Stein et al.,
2001), suggesting that diverse prokaryotes are present on
and/or within the ferromanganese oxides. Electron microscopic observation has shown that microorganism-like
structures are present on the oceanic ferromanganese oxides
(Wang et al., 2009). The presence of phylogenetically diverse
bacteria in the seafloor basalt covered with thin (o200 mm)
ferromanganese oxides on the East Pacific Rise has been
reported (Santelli et al., 2008). However, our knowledge of
the spatial distribution, diversity and abundance of microbial communities on oceanic ferromanganese oxides is still
limited.
Here, we report on the abundance, diversity and composition of the microbial community of an oceanic Mn crust
by a culture-independent molecular microbiological analysis. The Mn crust was carefully collected with on-site
observation using a remotely operated vehicle, enabling us
to investigate microorganisms on the undamaged surface of
the Mn crust that is exposed to overlying seawater by
molecular microbiological analysis. The Takuyo-Daigo Seamount of the sampling field is a flat-topped seamount that is
located approximately 150 km southeast of Minamitorishima Island, Japan, in the northwest Pacific Ocean (Supporting Information, Fig. S1). This area is one of the oldest
seafloors in the world (4150 million years, Müller et al.,
2008). No age determination has been carried out on the
Takuyo-Daigo Seamount, but the age of nearby seamounts
is around 80 million years. This seamount has a flat-top at a
depth of 810 m, elevating more than 4000 m from the
abyssal seafloor of 5300 m. The Mn crusts were collected
from the slope of the seamount at a water depth of 2991 m.
In addition to the Mn crust, we also sampled and analyzed
the overlying seawater and surrounding sandy sediment
using the same methods to assess the uniqueness of the
microbial communities of the oceanic Mn crust.
Materials and methods
Sample collection
The Mn crusts, sandy sediments and overlying seawater
samples were collected on the slopes of the Takuyo-Daigo
Seamount (Figs 1 and S1) at 2991 m water depth during the
NT09-02 cruise (February 8–23, 2009) of the R/V Natsushima (JAMSTEC, Japan) with the remotely operated vehicle
Hyper-Dolphin (JAMSTEC). The temperature, dissolved
oxygen concentration and salinity of the bottom ambient
seawater were 2 1C, 2.5 mL L1 and 34.0 practical salinity
units, respectively. The Mn crusts were carefully collected
using a manipulator on the vehicle while observing on TV
monitors. Samples of sandy sediments and seawater were
collected approximately 10 m from the sampling point of
the Mn crusts using a push-core and a Niskin bottle sampler,
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S. Nitahara et al.
(a)
(b)
Fig. 1. Photograph of on-site observation, a cross section image of a Mn
nodule and fluorescent microscopic image of the surface of a Mn nodule.
(a) On-site observation of Mn nodules and crusts at the sampling point.
(b) A representative image of the cross section of a Mn nodule. Fe–Mn
oxides of black materials coat a gray basement rock that contains orange
materials in spots.
respectively. Samples from 0 to 1 cm from the top of the
sediments, which were collected using a push-core sampler,
were used for analysis. Although the correct thickness of the
covering sediments is unknown, the thickness seemed to be
o 1 m judging from the depth of an iron stick inserted into
sediments at the sampling area. Once retrieved on-board,
the Mn crust samples were crushed using an autoclaved
hammer and chisel in a clean box. Parts of the Mn crusts and
sediments were transferred to a DNA/RNA-free plastic tube
and stored at 80 1C until DNA extraction. One liter of the
seawater sample was filtered with a 0.2-mm-pore-size polycarbonate membrane to trap the suspended particles
(Advantec, Tokyo, Japan) on board and then the filter was
stored in a DNA/RNA-free plastic tube at 80 1C until
DNA extraction.
16S rRNA gene analysis
Analysis of the 16S rRNA genes present in the collected solid
and liquid samples was performed as described previously
(Kato et al., 2009c, 2010). In brief, genomic DNA was
extracted from the samples using a Fast DNA kit for soil
(Qbiogene, Carlsbad, CA). Partial 16S rRNA genes were
amplified by PCR with the prokaryote-universal primer set,
Uni515F and Uni1406R. The PCR products were cloned
using a TOPO TA cloning kit (Invitrogen, CA). The nucleotide sequences of randomly selected clones were determined
using M13 forward and reverse primers (Invitrogen) on an
ABI PRISM 3130xl Genetic analyser (Applied Biosystems,
FEMS Microbiol Lett 321 (2011) 121–129
123
Microorganisms in Mn crusts
Quantitative PCR (Q-PCR)
Bacterial and archaeal rRNA gene copy numbers in DNA
extracts from each sample were determined by Q-PCR as
described previously (Kato et al., 2009b). For bacterial rRNA
genes, the bacterial-specific PCR primers, Bac1369F (5 0 -CG
GTGAATACGTTCYCGG-3 0 ) and Prok1492R (5 0 -GGWTA
CCTTGTTACGACTT-30 ), and the TaqMan probe, TM1389F
(5 0 -CTTGTACACACCGCCCGTC-30 ), were used. For archaeal rRNA genes, the archaeal PCR primers, Arc349F (5 0 -CCTA
CGGGRBGCASCAG-3 0 ) and Arc806R (50 -GGACTACNNG
GGTATCTAAT-30 ), and a TaqMan probe, Arc516F (5 0 -TGY
CAGCMGCCGCGGTAAHACVNRS-30 ), were used. The purified PCR products from the 16S rRNA gene of Escherichia coli
and environmental archaeal clones belonging to Marine group
I (MGI) were used as the standard DNA for bacterial and
archaeal analyses, respectively. All assays were performed in
triplicate. Regression coefficient (r2) values of the standard
curve were 0.994 and 0.999 for bacterial and archaeal analyses,
respectively.
Accession number
The nucleotide sequences of the phylotypes reported in this
paper have been deposited in the DDBJ database under the
following accession numbers: AB606678–AB606781 for Mn
crust clones, AB606782–AB606862 for sediment clones and
AB606863–AB606968 for overlying seawater clones.
Results and discussion
The thickness of the Mn oxides covering the basement rock
was 20 mm (Fig. 1b; a representative image of the Mn
crusts collected). The chemical composition of the Mn crust
sample (0–3 mm from the surface) was determined by
inductively coupled plasma-optical emission spectrometry,
FEMS Microbiol Lett 321 (2011) 121–129
1×1010
Bacteria
1×109
Archaea
1×108
Cell numbers (g or mL)
CA). Nucleotide sequences were aligned and distance matrices were generated from alignment data sets from each
clone library using ARB (Ludwig et al., 2004). Clones having
97% sequence similarity or higher were treated as the same
phylotype using DOTUR (Schloss & Handelsman, 2005).
Maximum-likelihood trees were constructed using PHYML
(Guindon & Gascuel, 2003) with non-gap homologous
positions in the alignment dataset. Bootstrap values were
estimated using 100 replicates. Rarefaction analysis, the
Shannon diversity index and Chao1 richness estimators
were estimated using DOTUR based on the distance matrices
generated from the alignment data sets of the clones from
each clone library. Chao1 species richness estimates of
shared phylotypes were calculated using SONS (Schloss &
Handelsman, 2006). The phylogenetic (P)-test and the
UniFrac significance test were performed using UniFrac
(Lozupone et al., 2006).
1×107
1×106
1×105
1×104
1×103
1×102
1×101
1×100
Sample ID:
Sample type:
Bac/Arc%:
953Mn
Mn crust
34.5/65.5
953Sed
Sediment
15.3/84.7
953Asw
Seawater
98.4/1.6
Fig. 2. Bacterial and archaeal cell numbers estimated based on the
average 16S rRNA gene copy numbers. Error bars indicate the SDs.
Percentages of bacterial and archaeal cell numbers of the total cells are
shown at the bottom of the figure.
which yielded the following results: (wt%) 17.4% Fe, 16.0%
Mn, 1.62% Ca, 0.834% Na, 0.715% Ti, 0.663% Mg, 0.661%
Al, 0.389% K, 0.386% Co, 0.323% P, 0.209% Ni, 0.134% Pb,
0.118% S, 0.111% Sr. This sample also contained o 0.1%
Ba, V, Zn, Cu, Y, Cr and Sc as minor components. Although
the chemical composition of the sediments was not determined, these sediments are likely to consist of calcareous
shells of foraminifers that are generally found on the
seafloor of open oceans.
Bacterial and archaeal cell densities were estimated based
on the 16S rRNA gene copy numbers determined by Q-PCR
(Fig. 2). In principle, the quantification of microorganisms
by Q-PCR provides more reliable data than by clone library
analysis (Smith & Osborn, 2009). Our estimation is based
on the assumption that the genomes of bacterial and
archaeal cells have on average 4.06 and 1.77 copies of the 16S
rRNA gene, respectively (Lee et al., 2009). The total prokaryotic cell numbers were estimated to be 7.27 107 cells g1,
1.29 109 cells g1 and 8.20 103 cells mL1 for the Mn
crust, sediment and ambient seawater, respectively. The cell
numbers of deep-sea water (42000 m depth) are generally
0.8–2.0 104 cells mL1 as shown by direct counting (Karner
et al., 2001; Herndl et al., 2005; Kato et al., 2009c). Our result
of the seawater from Q-PCR was within the range reported
previously. Bacteria were found to be dominant in the seawater sample (98.4% of the total prokaryotic cell number; Fig.
2). In contrast, Archaea were found to be dominant in the Mn
crust and sediment (65.5% and 84.7%, respectively; Fig. 2).
The percentage of archaeal clones in the libraries (Fig. 3) did
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124
S. Nitahara et al.
not quantitatively match that obtained from Q-PCR (Fig. 2)
and is probably due to PCR bias. In fact, the prokaryoteuniversal primer set that was used does not amplify 16S rRNA
genes from all Archaea (Baker et al., 2003). However, the
relative abundance of archaeal clones in the libraries (17.3%
for the Mn crust, 24.7% for the sediment and 5.7% for the
seawater, respectively; Fig. 3) showed the same trend as the
results obtained by Q-PCR (65.5%, 84.7%, 1.6%, respectively;
Fig. 2): the relative abundance of archaeal clones was much
higher in the Mn crust and the sediment than in the seawater.
Fig. 3. Community structures based on the 16S rRNA gene clone
libraries. Pie charts show the percentage of each taxa of clones in total
clone numbers.
Although Archaea dominate in marine sediments (Lipp et al.,
2008), Archaea are thought to be a minor component of the
microbial community of seafloor basaltic rocks (Einen et al.,
2008) and also that within thin ferromanganese oxides
(Santelli et al., 2008). Our results suggest that Archaea occupy
a significant portion of the prokaryotic communities in aged
Mn crusts and sandy sediments.
The microbial communities on/within basaltic glass and
rocks on the seafloor have been well studied (Fisk et al.,
2003; Lysnes et al., 2004; Mason et al., 2007; Einen et al.,
2008; Santelli et al., 2008); however, little is known about
those on well-developed Mn crusts on the aged seafloor. For
the first time, we analyzed the composition and diversity
of Archaea and Bacteria on an aged Mn crust (Fig. 3 and
Table 1). The archaeal clones recovered from the Mn crust were
affiliated with MGI Crenarchaeota (Delong, 1992; Fuhrman
et al., 1992) and with the pSL12-related group (Barns et al.,
1996) (Fig. S2a). MGI includes the chemolithoautotrophic
ammonia-oxidizing archaeon Nitrosopumilus maritimus
(Könneke et al., 2005). The pSL12-related group may also
include ammonia oxidizers as inferred by the analysis of 16S
rRNA and archaeal amoA genes (Mincer et al., 2007; Kato et al.,
2009b). Several microdiverse phylogenetic clusters within MGI
have been defined in previous reports (Massana et al., 2000;
Takai et al., 2004; Durbin & Teske, 2010). Our MGI clones
recovered from the overlying seawater were affiliated with the
MGI-g (Fig. S2a). Those from the Mn crust and sediment
samples were affiliated with other MGI clusters such as the a,
Z–k–u, ı and e–z–y clusters (Fig. S2a). In the case study of the
South Pacific Gyre (Durbin & Teske, 2010), the relative
abundance of the MGI-a in the archaeal clone libraries has
been high in the overlying seawater and those of the MGI-Z
and –u have been high in the libraries from the sediments.
Although it is unclear what kinds of factors are responsible for
the relative abundance of each MGI cluster among deep-sea
environments, these differences may reflect differences in
geography, environmental characteristics and/or experimental
procedures (such as the DNA extraction methods and the PCR
primers used).
Table 1. Summary of 16S rRNA gene clone analysis
Sample ID
Sample type
Domain
Total clone number
Phylotype number
Chao1
Shannon score
Coverage (%)
953Mn
Mn crust
953Sed
Sediment
953Asw
Seawater
Combined
Bacteria
Archaea
Combined
Bacteria
Archaea
Combined
Bacteria
Archaea
104
86
18
81
61
20
106
100
6
70
65
7
52
46
7
40
39
1
223 (139–411)
193 (123–347)
9 (7–22)
105 (74–175)
106 (71–190)
7 (7–7)
69 (50–123)
68 (49–122)
1 (1–1)
4.07 (3.90–4.24)
4.06 (3.89–4.23)
1.72 (1.37–2.08)
3.77 (3.58–3.95)
3.72 (3.52–3.92)
1.81 (1.52–2.10)
3.24 (3.04–3.44)
3.20 (2.99–3.42)
NP
49
41
83
56
43
95
79
78
100
Numbers in parentheses indicate 95% confidential intervals.
NP, Not possible to calculate.
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FEMS Microbiol Lett 321 (2011) 121–129
125
Microorganisms in Mn crusts
In contrast to Archaea, diverse bacterial phylotypes were
detected in the Mn crust, sediment and seawater samples.
All analyses, i.e., Chao1 species estimates and the Shannon
index (Table 1) and rarefaction curves (Fig. S3), indicated
that the community diversity of Bacteria in the crust sample
was comparable to or higher than that in sediment and
overlying seawater. In addition, the diversity of Bacteria was
higher in all samples than those of Archaea (Table 1). The
bacterial diversity of the Mn crust was comparable to or
higher than those of seafloor basaltic rocks reported previously (Lysnes et al., 2004; Mason et al., 2007; Santelli et al.,
2008), suggesting that aged Mn crusts provide a habitat for
diverse Bacteria as in basaltic rocks.
Bacterial phylotypes dominated in all libraries (75.3–94.3%
of the total clone numbers; Fig. 3). The bacterial phylotypes
recovered were affiliated with the following phyla or uncultured clone groups (Figs 3 and S2b–e): Acidobacteria, Actinobacteria, Bacteroidetes, ‘Caldothrix’, Chlamydiae, Chloroflexi,
Nitrospirae, Planctomycetes, Proteobacteria (Alpha, Beta, Gamma and Delta subdivisions), Verrucomicrobia, BRC1, KSB,
NKB19, OP11, OP3, SAR406 and SBR1093. Several phylotypes were affiliated with unclassified environmental clone
groups, UBSedI to VI and UBMnI and II, as defined in the
present study (Fig. S2e). Phylotypes in the Gammaproteobacteria were abundant in the clone libraries from the Mn crust
and sediment samples (24.0% and 23.5% of the total clone
numbers, respectively; Fig. 3). These phylotypes were related
to not yet cultivated environmental clones recovered from
seafloor basaltic rocks (Lysnes et al., 2004; Mason et al., 2007,
2008; Santelli et al., 2008) rather than cultured species
( o 95% similarity) (Fig. S2b). In contrast, phylotypes in the
Alphaproteobacteria were abundant in the clone libraries from
the seawater sample (44.3% of the total clone number). In
particular, most of them were related to Candidatus Pelagibacter (SAR11 cluster, Rappé et al., 2002) and Sphingomonadales (Fig. S2c), groups from which members have often been
recovered from deep-sea water of 4 1000 m water depth
(Garcı́a-Martı́nez & Rodrı́guez-Valera, 2000; Delong et al.,
2006; Kato et al., 2009a, c).
Comparative analysis showed that the microbial community composition of the Mn crust was different from those of
the sediment and overlying seawater. The differences among
the three communities were supported by the UniFrac
significance and P values (o0.01). To compare the microbial
community composition, the shared phylotype numbers
among the libraries from the crust, sediment and seawater
samples were estimated using SONS. The Mn crust and
sediment communities shared few or no phylotypes with
the seawater community (Fig. 4). The Mn crust community
contained a fraction of phylotypes recovered from the
sediment sample (20% of the total phylotype richness
estimates of the Mn crust; Fig. 4). Thus, 80% of the total
phylotypes richness estimates of the Mn crust community
FEMS Microbiol Lett 321 (2011) 121–129
953Mn
= 223 (139 – 411)
S
173
48
2
67
S
953Asw
= 69 (50 – 123)
57
S
953Sed
= 105 (74 – 175)
Fig. 4. Venn diagram comparing the communities of the samples.
Numbers in circles indicate Chao1 species richness estimates. Numbers
in parentheses indicate 95% confidential intervals.
were unique compared with the sediment communities. In
fact, unique phylotypes of the Mn crust were observed in the
phylogenetic trees (Fig. S2). Several phylotypes in MGI were
shared between the Mn crust and sediment, but not between
the Mn crust and seawater (Fig. S2a) as described above.
Phylotypes related to the genus Nitrosospira in the
Betaproteobacteria were unique in the Mn crust (Fig. S2b).
Representative clone 953Mn48u has 97% similarity to the
ammonia-oxidizing chemolithoautotrophic bacterium Nitrosospira multiformis (Watson et al., 1971). Phylotypes
related to the family Ectothiorhodospiraceae in the Gammaproteobacteria were also unique in the library of the Mn crust
(Fig. S2b). Representative clone 953Mn100u has 94% similarity to the arsenite-oxidizing chemolithoautotroph Alkalilimnicola ehrlichii (Hoeft et al., 2007) or sulfur-oxidizing
chemolithoautotrophic Thioalkalivibrio species (Sorokin
et al., 2001). These results suggest that putative ammonia(or in some cases, sulfur- and/or arsenite-) oxidizing chemolithoautotrophs are present on the Mn crust surface.
The detection of the phylotypes related to ammoniaoxidizing Archaea and Bacteria in the Mn crust suggests that
these putative ammonia oxidizers may play a role as primary
producers in the microbial ecosystem on Mn oxides that
coats old seamounts in western Pacific. Although the
ammonium concentration in the open ocean is generally
extremely low (o 5 mM) (Rees et al., 2006; Herfort et al.,
2007; Agogue et al., 2008), ammonia-oxidizing Archaea
belonging to MGI Crenarchaeota can grow under these
conditions using ammonium as the energy source (Martens-Habbena et al., 2009). Ammonia-oxidizing bacteria can
also grow at low concentrations of ammonium (Bollmann &
Laanbroek, 2001; Bollmann et al., 2002). In fact, we detected
both bacterial and archaeal amoA genes, which encode the
alpha subunit of the ammonia monooxygenase, from DNA
extracted from the Mn crust (the data will be published
elsewhere). Ammonia is the most likely chemical species to
be utilized as an electron donor for microbial growth on the
Mn crust. Dissolved organic carbon compounds in deep-sea
water may resist microbial growth (Barber, 1968). Buried
organic compounds from surface seawater may be limited
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126
on the Mn crust because little sandy sediment is formed
(Fig. 1a). Accordingly, H2, CH4, H2S, Fe21 and Mn21 from
the degradation of organic compounds by anaerobes and
fermenters would be limited on the Mn crust. Fe21 and
reduced sulfides contained in basaltic rocks are thought to
be energy sources for the microorganisms on the rocks
(Bach & Edwards, 2003; Santelli et al., 2008), but the
argument is still controversial (Templeton et al., 2009). Our
data suggest that ammonia in surrounding seawater is likely
to be an important energy source for sustaining the microbial ecosystem on the Mn crust. Furthermore, the presence
of ammonia-oxidizing bacteria on oceanic basaltic rocks has
been supported by the detection of 16S rRNA genes related
to these members such as Nitrosospira (Mason et al., 2008;
Santelli et al., 2008). These facts lead to the hypothesis that
the ammonia oxidizers play a role in the microbial ecosystem on outcrops of the global seafloor including bare young
basalts and aged Mn crusts.
One of the subjects in the study of oceanic Mn nodules
and crusts is the mechanism of their creation and growth.
Microorganisms may play a role in the accumulation of Mn
oxides by biofilm formation on rocks on the seafloor (Wang
& Müller, 2009). This notion is consistent with the detection
of abundant microorganisms, both Bacteria and Archaea,
within/on the Mn crust (Fig. 2). Mn-oxidizing bacteria,
which are thought to play a role in Mn precipitation in the
first step of the biomineralization model for Mn crusts as a
bioseed (Wang & Müller, 2009), have been isolated from
marine environments (Tebo et al., 2005). This model is
supported by the detection of phylotypes related to the
Leptothrix in the Betaproteobacteria (953Asw11u; Fig. S2b),
Erythrobacter and Aurantimonas in the Alphaproteobacteria
(953Asw97u and 953Asw05u; Fig. S2c) and Arthrobacter in
the Actinobacteria (953Asw07u; Fig. S2d), which includes
marine Mn-oxidizing bacteria (Tebo et al., 2005), from the
overlying seawater, but not from the Mn crust and sediment
samples. Although no phylotypes related to the known Mn- or
Fe-oxidizing bacteria were detected in the Mn crust and
sediment, there is a possibility that as yet uncultivated Mnor Fe-oxidizing bacteria are hidden in the diverse phylotypes
detected. Further analyses, for example, isolation and characterization of Mn- and Fe-oxidizing bacteria, quantification
of their abundance and determination of rates of Mn and Fe
oxidation by them are required to elucidate the significance of
their role in the formation of the Mn crusts. A recent study has
shown that manganese precipitation is promoted by superoxide that is produced by enzymatic activity of marine
bacteria (Learman et al., 2011). This biogenic superoxide is
also potentially related to the precipitation of Mn in overlying
seawater and on the surface of Mn crusts.
Two common features are found between the microbial
communities in the oceanic Mn crust shown in the present
study and those in the freshwater Mn nodules reported by
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S. Nitahara et al.
Stein et al. (2001). Firstly, many bacterial phylotypes
detected in the Mn crust and nodules have low similarity
( o 96%) to known cultured species. Secondly, the phylotypes relatively close to Hyphomicrobium in the Alphaproteobacteria and Leptothrix in the Betaproteobacteria, both of
which include Mn-oxidizing bacteria, and the phylotypes
close to MGI Crenarchaeota were detected in both environments. Our phylotypes related to these members were
detected in the Mn crust, sediment and/or overlying seawater (Fig. S2b and c). It is unclear how these phylotypes are
distributed among the Mn nodules, surrounding sediments
and overlying lake water in the freshwater environment
(Stein et al., 2001). Nevertheless, phylotypes related to these
genera (i.e. Hyphomicrobium and Leptothrix) may play a role
in Mn accumulation on solid surfaces in marine and freshwater environments.
Although numerous studies of microbial communities in
coastal sediments have been conducted, those in deep-sea
sediments in open oceans that are far from lands are poorly
understood. Deep-sea sediments in open oceans are nutrient-poor (i.e. oligotrophic) environments (D’hondt et al.,
2004), except for hydrothermal vents and cold seep areas.
Previous reports have suggested that there are diverse
uncultured species on the surface of such deep-sea sediments and the relative abundances of phylotypes belonging
to Gammaproteobacteria and MGI Crenarchaeota are high in
these environments (Li et al., 1999; Vetriani et al., 1999;
Bowman & Mccuaig, 2003; Schauer et al., 2009; Durbin &
Teske, 2010). These results are compatible with our results of
the sediment sample (Figs 3 and S2), suggesting that these
features of the microbial community structures may be
common on the surface of oligotrophic deep-sea sediments
in open oceans.
Acknowledgements
We would like to thank the crew of the R/V Natsushima and
the operation team of the ROV Hyper-Dolphin for their
cooperation in sample collection. We would like to thank Dr
Blair Thornton for providing the on-site photograph of the
Mn crust and for English language editing. We would like to
thank Ms Satomi Minamizawa for her technical assistant on
the cruise. We are also grateful to the scientists who joined
the NT09-02 cruise and to Dr Katsuhiko Suzuki and the
other members of the Project TAIGA for providing valuable
samples and for helpful discussions. We would like to thank
two anonymous reviewers for their helpful comments. 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).
FEMS Microbiol Lett 321 (2011) 121–129
127
Microorganisms in Mn crusts
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Supporting Information
Fig. S1. (a) Location of the Takuyo-Daigo Seamount and (b)
an enlarged view of the sampling point.
Fig. S2. Phylogenetic trees for 16S rRNA genes of (a)
Archaea, (b) Gammaproteobacteria and Betaproteobacteria,
(c) Alphaproteobacteria and Deltaproteobacteria, (d) other
bacterial phyla, and (e) uncultured clone groups.
Fig. S3. Rarefaction curves for (a) Bacteria and (b) Archaea.
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