Jaeschkeetal2012

Jaeschkeetal2012
Geobiology (2012)
DOI: 10.1111/gbi.12009
Microbial diversity of Loki’s Castle black smokers at the
Arctic Mid-Ocean Ridge
A. JAESCHKE,1* S. L. JØRGENSEN,2 S. M. BERNASCONI,1 R. B. PEDERSEN,3
I . H . T H O R S E T H 3 A N D G . L . F R Ü H - G R E E N 1
1
Department of Earth Sciences, ETH Zurich, Zurich, Switzerland
Department of Biology, Centre for Geobiology, University of Bergen, Bergen, Norway
3
Department of Earth Sciences, Centre for Geobiology, University of Bergen, Bergen, Norway
2
ABSTRACT
Hydrothermal vent systems harbor rich microbial communities ranging from aerobic mesophiles to anaerobic hyperthermophiles. Among these, members of the archaeal domain are prevalent in microbial communities in the most extreme environments, partly because of their temperature-resistant and robust membrane
lipids. In this study, we use geochemical and molecular microbiological methods to investigate the microbial
diversity in black smoker chimneys from the newly discovered Loki’s Castle hydrothermal vent field on the
Arctic Mid-Ocean Ridge (AMOR) with vent fluid temperatures of 310–320 °C and pH of 5.5. Archaeal
glycerol dialkyl glycerol tetraether lipids (GDGTs) and H-shaped GDGTs with 0–4 cyclopentane moieties
were dominant in all sulfide samples and are most likely derived from both (hyper)thermophilic Euryarchaeota and Crenarchaeota. Crenarchaeol has been detected in low abundances in samples derived from
the chimney exterior indicating the presence of Thaumarchaeota at lower ambient temperatures. Aquificales and members of the Epsilonproteobacteria were the dominant bacterial groups detected. Our observations based on the analysis of 16S rRNA genes and biomarker lipid analysis provide insight into microbial
communities thriving within the porous sulfide structures of active and inactive deep-sea hydrothermal
vents. Microbial cycling of sulfur, hydrogen, and methane by archaea in the chimney interior and bacteria
in the chimney exterior may be the prevailing biogeochemical processes in this system.
Received 13 April 2012; accepted 16 August 2012
Corresponding author. Andrea Jaeschke. Tel.: +41 44 6326349; fax: +41 44 6321636; e-mail: andrea.
[email protected]
INTRODUCTION
Deep-sea hydrothermal vents are unique environments that
support highly productive ecosystems driven by geochemical energy and have been proposed as a possible site for
the origin and early evolution of life (Baross & Hoffman,
1985; Martin et al., 2008). The distinctive black smoker
chimneys at submarine hydrothermal environments are
formed when sulfates and sulfides precipitate due to mixing
of hot, acidic, and anoxic vent fluids with cold oxic seawater, thus creating steep thermal and chemical gradients
along and inside the chimney (Tivey, 1995). Migrating fluids contain reduced inorganic and organic components
providing metabolic energy for diverse thermophilic microbial communities that inhabit either specific microniches in
different parts of the porous chimney structure, appear as
© 2012 Blackwell Publishing Ltd
free-living micro-organisms in vent fluids and plumes, or
occur as symbionts of vent macrofauna (Karl, 1995). These
micro-organisms are adapted to a habitat characterized by
extreme environmental conditions such as high temperature and pressure, low pH as well as elevated concentrations of dissolved gases (H2S, H2, CO2, CH4) and metal
sulfides (Miroshnichenko, 2004).
Since the discovery of deep-sea hydrothermal vent systems in the late 1970s (Corliss et al., 1979), enrichment
and isolation studies as well as culture-independent
approaches, which mainly involved molecular studies of the
16S rRNA gene, were performed. These studies revealed a
remarkable microbial diversity with numerous so far uncultivated organisms thriving in these extreme and unstable
habitats (Takai et al., 2001; Schrenk et al., 2003; Alain
et al., 2004; Kormas et al., 2006; Sogin et al., 2006).
1
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A. JAESCHKE et al.
populations. Here, we report first insights into the microbial assemblages inhabiting active and inactive sulfide
chimneys of the Loki’s Castle hydrothermal vent field based
on biomarker lipid analysis and 16S rRNA gene-based
taxonomy.
Hyperthermophilic archaea tend to be dominant in the
high temperature zones of the chimney interiors, whereas
both archaea and bacteria are present in the cooler exterior
parts where mixing with seawater is more prevalent
(Schrenk et al., 2003). Along with the apparent temperature zonation, where distinctive micro-organisms can
thrive, different metabolisms are thermodynamically available. For example, oxidation of methane, ammonia, and
sulfur are favored at low temperatures toward the chimney
exterior, while methanogenesis and reduction of sulfate or
sulfur is favored at higher temperatures toward the chimney interior (McCollom & Shock, 1997; Takai et al.,
2001; Schrenk et al., 2003; Kormas et al., 2006; Takai &
Nakamura, 2011).
In addition to molecular techniques, organic geochemical approaches can be employed to reveal microbial community structures by analyzing lipid biomarkers. Archaea
synthesize distinctive membrane lipids predominantly composed of diether lipids (i.e., archaeol, hydroxyarchaeol) and
isoprenoid glycerol dialkyl glycerol tetraethers (GDGTs).
GDGTs with an additional covalent bond, so-called
H-shaped GDGTs, have so far only been reported for cultivated isolates of (hyper-) thermophilic archaea (Morii
et al., 1998; Sugai et al., 2004; Koga & Morii, 2005;
Schouten et al., 2008). GDGTs are excellent biomarkers
to use in hydrothermal vents as they are community-specific and resistant to extreme environmental conditions
(Derosa & Gambacorta, 1988; van de Vossenberg et al.,
1998; Macalady et al., 2004). There are, however, only
few studies carried out on biomarker lipids in deep-sea
hydrothermal vents indicating possible microbial communities with their potential metabolisms (Blumenberg et al.,
2007; Blumenberg et al., 2012; Bradley et al., 2009).
Loki’s Castle is a deep-sea hydrothermal vent field
located at the ultra-slow spreading Arctic Mid-Ocean Ridge
(AMOR) in the Norwegian-Greenland Sea at 74°N. Discovered in 2008, it is the northernmost black smoker field
known to date and hosts a unique ecosystem (Pedersen
et al., 2010). It is a sediment-associated system, and therefore, hydrothermal fluids contain an unusual enrichment of
carbon dioxide, methane, and ammonia, providing potential for various energy metabolisms and diverse microbial
7°00E
The Knipovich Ridge is one of the AMOR spreading centers in the Norwegian-Greenland Sea, and the southernmost part of this ridge is one of the slowest spreading
ridge segments on Earth. The Loki’s Castle vent field is
located at 2400 m water depth on an axial volcanic ridge
(AVR) where the magma-starved end of the Mohns Ridge
migrates into the Knipovich Ridge through a sharp
northward bend in the direction of the spreading axis at
73°30′N and 8°E (Fig. 1) (Pedersen et al., 2010). At the
seafloor, black smoker fluids are discharging from four, up
to 13 m tall, chimneys. The chimneys are situated on two
hydrothermal mounds that are approximately 150 m apart
and are estimated to be 20–30 m high and about 150–
200 m across. The mounds of Loki’s Castle are comparable in size with the TAG-mound (Trans-Atlantic
Geotraverse) on the Mid-Atlantic Ridge (Pedersen et al.,
2010). The four active chimneys were named João,
Menorah, Camel, and Sleepy. João is the tallest of the four
chimneys, situated on the eastern sulfide mound.
The hydrothermal fluids from Loki’s Castle reach temperatures of 310–320 °C and have a pH of 5.5. The
vent fluid compositions are characterized by high concentrations of CH4, H2, and NH4, as well as elevated
concentrations of higher hydrocarbons, and are indicative
of a sediment-impacted hydrothermal vent system (Pedersen et al., 2010; Baumberger, 2011). Significant sediment accumulation is not present at the volcanic ridge
hosting the field. However, the rift valley of the southern Knipovich Ridge in the vicinity of Loki’s Castle is
partly buried by a thick sediment cover. These
sediments, derived from the nearby Bear Island fan,
likely underlie the AVR and influence hydrothermal
fluid compositions (Pedersen et al., 2010; Baumberger,
2011).
9°00E
e
8°00E
dg
Hydrothermal plume
STUDY AREA AND SAMPLING
ich
Ri
Sulfide deposit
ov
Extinct field
Kn
ip
Active field
73°40
Loki´s Castle
d
an
l
n
ee
r
AV
R
600 m
Bear Island Fan
A
VR
oh
ns
R
id
ge
73°30
M
G
10 km
3500 m
Fig. 1 Location of the Loki’s Castle vent field
at the Arctic Mid-Ocean Ridge (AMOR). AVR,
Axial Volcanic Ridge.
© 2012 Blackwell Publishing Ltd
Microbial communities in Loki’s black smokers
3
Table 1 Bulk data of chimney samples and microbial filaments from Loki’s Castle black smokers
Dive
Chimney
Sample material
Sample part
TOC (%)
GS08-ROV10(1)
GS08-ROV10(2)
GS09-ROV9
GS10-ROV9(1)
GS10-ROV9(2)
GS08-ROV11(1)
GS08-ROV11(2)
GS08-ROV11(3)
GS08-ROV11(4)
GS09-ROV6(1)
GS09-ROV6(2)
João
João
João
João
João
Menorah
Menorah
Menorah
Menorah
No name
No name
Active
Active
Filaments
Active
Active
Active
Active
Active
Active
Inactive
Inactive
Interior
Interior
Exterior
Middle
Middle
Interior
Interior
Interior
Interior
Exterior
Exterior
0.09
0.10
12.11
0.86
0.17
3.12
0.11
0.09
0.03
0.06
0.11
d13C (&)
19.0
24.9
27.1
36.0
39.3
14.2
24.3
22.7
15.3
6.8
6.0
d34S (&)
Major mineral composition, color, texture
4.3
N.a.
n.a.
1.0
1.7
2.1
2.3
2.5
n.a.
18.5
21.0
Amorphous silica rich; gray, relatively hard material
Anhydrite; gray, soft material
Thin, white filaments
Marcasite, sphalerite; black soft material
Marcasite, sphalerite, pyrite; black soft material
Sphalerite, pyrrhotite, pyrite; black soft material
Pyrite; black soft material
Sphalerite, pyrrhotite; black soft material
Sphalerite, pyrrhotite; black soft material
Anhydrite, gypsum, talc; gray, relatively soft material
Anhydrite, gypsum; gray, relatively soft material
n.a. not analyzed. Numbers in brackets indicate subsamples taken from same chimney wall.
Table 2 Taxonomic affiliation, abundances and 16S rRNA gene numbers of
microbial populations of Menorah bulk chimney material (GS08-ROV11)
MATERIALS AND METHODS
Taxonomic level
Phylum
% of prokaryotic SSU
454 sequence reads
Rock analyses
Taxonomic level Class
Proteobacteria
Proteobacteria
Proteobacteria
Proteobacteria
Firmicutes
Deferribacteres
Thermotogae
Aquificae
Thermodesulfobacteria
Candidate division SR1
Chloroflexi
Crenarchaeota
Euryarchaeota
Euryarchaeota
Euryarchaeota
Euryarchaeota
Thaumarchaeota
Gammaproteobacteria
Betaproteobacteria
Epsilonproteobacteria
Deltaproteobacteria
Clostridia
Deferribacterales
Thermotogae
Aquificae
Thermodesulfobacteria
–
Dehalococcoides
Thermoprotei
Thermoplasmata
Methanococci
Archaeoglobi
Thermococci
Marine Group I
0.2
0.1
36.1
0.1
0.1
0.1
0.6
26.1
0.8
0.8
0.1
0.9
0.3
2.8
1.8
28.4
0.1
Bold font indicates taxonomic groups represented by more than 1% of the
total 16S rRNA gene pool.
The chimney samples analyzed in this study were collected during R/V G.O. Sars cruises in 2008, 2009, and
2010 using a Bathysaurus XL remotely operated vehicle
(ROV) equipped with a hydraulically operated box sampler. In total, eleven samples recovered from two active
and inactive sulfide structures (Menorah, João, and one
unnamed chimney) at Loki’s Castle hydrothermal vent field
were subsampled and analyzed for mineral composition
and lipid biomarkers (Table 1). From Menorah, a bulk
sample from the chimney wall was sampled for 16S rRNA
gene analysis (sample GS08-ROV11; Table 2). In addition,
microbial filaments at the surface of the João structure were
sampled by a suction sampler and analyzed for lipid biomarkers. All samples were stored at -20°C until used for
analysis.
© 2012 Blackwell Publishing Ltd
Freeze-dried, crushed, and powdered chimney samples
were analyzed by X-ray diffraction (Bruker, AXS D8
Advance) to determine the mineralogy. Total organic carbon (TOC), carbon isotope ratios (d13C), and sulfur isotope ratios (d34S) of the bulk rock samples were measured
after decarbonation on a ThermoFisher Flash-EA 1112
elemental analyzer coupled via a Conflo IV interface to a
ThermoFisher Delta V isotope ratio mass spectrometer. The
system was calibrated with the reference materials NBS22
(d13C = 30.03) and IAEA CH-6 (d13C = 10.46) for
carbon and IAEA –S-1 (d34S = 0.3), IAEA –S-2
(d34S = +22.67), IAEA –S-3 (d34S = 32.55) for sulfides
and NBS 127 (d34S = +21.1), IAEA-SO-5 (d34S = +0.49)
and IAEA-SO-6 (d34S = 34.05) for sulfates. All analytical
results are reported in the conventional d notation, in per
mil relative to the Vienna Pee Dee belemnite (VPDB) standard for carbon and the Vienna Canon Diablo Troilite
(VCDT) standard for sulfur. Reproducibility of the measurements was better than 0.2&.
Molecular techniques and taxonomic analysis
DNA extraction and PCR amplification
Ten-gram of bulk chimney wall material from the active
Menorah structure (GS08-ROV11) was pulverized in a
sterilized steel mortar, and DNA was extracted from
this homogenized material (approximately 0.5 g) using a
FastDNA® spin kit for soil in conjunction with the
FastPrep instrument (MP Biomedicals, Santa Ana, CA) following manufactures protocol and applying the modifications described by Hugenholtz et al. (1998). The
extracted DNA was PCR amplified in triplicates using the
prokaryotic primer set 787F (5′ATTAGATACCCNGG
TAG3′) (Roesch et al., 2007) and Uni1391R (5′ACGGG
CGGTGWGTRC3′) modified from Lane et al. (1985), as
4
A. JAESCHKE et al.
described by Lanzén et al. (2011). The resulting amplicons
were purified and sequenced using multiplex GS FLX
pyrosequencing (without Titanium chemistry) at the
Norwegian High-Throughput Sequencing Centre (NSC)
in Oslo, Norway.
Filtering, removal of noise and taxonomic assignment of
16S rRNA gene amplicon sequence data
The dataset (5485 reads) was filtered and cleaned from
noise by using the software AmpliconNoise (Quince et al.,
2011). The protocol has been described previously
(Lanzén et al., 2011). In short, bad-quality reads are
removed (flow intensity 0.5–0.7), so are sequences not
matching the applied primer sequence as well as chimeric
reads. The resulting 3646 high-quality reads distributed on
122 unique reads with an average read length of 231 bp
were used for taxonomic evaluation. To assign each
sequence read to a taxon, we compared our reads with the
SILVA SSUref database release 100 (Pruesse et al., 2007),
using blastn. A manual revision of this database was conducted, and the taxonomy updated with respect to Epsilonproteobacteria, Acidobacteria, Chloroflexi, and the Archaea
as well as a more restrictive quality filter (pintail score >75,
alignment quality >75 and length >1200 bp; database
available at http://services.cbu.uib.no/supplementary/
community-profiling/). Taxomomical assignments were
then evaluated using the software MEGAN version 3.7
(Huson et al., 2007) by applying a last common ancestor
algorithm (for details see Lanzén et al., 2011). Taxonomic
affiliation of sequence reads and the relative abundances
within the bulk chimney wall of the Menorah structure are
given in Table 2. Pyrosequencing flowgrams (SSF files)
have been deposited in the NCBI Sequence Read Archive
under the accession number SRA052614.
Lipid extraction, derivatization, and fractionation
About 6–12 g of each chimney sample was freeze-dried,
crushed to a fine powder, and ultrasonically extracted using
methanol (MeOH), dichloromethane (DCM)/MeOH
(1:1 v/v), and DCM (three times). The extracts were combined and the bulk of the solvent subsequently removed by
rotary evaporation under vacuum. Elemental sulfur was
removed from the total lipid extract (TLE) by flushing with
n-hexane over a small pipette filled with HCl-activated copper. The TLE was further transmethylated with MeOH/
HCl (10% w/v) at 70 °C for 2 h to convert free and esterbound fatty acids into their corresponding methyl esters
(FAMEs), and silylated with bis (trimethyl) trifluoroacetamide (BSTFA) in pyridine at 60 °C for 20 min to convert
alcohols in trimethylsilyl (TMS) ether derivatives. An aliquot of the TLE was chromatographically separated into
apolar and polar fractions using a column with activated silica as stationary phase. Apolar compounds were obtained
using n-hexane/DCM (9:1 v/v) as eluant. Polar fractions
containing the GDGTs (i.e., structures I-XII, Fig. 3) were
eluted with DCM/MeOH (1:1 v/v, 3 column volumes).
After solvent evaporation, the polar fractions were redissolved in 200 lL of HPLC-grade n-hexane/isopropanol
(99:1 v/v) and were filtered through a 0.45-lm PTFE filter
prior to HPLC/APCI/MS analysis. Microbial filaments
were extracted using a modified Bligh-Dyer procedure
(Bligh & Dyer, 1959). A solvent mixture of phosphate-buffer (0.05 M, pH 7.4)/methanol (MeOH)/dichloromethane (DCM) 0.8/2/1 (v/v) was added to the frozen
cell material. The mixture was sonicated for 10 min after
which further DCM and phosphate-buffer were added to a
volume ratio of 0.9/1/1. After centrifuging (5 min at
1120 g), the DCM layer was collected. The residue was
re-extracted twice following the same procedure. The
extracts were combined and the bulk of the solvent subsequently removed by rotary evaporation under vacuum. An
aliquot of the extract was further hydrolyzed in 2 M HCl/
MeOH (1/1, v/v) for 3 h at 75 °C. The pH of the hydrolyzed extract was adjusted to pH 3 using 1 M KOH
(MeOH 96%). The extract was derivatized as described previously. The position of the double bonds in the fatty acids
was determined by analysis as their dimethyl disulfide
(DMDS) adducts according to the method of Nichols et al.
(1986). Briefly, an aliquot of the sample dissolved in 50 lL
of n-hexane was treated with 100 lL of DMDS and 20 lL
of iodine solution (6% w/v in diethyl ether). The reaction
was carried out in 2-mL screw-cap glass vials at 50 °C for
48 h. The mixture was cooled and diluted with 500 lL of
n-hexane. The excess of iodine was reduced by addition of
500 lL of sodium thiosulfate (5% w/v in MilliQ water).
The organic phase was removed, and the aqueous phase
extracted twice with 500 lL of n-hexane. Combined
organic phases were evaporated under a stream of nitrogen
and diluted with 100 lL of n-hexane prior to GC-MS
analysis.
Analysis and identification of biomarkers
High-performance liquid chromatography-mass
spectrometry (HPLC-MS)
Glycerol dialkyl glycerol tetraethers analysis was performed
at the Geological Institute of the ETH Zurich using highperformance liquid chromatography/atmospheric pressure
chemical ionization–mass spectrometry (HPLC/APCI–MS)
with a Thermo Surveyor LC system coupled to an LCQ
Fleet ion trap mass spectrometer equipped with a PAL LC
autosampler and Xcalibur software, as described by
Hopmans et al. (2000). Normal phase separation was
achieved with an Alltech Prevail Cyano column
(150 mm 9 2.1 mm; 3 lm) maintained at 30°C. Flow rate
of the n-hexane/isopropanol (IPA) (99:1) mobile phase was
0.3 mL min 1, isocratically for the first 5 min, thereafter
© 2012 Blackwell Publishing Ltd
Microbial communities in Loki’s black smokers
with a linear gradient to 2% IPA in 30 min, and a column
cleaning step with 10% IPA in n-hexane. Injection volume
was 20–50 lL. Scanning was performed over the m/z
ranges 740–746, 1016–1054, and 1280–1318. Relative
abundances of GDGTs were calculated using peak areas of
the [M+H]+ ions vs. those of the C20-diol internal standard
(m/z 743). GDGTs were identified and distinguished via
their MS2 spectra. MS2 experiments were performed with
conditions according to Knappy et al. (2009). Briefly, eluting species were monitored using the positive ionization
mode of the APCI source. Conditions for APCI-MS were as
follows: vaporizer temperature 300 °C, sheath gas (N2) flow
rate 40 (arb. units), auxiliary gas (N2) flow rate 5 (arb.
units), capillary temperature 200 °C, capillary voltage 23 V,
and corona discharge current 5 lA. Positive ion MS spectra
were obtained by scanning a narrow mass range from m/z
1220 to 1350. MS2 spectra were recorded using the data
dependent ion scan feature, in which the base peak of an
MS scan is selected for collision induced dissociation (CID)
in MS2 (collision energy was set at 30%).
Gas chromatography-mass spectrometry (GC-MS)
Compound identification was done by combined GC-MS.
GC-MS was conducted using a Hewlett Packard 6890 gas
chromatograph equipped with an on-column injector.
A fused silica capillary column (HP-5, 30 m length,
0.25 mm inner diameter, 0.25 lm film thickness) with
helium as a carrier gas was used. The gas chromatograph
was interfaced to a HP 5973 mass selective detector
(MSD) with a mass range of m/z 50–800. The samples
were injected at 60 °C. The GC oven temperature was
subsequently raised to 120 °C at a rate of 10°C min 1 and
then to 320 °C at 4°C min 1. The temperature was then
held constant for 20 min. The structural characterization
of lipids was evaluated by comparing their mass spectral
fragmentation pattern with published spectra.
RESULTS AND DISCUSSION
5
porous sulfide samples mainly consisted of pyrite, pyrrhotite, sphalerite, and marcasite (Table 1), reflecting intermediate temperatures of formation (<240 °C for marcasite)
(Haymon, 1983). A number of studies have shown that
once the anhydrite walls of a chimney are in place, and
hydrothermal fluid is protected from extensive mixing with
seawater, which subsequently leads to the precipitation of
Zn-Cu-Fe sulfides toward the chimney interior. During
this stage, anhydrite is partially dissolved again and
replaced by sulfides (Haymon, 1983).
d34S values generally ranged from 1& to 2& in the sulfide samples (Table 1) indicating a mid-ocean ridge basalt
(MORB) source (Shanks & Seyfried, 1987). The sulfate
samples (i.e., anhydrite) showed d34S values of 19& and
21&, reflecting seawater sulfate values. Total organic
carbon (TOC) was generally low in the chimney samples
ranging from 0.03% to 0.9%; however, GS08-ROV11(1)
of active Menorah revealed an extraordinarily high TOC
content of 3% (Table 1). These strong variations in the
organic content of samples from the same chimney indicate
that hydrothermal vents provide small, patchy, and unstable habitats for microbes. The d13C of organic carbon varied between
6 and
7& for the inactive chimney
samples which is in the range of the isotopic composition
observed for mantle derived CO2. d13C values of organic
carbon in the active chimney samples varied from 14 to
39&, yielding a range of 25& (Table 1). The low d13C
values in the marcasite-bearing samples GS10-ROV9
(1 + 2) of João may indicate the presence of chemolithoautotrophs preferentially using 12C in a distinct layer
within the middle/outer chimney wall where metastable
pyrrhotite is being rapidly replaced by either pyrite or marcasite, depending on the pH of the fluid (for marcasite
pH < 5) (Murowchick & Barnes, 1986). Thus, besides
variations in temperature and availability of reduced chemical species, the mineralogy and habitat type could be
important factors affecting the composition of microbial
communities (Kato et al., 2010).
Mineralogy and bulk isotope data
Microbial diversity
The mineralogy of the samples recovered from active (João
and Menorah) and inactive sulfide chimneys (Table 1) indicated that they were mostly derived from the interior zones
of the chimney wall. The sulfide-poor samples GS09ROV6(1 + 2) were dominated by abundant anhydrite, and
less gypsum and talc. In general, during chimney growth,
anhydrite precipitates around a black smoker vent at the
leading edge of chimney growth, where hot hydrothermal
fluids first encounter cold seawater (Haymon, 1983), talc
forms in hot chimneys from seawater magnesium and
hydrothermal silica (Haymon & Kastner, 1981). Thus, we
can assume that our samples containing anhydrite were
derived from the exterior zones of the chimney wall. The
The sulfide chimney matrix of the active Menorah structure
(GS08-ROV11) harbored a diverse range of thermophilic
and hyperthermophilic archaea and bacteria, as shown both
by our 16S rRNA gene-based taxonomy (Table 2, Fig. 2)
and lipid analysis (Fig. 3). However, lipids such as GDGTs
are common lipids of many archaea, and therefore, it is difficult to link them with a specific archaeal group without
additional microbial- or compound-specific isotope data.
In the following sections, we use the information obtained
from the 16S rRNA gene amplicon library to discuss the
link between possible source organisms and different compound classes detected in samples originating from the
same chimney wall.
© 2012 Blackwell Publishing Ltd
6
A. JAESCHKE et al.
Fig. 2 Phylogenetic Neighbor-joining tree
based on archaeal 16S rRNA gene sequence
information. The taxonomic affiliation of the
sequences obtained from the Menorah active
sulfide chimney is highlighted in bold. Further
the group to which these sequences could be
assigned at a lower taxonomic level is given
below the phylum name in italic. The relative
abundance of each group can be found in
Table 2. THSCG, Terrestrial hotspring crenar
chaeotic group; MCG, Miscellaneous crenar
chaeotic group; MBG, Marine Benthic Group;
SCG, Soil crenarchaeotic group; SAGMCG,
South African gold mine crenarchaeotic group;
MG I, marine group I; SAGMEG, South
African gold mine Euryarchaeotic group.
Archaea
Distribution and origin of archaeal GDGTs (I–VII)
All samples from the chimney structures contained a range
of isoprenoid GDGTs (Fig. 3), indicating that archaea are
present throughout the chimney walls. GDGT I, a trialkyltype GDGT (Fig. 3), was detected as a minor constituent
(<1%) of the total GDGT pool from Menorah and João
active sulfide chimneys (Fig. 4). GDGT I has been
reported in a number of cultivated thermophilic and hyperthermophilic Crenarchaeota (Gulik et al., 1988; de la
Torre et al., 2008) and has been proposed as an intermediate in the biosynthesis of GDGT II from archaeol (Koga
& Morii, 2007). GDGT II (Fig. 3) was detected in all
samples analyzed and was also the most abundant compound with relative abundances between 29 and 61% of all
GDGTs, followed by GDGTs containing 1–4 cyclopentane
rings (Fig. 3 structures III-VI) with relative abundances
between 1 and 19% (Fig. 4). Methanogenic archaea and
members of the family Archaeoglobaceae have been found
to produce predominantly GDGT II (Koga & Morii,
2005). Sequences related to both groups were found in
our taxonomic data, namely members of the family
Methanococcales and of the genus Ferroglobus belonging to
the family of Archaeoglobales. Methanococcales (2.8% of the
total community) is a group that constitutes strictly anaerobic autotrophs that gain energy by the reduction of CO2
with H2, generating CH4. Ferroglobus made up 1.8% of
the total community (Fig. 2) and is a hyperthermophilic
(growth between 65 and 95 °C) member of the Archaeoglobales that oxidizes ferrous iron (Fe2+) but also molecular
hydrogen, and sulfide under strictly anaerobic conditions
(Hafenbradl et al., 1996). Nitrate and thiosulfate (S2O32 )
are used as electron acceptors that are known from a variety of hyperthermophiles (Stetter et al., 1987; Stetter,
2002). Although it seems likely that the origin of the
detected GDGT II stems from the above-mentioned
groups, we cannot exclude that there are other, still uncultivated archaeal groups that also produce this as a major
lipid.
Detection of 16S rRNA genes related to hyperthermophilic members of the family Thermoproteaceae (Crenarchaeota) and Thermoplasmataceae (Euryarchaeota)
(Table 2, Fig. 2) suggest that, at least partly, they may be
the source organisms for GDGT III–VI containing 1–4
cyclopentane rings (Fig. 3). As reported by Koga & Morii
(2005), these groups have been found to predominantly
synthesize GDGT II-VI in culture. Indeed, members of
the Desulfurococcus, a group composed of hyperthermophilic heterotrophs, growing at temperatures up to 95°C
(no growth is reported for temperatures of 65 °C) and
gaining energy by oxidation of hydrogen using elemental
sulfur (Huber & Stetter, 2006; Stetter, 2006), were
detected as minor components representing 0.9% of the
microbial community (Fig. 2). It is interesting to know
that we found GDGTs with a maximum of only four rings
in such high temperature environment. In general, the
number of cyclopentane rings incorporated in tetraether
lipids increases with increasing growth temperature (i.e.,
Uda et al., 2001; Schouten et al., 2002; Boyd et al.,
2011). GDGTs with up to eight rings were reported from
Yellowstone hot springs (Schouten et al., 2007), where
© 2012 Blackwell Publishing Ltd
Microbial communities in Loki’s black smokers
GTGT
GDGT
7
H-GDGT
Relativ eabundance
II
VIII
IV
III
V VI
IX
X
I
XI XII
Retention time (min)
Fig. 3 High-performance liquid chromato
graphy (HPLC)/APCI/MS base peak chromato
gram showing the distribution of GDGTs in
Menorah active black smoker material GS08ROV11(1), and structures of GDGTs present
in the black smokers of Loki’s Castle. The
position of the covalent bond between the
isoprenoid hydrocarbon chains in GDGTs VII–
XI is tentative (Morii et al., 1998). Cren,
Crenarchaeol; GTGT, glycerol dialkyl glycerol
tetraether; GDGT, glycerol dialkyl glycerol
tetraether; H-GDGT, H-shaped GDGT (see
text).
I.
VII.
II.
VIII.
III.
IV.
V.
VI.
temperatures were generally lower than in black smokers.
Thus, there are possibly other parameters controlling the
GDGT lipid composition in archaea, for example, pH,
pressure, heavy metal content as well as biological factors.
For the thermoacidophilic archaea Sulfolobus acidocaldarius, it has been shown that the incorporation of cyclopentane rings leads to a more tightly packed membrane than
one without rings, thus regulating membrane behavior,
that is, fluidity or proton permeability (Gabriel & Chong,
2000).
Crenarchaeol (GDGT VII, Fig. 3) was found in the
middle and exterior zones of the chimneys, and only
traces could be detected in the interior parts of the chim-
© 2012 Blackwell Publishing Ltd
IX.
X.
XI.
XII.
ney wall at Menorah GS08-ROV11(1) (Fig. 5A). In the
João sample GS10-ROV9(2), crenarchaeol and the regioisomer accounted for 5% and <0.1%, respectively, of the
total GDGTs (Fig. 4) (concentration was 1 ng/g chimney
material). The mineralogy of this sample suggests that it
is derived from the chimney middle to exterior where
marcasite precipitated during chimney growth (Haymon,
1983). As for GDGT II–VI, very low concentrations of
about 20 pg/g chimney material were detected in an
anhydrite-dominated chimney sample GS09-ROV6(2),
clearly showing a seawater sulfate signal (Table 1). Here,
crenarchaeol accounted for 25% of all GDGTs (Fig. 4).
Crenarchaeol was originally thought to be a specific
8
A. JAESCHKE et al.
Active
Inactive
100
% Archaeal lipids
80
XII
XI
X
IX
VIII
VII
VI
V
IV
III
II
I
60
40
20
João
Menorah
H-GDGT
GDGT
GTGT
No name
Interior
9R
O
V6
(2
)
(1
)G
O
V6
9R
S0
1(
4)
G
S0
8R
O
V1
1(
3)
G
S0
8R
O
V1
1(
2)
O
V1
8R
O
V1
8R
G
S0
G
S1
G
S0
Interior
G
S0
1(
1)
(2
)
O
V9
(1
)
Middle
0R
O
V9
0R
G
S1
8R
G
S0
G
S0
8R
O
V1
O
V1
0(
2)
0(
1)
0
Exterior
Fig. 4 Relative abundances of archaeal GDGTs present in samples derived from different parts of active and inactive chimneys. Roman numerals refer to
structures in Fig. 3. Numbers in brackets indicate subsamples taken from same chimneys.
biomarker for mesophilic Crenarchaeota (recently, the
phylum has been split up into two phyla; Crenarchaeota
and Thaumarchaeota: Brochier-Armanet et al., 2008;
Spang et al., 2010). This group is composed of putative
aerobic ammonia oxidizers (AOA) (Könneke et al., 2005;
Wuchter et al., 2006) and is ubiquitously found in marine
systems between 2 and 30 °C (Sinninghe Damsté et al.,
2002). However, thermophilic members of the ammoniaoxidizing archaea have also been found to synthesize crenarchaeol at temperatures up to 87 °C (Pearson et al.,
2004; Zhang et al., 2006; de la Torre et al., 2008).
Loki’s Castle is a sediment-impacted hydrothermal system
with high ammonium concentrations (6.1 mmol kg 1; Pedersen et al., 2010), suggesting that AOA may be active
at this site as well. Moreover, Zhang et al. (2006) suggested that crenarchaeol could be an original and ancient
biochemical property of the thermophilic Crenarchaeota,
which occupy a deeply branching point in the phylogenetic tree of life (Forterre et al., 2002). Our lipid and
16S rRNA gene-based data, however, suggest that hyperthermophilic Crenarchaeota only account for a small fraction of the total microbial community in the chimney (see
discussion previously; Table 2, Fig. 2). Low relative abundances of 16S rRNA genes related to marine group I
Thaumarchaeota (Table 2, Fig. 2) indicate the presence of
AOA. However, chimney walls are sufficiently permeable
to allow the influx of small amounts seawater; therefore,
our data could also be interpreted as an introduction of
Thaumarchaeal cells from the ingression of ambient
seawater where they have been found to be ubiquitous
(Takai et al., 2004).
Distribution and origin of archaeal H-GDGTs (VIII–XII)
In addition to GDGTs I-VII, another group of later-eluting compounds was present in the HPLC/MS chromatogram (VIII–XII, Fig. 3). These compounds showed mass
spectra characteristic of GDGTs with base peak ions of
1300, 1298, 1296, 1294, and 1292, respectively, which
are the [M+H]+ ions (Fig. 5A). The distribution of these
compounds is similar to that of GDGTs II-VII, and they
were further identified and distinguished via their MS2
spectra. MS2 experiments revealed that GDGTs VIII-XII
have a different fractionation pattern than GDGT II-VII,
exhibiting a far less pronounced degree of dissociation
(Fig. 5B,C). This is due to the covalent bond between the
two hydrocarbon chains in these compounds, which stays
intact during the dissociation, generating a product ion
that maintains the C80 hydrocarbon core, and two small
fragments resulting from the loss of an OH and a glycerol
group. The MS2 spectra are identical to those previously
published by Knappy et al. (2009) who analyzed cells from
a pure culture of Methanobacter thermoautotrophicus grown
at 70 °C. The specific MS2 spectrum indicates that compounds VIII-XII are GDGTs with an additional covalent
bond between the isoprenoid chains, so-called H-shaped
GDGTs (H-GDGTs) (Morii et al., 1998). Abundances of
H-GDGTs VIII-XII are generally lower than those of regular GDGTs (Fig. 3), and contribute with 3–21% to the
total lipid pool (Fig. 4). H-shaped isoprenoid GDGTs have
so far been identified in several cultivated archaea, for
example, in the above-mentioned hyperthermophilic Methanobacter thermoautotrophicus, in the hyperthermophilic
methanogen Methanothermus fervidus (Morii et al., 1998),
© 2012 Blackwell Publishing Ltd
Microbial communities in Loki’s black smokers
A
GDGT
B
H-GDGT
9
GDGT III
m/z 1300
II
40%
%
m/z 1302
VIII
14%
III
7%
IV
12%
m/z 1300
[M+H]+
IX
4%
m/z 1298
m/z
C
VI
5%
XI
1%
VII
XII
1%
H-GDGT VIII
m/z 1300
m/z 1296
m/z 1294
%
X
8%
V
7%
m/z 1292
Relative retention time
[M+H]+
m/z
Fig. 5 (A) Individual ion chromatograms for glycerol dialkyl glycerol tetraethers (GDGTs) II-X detected in Menorah active chimney material GS08-ROV11(1)
in the positive ion MS base peak chromatogram. The percentage of the total extract contributed by each lipid is also shown. MS2 spectra for (B) GDGT II
and (C) H-GDGT VIII with [M+H]+ at m/z 1300.2.
as well as in different species of the order Thermococcales
growing at neutral pH with optimal growth temperatures
>80 °C (Godfroy et al., 1997; Sugai et al., 2004). In congruence with this, we found a high abundance of Thermococcales-related 16S rRNA gene sequences (28.4% of total
community) (Table 2, Fig. 2), suggesting that this group
is the main source of H-GDGTs. Thermococcus is an obligate anaerobic, sulfur-reducing heterotroph belonging to
the Euryarchaeota, which is often detected as a member of
vent communities (Takai et al., 2001; Schrenk et al.,
2003; Kormas et al., 2006; Takai & Nakamura, 2011).
Sulfur reduction has been suggested to be the thermodynamically favored reaction at higher temperatures
(>38 °C), while sulfur oxidation is more viable at lower
temperatures (McCollom & Shock, 1997). Elemental sulfur is either stimulatory or is required for growth of Thermococcus. Elevated hydrogen concentrations measured in
the vent fluids (Pedersen et al., 2010) can serve as an electron donor for the reduction of elemental sulfur to H2S.
© 2012 Blackwell Publishing Ltd
Another candidate group for the high abundance of
H-GDGTs was identified by Schouten et al. (2008) who
detected H-shaped GDGTs with up to four cyclopentane
rings in Aciduliprofundum boonei, a cultivated thermoacidophilic sulfur- and iron-reducing Euryarchaeota from a
deep-sea hydrothermal vent. This organism belongs to the
DHVE2 cluster and is capable of growing from pH 3.3 to
5.8 and between 55 and 75 °C (Reysenbach et al., 2006).
However, at Loki’s Castle, 16S rRNA genes related to the
DHVE2 cluster contributed only 0.3% to the total microbial community (Table 2, Fig. 2), indicating that only a
minor part of the H-GDGTs originates from those archaea.
Our finding of high abundances H-shaped GDGTs is
not surprising as the introduction of an additional covalent
cross-link between the isoprenoid chains is thought to help
maintain membrane structure at high temperatures (Morii
et al., 1998; Schouten et al., 2008). Our results indicating
the prevalence of archaeal communities near the warm
interior of black smoker chimneys is also in agreement with
10
A. JAESCHKE et al.
earlier findings by Schrenk et al. (2003). Higher rigidity
and stability of archaeal tetraether lipids that form
monolayer membranes are better suited to extreme environments than the ester type of bilayer lipids of bacteria or
eukarya (van de Vossenberg et al., 1998). Moreover,
relatively high abundances of both GDGTs and H-GDGTs
with 1–4 cyclopentane rings detected in our samples is
compatible with culture studies showing that (hyper)thermophilic Crenarchaeota and Euryarchaeota produce
GDGTs containing more cyclopentane moieties with
increasing growth temperature (Uda et al., 2001, 2004).
As discussed previously, the distribution of archaeal
GDGTs and H-GDGTs could also be controlled by other
parameters such as pH, pressure, heavy metal content or it
could be related to a specific metabolism (Uda et al.,
2004; Boyd et al., 2011). GDGT abundances in samples
derived from the exterior wall of the inactive chimney
GS09-ROV6 (1 + 2) were about three orders of magnitude lower than those derived from active chimneys, suggesting that once a chimney stops venting, conditions are
no longer favorable for archaeal communities. In our sample of the exterior wall of the inactive chimney, high abundances of unsaturated (and branched) fatty acids were
detected and may indicate the presence of sulfur-oxidizing
Table 3 Concentration of distinctive compounds extracted from active and
inactive chimney material and chimney-associated filaments
Compound
Fatty acids
i-C15:0
ai-C15:0
ai-C16:0
C16:1x7c
C16:1x7t
C16:1x5c
C16:0
10-Me-C16:0
C18:1x9c
C18:1x7c
C18:0
C22:1x9c
Alcohols
Archaeol
sn-2-hydroxyarchaeol
Hydrocarbons
Crocetane
PME
PME:4
Hopanoids
diploptene
diplopterol
Active
GS08ROV11(1)
Menorah
(ng g 1)
Filaments
GS09ROV9(2)
João
(lg g 1)
Inactive
GS09ROV6(1)
unknown
(ng g 1)
Inactive
GS09ROV6(2)
unknown
(ng g 1)
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
778
549
236
17577
1985
2485
5350
nd
nd
6361
266
nd
nd
nd
nd
192.9
26.3
26.2
37.1
nd
15.6
31.6
nd
732.2
nd
nd
12.5
1074.2
114.2
77.7
314.0
10.8
24.9
100.2
nd
282.1
102.2
68.0
3.1*
nd
nd
nd
nd
nd
3.1
6.8
25.9
nd
nd
nd
nd
nd
nd
nd
nd
nd
29.2
15.2
nd
nd
nd
nd
nd
nd
(and sulfate-reducing) members of the e subclass of the
Proteobacteria (the e-Proteobacteria) (Table 3; see section
on bacteria below; H. Dahle, unpublished data).
Isoprenoid diether lipids and hydrocarbons
Isoprenoid compounds of archaeal origin, such as archaeol,
were also found in the Menorah sample GS08-ROV11(1).
Archaeol is produced by a variety of different archaeal
groups, such as halophiles, thermophiles, and acidophiles
(Koga & Morii, 2005), and was present with a concentration of 102 ng/g (Table 3). Another isoprenoid
compound, sn-2-hydroxyarchaeol, was detected as both
mono- and di-trimethylsilyl (TMS) derivatives with a concentration of 68 ng/g. sn-2-hydroxyarchaeol is a diagnostic
biomarker for the thermoacidophilic archaeal order Thermoplasmatales and the methanogenic order Methanococcales
(Koga et al., 1998), which is consistent with the findings of
abundant GDGTs with cyclopentyl moieties as well as 16S
rRNA gene-based taxonomy data (see section above;
Table 2). Concentrations of crocetane, irregular isoprenoids
pentamethyleicosane (PME) and PME 4 were 3, 7, and
26 ng/g chimney material, respectively. These compounds
have been attributed to anaerobic, methanotrophic archaea
(ANME) (Elvert et al., 1999; Bian et al., 2001), although
PME was originally believed to be synthesized by methanogenic archaea (Holzer et al., 1979; Risatti et al., 1984).
Whereas the presence of methanogenic archaea is supported
by our 16S rRNA gene data (methanococci), no sequences
related to any of the ANME groups were detected.
Bacteria
nd, not detected.*sample contained small amounts of rock debris. Also
glycerol dialkyl glycerol tetraethers (GDGTs) and H-GDGTs were detected
in low abundances (<1 lg g 1).
The lipid composition of large filamentous bacteria collected
from the João active sulfide chimney (GS09-ROV9) revealed
the dominance of C16:1x7c (47%), C18:1x7c (17%), and C16:0
(14%) fatty acids. Concentrations of these compounds are
about three orders of magnitude higher in this sample as in
the chimney material (Table 3). The lipid pattern is consistent with lipid profiles of known sulfur-oxidizing bacteria
from sediments and hydrothermal vents (Jacq et al., 1989;
Guezennec et al., 1998; Zhang et al., 2005). The filaments
attached to the outer chimney wall have been found to be
related to sulfur-oxidizing bacteria Sulfurovum, a group
belonging to the e-Proteobacteria that thrive at lower ambient temperatures (H. Dahle, unpublished data). Minor abundances of C16:1x5c and C18:1x9c detected in the filaments as
well as in the inactive chimney samples GS09-ROV6(1 + 2)
may be derived from sulfate-reducing bacteria (Dowling
et al., 1986; Elvert et al., 2003; Londry et al., 2004).
16S rRNA gene analysis with subsequent taxonomic analysis of Menorah active chimney GS08-ROV11 revealed that
members of the Aquificales and e-Proteobacteria were the
dominant bacteria accounting for 26 and 36% of the total
© 2012 Blackwell Publishing Ltd
Microbial communities in Loki’s black smokers
prokaryotic community (Table 2). The Aquificales is
thought to be the earliest branching lineage within Bacteria
and have often been detected in hot springs (Spear et al.,
2005; Purcell et al., 2007) and deep-sea vent ecosystems
(Kormas et al., 2006; Blumenberg et al., 2012). Among
the bacteria, Aquificales exhibit one of the highest growth
temperatures (95 °C). They are anaerobic lithoautotrophs,
gaining metabolic energy from the oxidation of molecular
hydrogen (H2) or sulfur compounds. Lithotrophic sulfur
reduction by oxidation of H2 is believed to be one of the
most ancient types of catabolism (Fischer et al., 1983), and
the preferred source of energy at temperatures >38 °C
(McCollom & Shock, 1997). The apparent lack of dialkyl
glycerol diethers (DAGE) specific for the Aquificales
(Jahnke et al., 2001) at Menorah may be because the
distinct zones where the Aquificales thrive within the chimney structure were not present in our subsamples for lipid
analysis. Another possibility of the observed discrepancy
could be a primer bias in the 16S rRNA gene approach,
leading to a skewed relative abundance estimate. The same
may be the case for the deep-branching e-Proteobacterial
group Nautiliales that were detected as one of the main
organisms within the chimney wall of Menorah (Table 2).
Nautiliales is believed to be constituted of thermophilic
sulfur-reducing bacteria that are found to be key players in
sulfidic habitats (Alain et al., 2004; Campbell et al., 2006).
Other members of the e-Proteobacteria, for example, the
Campylobacteriales, made up only a very small fraction of
the total population (data not shown). The dominant lipids
of Nautilia profundicola have been shown to be C18:1x7c
and C16:1x7c fatty acids (Smith et al., 2008). High abundances of branched and monounsaturated fatty acids
detected in samples derived from the outer chimney wall
GS09-ROV6 (1 + 2) (Table 3) may be produced by sulfur-oxidizing and sulfate-reducing bacteria. In general,
lower growth temperatures of 45–53 °C indicate that bacteria mainly thrive within the exterior chimney walls, which
has also been reported for a white smoker by Kormas et al.
(2006). The hopanoids diploptene and diplopterol with
low concentrations (Table 3) have predominantly been
found in aerobic bacteria, that is, methanotrophs, heterotrophs, and cyanobacteria. Hopanoids also occur in anaerobic bacteria, for example, members of the Planctomycetes
capable of anaerobic ammonium oxidation (Sinninghe
Damsté et al., 2004), in Geobacter species (Fischer et al.,
2005; Härtner et al., 2005), and in sulfate-reducing bacteria of the genus Desulfovibrio (Blumenberg et al., 2006).
Concentrations of these lipids, however, are about 1–2
orders of magnitude lower than measured for GDGTs.
CONCLUSIONS
In the present study, we gave first insights into the diversity of microbial communities present in sulfide structures
© 2012 Blackwell Publishing Ltd
11
of active and inactive vents from the newly discovered
Loki’s Castle black smoker field at the Arctic Mid-Ocean
Ridge. Evidence for both archaea and bacteria was provided by a combination of lipid biomarker and 16S rRNA
gene-based techniques. The specific lipid distribution
observed in samples derived from different parts of the
active and inactive chimneys indicate the presence of
diverse consortia of (hyper)thermophilic Euryarchaeota and
Crenarchaeota within the warmer interior zones, while bacterial lipids were only a minor constituent. Our study particularly showed that H-GDGTs, which have to date not
been reported from archaea under environmental conditions, were abundant in all samples. With additional 16S
rRNA gene data available, these compounds could also be
linked with their potential source organisms which may be
(hyper)thermophilic members of the Thermococcaceae.
Based on biomarker lipid and 16S rRNA gene analyses, we
conclude that sulfur reduction by (hyper)thermophilic
archaea and bacteria as well as archaeal methanogenesis are
the most likely metabolic activities within the interior
zones of the black smoker chimney walls at Loki’s Castle.
Bacterial sulfur oxidation and sulfate reduction as well as
ammonia oxidation are favorable metabolisms in the exterior zones of the chimney walls.
ACKNOWLEDGMENTS
We would like to thank the crew of R/V G.O. Sars for shipboard support and Tamara Baumberger for help with sampling. We also greatly appreciate the help with the
bioinformatics from Anders Lanzén. Our special acknowledgements go to Carme Huguet and one anonymous
reviewer for comments and suggestions considerably
improving the quality of the manuscript. The Norwegian
High-throughput sequencing Centre at the University of
Oslo performed the sequencing. (http://www.sequencing.
uio.no). This work was supported by the Swiss National
Science Foundation (SNF projects 20MA21-115916 and
200020-132804).
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