Download Microbial community diversity in seafloor basalt from the Arctic

FEMS Microbiology Ecology 50 (2004) 213–230
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Microbial community diversity in seafloor basalt from the
Arctic spreading ridges
Kristine Lysnes
a
a,*
, Ingunn H. Thorseth b, Bjørn Olav Steinsbu a, Lise Øvreås
Terje Torsvik a, Rolf B. Pedersen b
a,b
,
Department of Microbiology, University of Bergen, Jahnebakken 5, N-5020 Bergen, Norway
Department of Earth Science, University of Bergen, Allegaten 41, N-5007 Bergen, Norway
b
Received 9 February 2004; received in revised form 28 June 2004; accepted 29 June 2004
First published online 26 July 2004
Abstract
Microbial communities inhabiting recent (61 million years old; Ma) seafloor basalts from the Arctic spreading ridges were analyzed using traditional enrichment culturing methods in combination with culture-independent molecular phylogenetic techniques.
Fragments of 16S rDNA were amplified from the basalt samples by polymerase chain reaction, and fingerprints of the bacterial and
archaeal communities were generated using denaturing gradient gel electrophoresis. This analysis indicates a substantial degree of
complexity in the samples studied, showing 20–40 dominating bands per profile for the bacterial assemblages. For the archaeal assemblages, a much lower number of bands (6–12) were detected. The phylogenetic affiliations of the predominant electrophoretic
bands were inferred by performing a comparative 16S rRNA gene sequence analysis. Sequences obtained from basalts affiliated with
eight main phylogenetic groups of Bacteria, but were limited to only one group of the Archaea. The most frequently retrieved bacterial sequences affiliated with the c-proteobacteria, a-proteobacteria, Chloroflexi, Firmicutes, and Actinobacteria. The archaeal sequences were restricted to the marine Group 1: Crenarchaeota. Our results indicate that the basalt harbors a distinctive microbial
community, as the majority of the sequences differed from those retrieved from the surrounding seawater as well as from sequences
previously reported from seawater and deep-sea sediments. Most of the sequences did not match precisely any sequences in the database, indicating that the indigenous Arctic ridge basalt microbial community is yet uncharacterized. Results from enrichment cultures showed that autolithotrophic methanogens and iron reducing bacteria were present in the seafloor basalts. We suggest that
microbial catalyzed cycling of iron may be important in low-temperature alteration of ocean crust basalt. The phylogenetic and
physiological diversity of the seafloor basalt microorganisms differed from those previously reported from deep-sea hydrothermal
systems.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Deep biosphere; 16S rRNA gene; Cultivation; Basalt; Arctic ridges; Bacteria; Archaea
1. Introduction
Bacterial communities and processes in the subsurface have received increasing scientific attention over
the last decade. The deep biosphere is estimated to con*
Corresponding author. Tel.: +47-55588187; fax: +47-55589671.
E-mail address: [email protected] (K. Lysnes).
tain a biomass in the same order of magnitude as that of
the surface of this planet [1,2], with the major fraction of
this biomass residing in the marine subsurface. Microbial
surveys of ocean sediments have shown that high microbial diversities and large bacterial populations are present in marine sediment deposits [3]. Microorganisms
have also been shown to inhabit the upper volcanic layer
(500 m) of the oceanic crust. The presence of microbial
0168-6496/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsec.2004.06.014
214
K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
DNA in characteristic alteration textures in the glassy
margins of the basaltic lava, as well as biological carbon
isotope signatures of disseminated carbonate, indicate
that microorganisms participate in the alteration of the
glass and the chemical exchange between oceanic crust
and seawater [4–8]. As the phylogenetic and physiological diversity of these microorganisms is still largely unknown, the mechanisms for the suggested microbial
influenced dissolution and alteration are not known.
Several studies of deep-sea hydrothermal vents have
noted distinctive differences in the microbial communities inhibiting the hydrothermal fluids and the background seawater, indicating that vent fluids transport
microorganisms from the subsurface to the seafloor [9–
12]. In the recent studies of diffuse flow vent fluids
(<50 C) following a volcanic eruption on the Juan de
Fuca ridge [11,12], the most frequently retrieved DNA
sequences were affiliated with the e-proteobacteria [11].
In the study by Cowen et al. [13] of 65 C subseafloor
hydrothermal fluids from 3 Ma sediment covered crust
on the flank of the Juan de Fuca ridge, most of the retrieved DNA clones were related to the ammonia-producing bacterium Ammonifex degensii. The reported
data indicate the presence of a diverse bacterial and archaeal community in these hydrothermal regions of the
ocean crust, where the most important microbial processes appear to be reduction and oxidation of sulfur
and nitrogen compounds [11,13].
In contrast, psychrophilic iron oxidizers were detected
and isolated from low-temperature (4 C) seafloor
habitats of sulfide minerals and metalliferous sediments
at the Juan de Fuca ridge [14]. Two groups of ironoxidizing bacteria were isolated; one group was related
to the c-proteobacterium Marinobacter aquaeolei and
the other to the a-proteobacterium Hyphomonas jannaschiana. The iron oxidizing bacteria were isolated from
surfaces of weathered rock, and data suggest that these
microorganisms participate in rock weathering at the
seafloor. The microbial populations found in these
low-temperature habitats differed from those of hydrothermal habitats from the same ridge [11–13].
Evidence for a diverse microbial community and microbial iron oxidation in young (<1 Ma) non-hydrothermal seafloor basalts from the Knipovich ridge has also
recently been reported [15]. In this study, the presence
of numerous endolithic microbial cells of various
morphologies (e.g. cocci, rods, filaments, star-shaped
cells, and twisted stalks resembling those of Gallionella
ferruginea) was revealed by scanning electron microscopy (SEM). 16S rRNA gene amplification, denaturing
gradient gel electrophoresis (DGGE) and sequencing
analysis showed that the microbial community comprised the phylogenetic groups Bacteroidetes, c- and eproteobacteria, and marine Group 1: Crenarchaeota.
Even though cell morphologies resembling Gallionella
were observed in these samples, G. ferruginea was not
detected by molecular biology methods. However, a later
study of basalts collected from shallow areas around
Jan Mayen retrieved 16S rDNA sequences matching
Gallionella [16]. The bacterial populations appeared to
be characteristic and unique for the rock environment
and differed from those found in associated sediments
and seawater [15]. Investigation of 14–28 Ma nonhydrothermal subseafloor basalt from the northern
flank of the Southeast Indian ridge also showed the presence of microbial populations characteristic for the rock
environment, with Actinobacteria, Chloroflexi, Bacteroidetes, Firmicutes, and b- and c-proteobacteria as
the dominating phylogenetic groups [17]. Enrichment
studies showed that iron reducing bacteria and autolithotrophic methanogens were present.
SEM observations of microbial cells in seafloor basalts from different ridges suggest that endolithic microbial growth is a general feature of ocean ridges
[15,18,19]. From analysis of the organic carbon content
in recent lava flows, a biomass of 106 cells/cm3 seafloor
basalt has been estimated [19]. With time, the colonized
seafloor basalt becomes buried by younger lava flows
and sediments and brought to deeper levels of the oceanic
crust. Consequently, a significant proportion of the alteration observed in subsurface lavas may have developed before burial [18]. More knowledge of microbial
phylogeny and processes in young ocean ridge basalt is
thus important when studying the deep biosphere
and evaluating the function and impact this has on the
alteration of basalt.
In the present study, samples of recent, non-hydrothermal seafloor basalt from various depths in the axial
valleys of the Kolbeinsey, Mohns, and Knipovich ridges
were investigated. The phylogenetic and functional diversity of microorganisms were studied by PCR-based
fingerprinting methods in combination with enrichment
techniques. Together with physical parameters of the
samples and their original habitats, these results were
used to draw attention to biogeochemical and microbial
processes involved in basalt alteration. Samples of bottom and surface seawater, as well as one sample of basalt-associated sediment, were analyzed for comparison.
2. Materials and methods
2.1. Sample collection and preparation
During three cruises with R/V Håkon Mosby to the
Norwegian-Greenland Sea in July–August 1999, August–September 2000, and July–August 2001, a total
of 22 samples of recent basalt flows were collected from
the Kolbeinsey, Mohns, and Knipovich ridges (Fig. 1,
Table 1). Sampling was done either by dredging or by
use of a remote operated vehicle (ROV). In addition,
one sample of sediment attached to dredged basalt, four
K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
215
Fig. 1. Location of sampling sites on the Arctic spreading ridges.
bottom seawater samples and two surface seawater samples were collected for comparison and for control of
contamination (Table 1). The bottom seawater was sampled by Niskin bottles attached to a CTD (conductivity,
temperature, density) rosette, whereas the surface seawater was sampled with a water hose.
The water depth at the sampling sites ranged from
716 to 3385 m, and the ambient bottom seawater temperature between –0.4 and –0.8 C, respectively. Based
on the very low degree of alteration, the samples from
the rift valley of Knipovich ridge were estimated to be
less than 20 years old. The oldest sample (SM00-5R)
was collected from a seamount north of the rift valley
of the Mohns ridge, and is estimated, based on paleomagnetic data, to be around 1–2 Ma. The other samples
were all collected from relatively young volcanoes within
the neovolcanic zones of the Mohns and Kolbeinsey
ridges, and are roughly estimated to be less than
100,000 year, since the volcanoes were yet not covered
by deep-sea sediments.
Visual inspection by cameras attached to the ROV
showed that the lava flows were covered by a thin layer
of sediment. At shallow sites (61200 m below sea level;
mbsl) the basalt samples were colonized by a sessile
macrofauna. At deeper sites, only few of the basalt samples were colonized by a macrofauna, and samples without macrofauna were chosen from these sites.
The samples were processed immediately after collection. Basalt was split with hammer and chisel and
crushed in a mortar. Fragments of glassy margins with
visible alteration on the surface and along fractures
and cracks were preferred. All tools for rock crushing,
as well as aluminum foil covering the workbench, were
flame-sterilized. Two alternative applications for ex-
tracting cell material for the molecular phylogenetic
techniques were used: (1) cells in the basalt samples were
extracted by rinsing cracks- and fracture surfaces with
sterile water; (2) crushed basalt or sediment were mixed
thoroughly with phosphate buffer saline (PBS: 137 mM
NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM
KH2PO4 (pH 7.3)) in sterile 50-ml plastic centrifuge
tubes. After sedimentation of the visible solid particles,
5–10 ml of the supernatant was centrifuged to achieve
a cell pellet. Seawater samples, also 5–10 ml, were centrifuged directly. The cell suspensions and cell pellets were
frozen (20 C) for later onshore DNA amplification.
2.2. 16S rRNA gene amplification, DGGE and sequencing
PCR amplification of the hyper variable V3-region of
16S rDNA using HotStar Taq DNA Polymerase (Qiagen, Germany) and the bacterial primers PRBA338f
and PRUN518r [20], the first with a 40-nucleotide long
GC clamp [21], was performed in a GeneAmp2400 thermal cycler (Perkin–Elmer Applied Biosystems, USA).
The reaction mixture was prepared as recommended
by the manufacturer [22]. The PCR program was: 95
C, 15 min; 30 (up to 40 for 1999 samples) cycles of denaturation at 94 C, 1 min; annealing 55 C, 30 s; extension 72 C, 1 min; and a final extension 72 C, 10 min.
The sample material from the 1999-cruise was limited,
so to achieve sufficient PCR product for further analysis
up to 40 cycles of amplification had to be run for these
samples.
Amplification of archaeal 16S rDNA V3-region was a
nested PCR protocol modified from the method of
Øvreås et al. [20], with the primers mPRA46f (5 0 -C/
TTA AGC CAT GC/TA/G AGT-3 0 ) and mPREA1100r
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K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
Table 1
Location and depth (meters below sea level: mbsl) of sampling sites and character of samples
Sample
Sample
mode
Location
Ridge
Depth (mbsl)
Sample
character
Age (Ma)
SM99-1R
SM99-2R
SM99-5R
SM99-6R
SM99-7R
SM00-2D
ROV
ROV
ROV
ROV
ROV
Dredged
Kolbeinsey
Kolbeinsey
Kolbeinsey
Kolbeinsey
Kolbeinsey
Mohns
861
1212
1191
1191
730
2291–2459
Glassy
Glassy
Glassy
Glassy
Glassy
Glassy
0.1
0.1
0.1
0.1
0.1
0.1
SM00-4D
Dredged
Mohns
1216–1404
Basalt
0.1
B
SM00-5R
SM00-6D
ROV
Dredged
Mohns
Mohns
716
2090–2201
Basalt
Glassy basalt
1
0.1
B
B
SM00-7D
Dredged
Mohns
920–950
Basalt
0.1
SM00-10D
Dredged
Mohns
944–1600
Glassy basalt
0.1
B
SM00-17D
Dredged
Mohns
2201–2263
Glassy basalt
0.1
A+B
SM00-18D
Dredged
Mohns
2562–2598
Glassy basalt
0.1
A+B
SM00-19D
Dredged
Mohns
2523–2751
Glassy basalt
0.1
B
SM00-20D
Dredged
Mohns
2475–2681
Glassy basalt
0.1
A+B
SM00-22D
Dredged
Mohns
2240–2716
Glassy basalt
0.1
B
SM00-25D
Dredged
Mohns
2506–2695
Glassy basalt
0.1
B
SM00-27D
Dredged
Mohns
2642–2853
Glassy basalt
0.1
B
SM00-47D
Dredged
Mohns
2500–2700
Glassy basalt
0.1
B
SM00-52D
Dredged
Knipovich
3204–3385
Glassy basalt
2 · 10
SM00-54D
Dredged
Knipovich
3132–3152
Glassy basalt
2 · 105
B
SM00-55D
Dredged
Knipovich
3150–3344
Glassy basalt
2 · 105
B
SM00-2DS
Dredged
Mohns
2291–2459
Sediment
A+B
SM00-50SW
SM00-65SW
SM00-SSW
SM01-8SW
SM01-45SW
SM01-SSW
Bottle
Bottle
Water hose
Bottle
Bottle
Water hose
N69 06.87 0 –W16 04.46 0
N68 56.68 0 –W17 13.00 0
N68 56.89 0 –W17 11.70 0
N68 56.89 0 –W17 11.70 0
N68 23.00 0 –W18 00.00 0
N72 19.90 0 –E1 29.57 0 –N72
19.75 0 –E1 29.87 0
N72 40.14 0 –E2 53.70 0 –N72
40.26 0 –E2 53.20 0
N72 40.95 0 –E2 50.83 0
N72 38.12 0 –E2 42.00 0 –N72
38.35 0 –E2 41.68 0
N72 39.33 0 –E2 40.87 0 –N72
39.44 0 –E2 40.86 0
N72 40.69 0 –E2 42.41 0 –N72
40.41 0 –E2 42.53 0
N72 29.03 0 –E2 40.19 0 –N72
28.81 0 –E239.68 0
N72 26.04 0 –E2 31.35 0 –N72
2.85 0 –E232.03 0
N72 29.95 0 –E2 45.70 0 –N72
30.05 0 –E244.90 0
N72 31.74 0 –E2 32.30 0 –N72
31.84 0 –E229.99 0
N72 28.71 0 –E2 26.59 0 –N72
29.48 0 –E226.07 0
N72 23.08 0 –E1 43.24 0 –N72
23.24 0 –E142.04 0
N72 16.52 0 –E1 19 0 –N72
16.51 0 –E120.81 0
N72 44.53 0 –E3 56.31 0 –N72
44.92 0 –E357.79 0
N76 47.68 0 –E7 22.60 0 –N76
48.42 0 –E722.70 0
N76 48.12 0 –E7 24.62 0 –N76
48.92 0 –E7 24.74 0
N76 48.20 0 –E7 25.01 0 –N76
49.88 0 –E7 25.28 0
N72 19.90 0 –E1 29.57 0 –N72
19.75 0 –E1 29.87 0
N72 50.99 0 –E4 15.86 0
N76 57.03 0 –E7 15.04 0
Mohns
Knipovich
Knipovich
Mohns
Mohns
Mohns
2326
3186
0
950 m
650 m
0
Seawater
Seawater
Surface seawater
Seawater
Seawater
Surface seawater
A+B
A+B
N71 09.89 0 –W7 51.09 0
N71 10.33 0 –W7 52.87 0
basalt
basalt
basalt
basalt
basalt
basalt
DNA
seq.
B
A+B
A+B
5
B
A+B
A+B
B
For the dredged samples, the coordinates and depths given are the start and end point of dredging. ‘‘DNA seq.’’ indicates that native DNA sequences
(Figs. 4 and 5) are presented for Archaea (a) and Bacteria (b), respectively.
(5 0 -C/G/TGG GTC TCG CTC GTT A/GCC-3 0 ) used
for the outer reaction and the primers mPARCH340f
(5 0 -TAC/T GGG GC/TG CAC/G CAG-3 0 ) and
PARCH519r [20], the first with a 40-nucleotide long
GC clamp [21], for the inner reaction. The reaction mixture used was as for the bacterial DNA amplification. In
addition, 5% acetamide was added to the mixture for the
outer PCR reaction. The PCR program used for both
reactions was: 95 C, 15 min; 35 cycles of denaturation
at 94 C, 30 s; annealing at 53 C for the outer reaction
and 54 C for the inner reaction, 30 s; extension 72 C, 1
min; and a final extension 72 C, 10 min. In order to
maintain an appropriate control reaction during the amplification process, several controls were processed to
avoid confusion with false-positive or false-negative results. DNA from Archaeoglobus fulgidus, Methanococcus voltae, and Halobacterium sp. was used as negative
controls for bacterial primers and positive controls for
archaeal primers, and DNA from Escherichia coli as
negative controls for archaeal primers and positive con-
K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
217
trols for bacterial primers. A non-template control
(added distilled water instead of template) was always
included, as control for contamination from air or
PCR reagents.
Amplification products were verified by visualization
on 1.5% agarose gels stained with ethidium bromide.
The amplified products of microbial community were
further analyzed on 1 mm thick, 8% polyacrylamide
gels, as described previously [20]. The linear gradient
of urea and formamide ranged from 25% to 65%. Electrophoresis was carried out at 65 C, 70 V, and 18 h in
0.5X TAE buffer. After electrophoresis, the gels were
stained with SYBR Gold (Molecular Probes, Eugene,
OR) in 1X TAE buffer for 30 min and photographed.
Several replicate PCR amplifications and DGGE gels
were run in order to consider the reproducibility of the
community profiles, which were all highly reproducible.
Images of DGGE gels were analyzed using the Gel2k
image-analyzing software program developed by Svein
Norland (Department of Microbiology, University of
Bergen). The program was used to recognize the number
of visible DNA bands.
DNA fragments to be sequenced were excised from
the gel and processed as described earlier [20]. The sequences were analyzed using the BLAST tool [23] at
the National Center for Biotechnology Information
(NCBI). Sequences with a low degree of similarity to
the NCBI database sequences were investigated for possible chimera using the CHECK_CHIMERA program
[24], which is available through the Ribosomal Database
Project (RDP). Evolutionary distances of the DNA
sequences were calculated using Clustal_X [25] and phylogenetic trees were constructed using the neighbor-joining algorithm. In order to simplify the graphic
presentation of the results, identical or near-identical
(P98% similarity) sequences are presented as one operational taxonomic unit (OTU) in the phylogenetic trees.
Pair-wise comparisons of similar DNA partial sequences
were performed using the BLAST 2 Sequences tool [26]
at the NCBI.
The sequences reported in this paper have been deposited in the GenBank database under accession numbers AY463804 to AY463909, AY505431 to AY505438,
AY505441 to AY505443, and AY505445 to AY505455.
dition of an organic carbon source (formate, methanol,
ethanol, acetate, lactate, butyrate, succinate, or caproate), in concentrations of 10–20 mM in the final medium, was prepared with 0.2 lm filtered and autoclaved
aged seawater buffered with 30 ml 1 M sodium bicarbonate (NaHCO3) per liter medium. For enrichment of anaerobes, media were flushed with a mixture of N2:CO2
(90:10). Enrichment medium (30 ml) was added to each
50 ml serum bottle with 20 ml headspace, except for
media amended with H2 or CH4, which were added to
100 ml bottles with 70 ml headspace.
Approximately 1 g of crushed basalt or sediment was
added to each bottle containing enrichment medium.
For anaerobic media, this inoculation was performed inside a disposable glove bag (Glove Bag model S30-20,
Instruments for Research and Industry Inc., Cheltenham, PA, USA) flushed and filled with N2. Seawater
samples were introduced into the bottles in 3 ml portions
using a syringe. A total of 171 basalt enrichment cultures were inoculated onboard the ship. Identical culture
media were inoculated with either sediment or seawater.
A total of 35 seawater enrichments and 18 sediment enrichments were prepared. The enrichment cultures were
incubated at 4 C. Cultures were examined on shore by
phase contrast microscopy after 1, 2, and 4 monthsÕ incubation in order to determine occurrence of growth.
From cultures with observed growth, a subsample was
centrifuged and the cell pellet frozen for 16S rRNA gene
amplification, DGGE and sequencing.
Sulfide was measured in enrichment cultures after 1
month incubation according to the method described
by Cord–Ruwisch [37]. Reduction of ferric iron (Fe3+)
to ferrous iron (Fe2+) was observed as a color-change
from reddish brown to gray after 1, 2, and 4 months.
Methane in the gas phase of the methanogenic enrichment cultures was measured after 4 months of incubation using a gas chromatograph (Hewlett Packard
6890) with helium as carrier gas, as described by the
manufacturer [38]. The gas chromatograph was
equipped with a thermal conductivity detector (TCD)
and a HaySep R packed column.
2.3. Primary enrichment cultures
3.1. 16S rDNA amplification and DGGE community
profiles
Subsets of different media were used in order to enrich for microorganisms participating in the cycling of
iron (FePPi medium [27]; Fe-reducer medium [28]; FeTSB medium [29]), manganese (PYGV [30]; PC medium
[30]), sulfur (thiosulphate medium [31]; synthetic seawater medium designated ‘‘W20’’ modified from [32]
as described by [33]; PM1 [34]), and methane (methanogenic medium 2 [35]; NMS [36]). In addition, natural
seawater enrichment medium, with and without the ad-
3. Results
A total of 22 basalt samples, one sediment sample
and six water samples were processed for microbial community analyses. DNA was successfully amplified using
bacterial primers from all basalt, sediment and seawater
samples (Table 1), with the exception of one basalt sample (SM99-7R). Due to complexity of banding patterns
on bacterial DGGE gels, excised bands from the basalt
samples SM99-1R, -5R, and SM00-7D, and the surface
218
K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
Fig. 2. (a) DGGE banding patterns of a selection of samples (one surface seawater, three basalts) of PCR-amplified partial 16S rDNA with specific
primers for Bacteria and (b) Archaea (all samples with a PCR product). Sample number is indicated for each lane. M, marker.
seawater sample SM00-SSW did not result in clean 16S
rDNA sequences. Archaeal primers amplified DNA
from six of the 22 basalt samples (SM99-6R, SM002D, -17D, -18D, -20D, and -54D), the sediment sample,
and the four bottom seawater samples, but we were unable to retrieve any archaeal PCR products from the two
surface seawater samples and the remaining basalt samples. For amplification of archaeal DNA, visible PCR
products appeared only after the second round of the
nested PCR procedure. All excised bands from the archaeal DGGE gel resulted in clean sequences. No visible
PCR products were detected in any of the blanks, either
with bacterial or archaeal primers, containing distilled
water instead of DNA template.
Microbial community profiles for bacterial and archaeal populations inhabiting the basalt, sediment, and
seawater samples were obtained as banding patterns
on DGGE gels (Fig. 2). The bacterial DNA banding
patterns obtained for the basalt samples were complex
and 20-40 bands were generally detected, using the
Gel2k image-analyzing software program. For the sediment and seawater samples, 17 and 21–28 bands were
detected, respectively. Pronounced changes in relative
brightness of DGGE bands were observed between the
different samples. Fragments of the 16S rDNA amplified
from different microbial communities showed varying
degree of sharpness on the DGGE gels. Most of the
DGGE profiles appeared to have bands in common,
but no banding patterns were identical to each other,
suggesting that the basalts were inhabited by similar,
but not identical, bacterial populations. Most basalt
samples were observed to have 1–2 dominant bands
(e.g., bands 119 and 121, Fig. 2(a)). Sediment and seawater samples were observed to have between 1 and 6
dominant bands.
The archaeal DNA band patterns were unique for
each individual sample, except for the four bottom
seawater samples that showed pair wise (SM00-50SW
and -65SW; SM01-45SW and -8SW) similar DNA profiles to each other, as shown in Fig. 2(b). The PCR amplifications were composed of 6–12 resolvable bands on
the gel, and the bands were mainly positioned in the
middle of the gel.
3.2. Phylogenetic diversity of bacteria
Sequencing analysis of the individual bacterial
DGGE fragments were carried out on the 18 environmental basalt samples SM99-2R, -6R, SM00-2D, -4D,
-5R, -6D, -10D, -17D, -18D, -19D, -20D, -22D, -25D,
-27D, -47D, -52D, -54D, -55D, the sediment sample
SM00-2DS, the four bottom seawater samples SM0050SW, -65SW, SM01-8SW, -45SW, and the surface seawater sample SM01–SSW. The partial DNA sequences
retrieved from basalt microbial communities (a total of
65 sequences) were phylogenetically affiliated with eight
main groups of the domain Bacteria: Firmicutes, Chloroflexi, Actinobacteria, Bacteroidetes, and the a-,
c-, d-, and e-proteobacteria (Figs. 3,4). In addition, 1
sequence (SM00-19D-300N, Fig. 4(a)) could not be
K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
219
Fig. 3. Composition of the bacterial populations in basalt samples of different crustal age based on distribution of 16S rRNA gene sequences into
different main bacterial divisions. The composition of the background seawater is shown as comparison. The figure is drawn based on 3 samples, 7
sequences for the 20 Yr basalts; 14 samples, 52 sequences for the 0,1 Ma basalts; 1 sample, 6 sequences for the 1 Ma basalts; and 5 samples, 19
sequences for the background seawater.
Fig. 4. Phylogenetic tree based on partial 16S RNA gene sequences from bacteria inhabiting environmental basalt (in bold, sequence names ends
with N), sediment, and seawater samples compared to reference strain sequences from GenBank. Reference sequences were cut to the same length as
the sample sequences. The scale bar corresponds to 0.1 change per nucleotide, and GenBank accession numbers are in parenthesis. OTU1N = SM002D, -25D, -47D; OTU2N = SM00-4D, -5R, -6D, -18D, -20D; OTU3N = SM00-10D, -19D; OTU4N = SM00-20D, -55D; OTU5N = SM00-10D, 20D, -22D, -47D, -55D; OTU6N = SM00-2D, -22D; OTU7N = SM99-2R, SM00-2D, -27D. a- and c-proteobacteria are presented in a separate
phylogenetic tree (b).
220
K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
Fig. 4 (continued).
assigned to any known phylum. The closest match to
this sequence was a candidate division SBR1093 [39],
with 90% sequence similarity. The SM00-19D-300N sequence was not found to be a chimera, using the
CHECK_CHIMERA program.
A summary of the phylogenetic data from basalt, divided into different crustal age (20 Yr, 0.1 Ma, and 1
Ma) together with data retrieved from the seawater samples is shown in Fig. 3. The most frequently retrieved 16S
rRNA gene sequences from basalts grouped within
c-proteobacteria (18 sequences), a-proteobacteria (11 sequences), Chloroflexi (11 sequences), Firmicutes (10 sequences), and Actinobacteria (9 sequences). The
c-proteobacteria and the Firmicutes were the only phylogenetic groups that were detected in basalt from all three
age groups. Sequences affiliated with a-proteobacteria
and Bacteroidetes (3 sequences) were found in 20 Yr
and 0.1 Ma basalts, but not in the oldest basalt. Actinobacteria were found in 0.1 and 1 Ma basalt, but not in the
youngest basalt. Chloroflexi, e-proteobacteria (1 sequence) and c-proteobacteria (1 sequence) were only obtained from the 0.1 Ma basalts. The sequences retrieved
from the basalt samples show distinct differences in phylogenetic affiliation compared to those derived from seawater. In the seawater samples, two of the six different
phylogenetic groups documented, Verrucomicrobia and
the plastids, were not present in basalts. Two sequences
were closely related to phytoplankton plastids derived
from photosynthetic organism, both most closely related
to the plastid of the Haptophyceae Ochrosophaera neapolitana. Also in seawater, the abundance of d-proteobacteria was high, whereas only one sequence affiliating
with this group was retrieved from basalt.
Fig. 4 shows the phylogenetic position among the
bacterial sequences from basalt and seawater. The most
abundant sequences retrieved in this study belonged to
the c-proteobacteria (Fig. 4(b)). Most sites (12 of 18
sites) studied had representatives from this group, and
a high heterogeneity of sequences was detected. One sequence (SM00-52D-119N), which was recovered from a
dominant band (Fig. 2(a)), showed phylogenetic affiliation to the marine oligotrophic bacterium Hyphomicrobium indicum (98%). One sequence from one of the
youngest lava flows (SM00-52D-121N, corresponding
to dominant band 121 in Fig. 2(a)) was similar to a sequence from the iron reducer Shewanella frigidimarina
(95%). Three sequences clustered together with Marinobacter sp. Five sequences from the young basalt
(OTU5N), and one sequence from the 0.1 Ma basalt
grouped together with Acinetobacter junii and an uncultured c-proteobacteria. One sequence from the 1 Ma basalt was affiliated to Pseudomonas stutzeri, whereas
several of the sequences retrieved from seawater were affiliated to Pseudoalteromonas. One sequence from the
K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
0.1 Ma basalt showed phylogenetic affiliation to a novel
moderately halophilic bacterium, Salinisphaera shabanense, isolated from the Shabana Deep, which represent
a new deeply branching lineage within the c-proteobacteria. Also among the a-proteobacteria several different
sequences were retrieved (Fig. 4(b)). However, the 11 sequences retrieved originate from only five different sites.
Three sequences (OTU6N (2 sequences) and SM00-22D295N) showed phylogenetic affiliation to the marine
oligotrophic prosthecate bacteria Brevundimonas sp.
(100%), and Hyphomonas oceanitis (100%), respectively.
The Chloroflexi sequences from this study were all
from the 0.1 Ma basalts (Fig. 4(a)). Almost half of the
sequences (5 of 11) affiliating with Chloroflexi were from
one sample (SM00-10D) from the Mohns ridge. There
was a high heterogeneity of sequences within this group
and, with the exception of one sequence resembling Dehalococcoides sp., all retrieved sequences resembled previously uncultured strains. Within Firmicutes, there was
a much lower heterogeneity, even though this group
contained representatives of all three age groups (20
221
Yr, 0.1 and 1 Ma basalts) and all 10 sequences were
from different samples. One sequence affiliated with
the psychrophilic bacterium Clostridium estertheticum
and the other nine sequences matched an uncultured
bacterium (AF365631), which was sampled from coral
microfauna. The Actinobacteria sequences retrieved
comprised nine basalt sequences and two seawater sequences grouping into roughly four different clusters:
one cluster of microbial populations from the 0.1 and
1 Ma basalts affiliating with uncultured bacteria from
a metal contaminated soil (AY124390) and a subseafloor basalt environment (AY129940); one cluster of
two seawater sequences affiliating with Actinobacterium
K20-72, also a metal-contaminated soil clone; one cluster of 0.1 and 1 Ma basalts affiliating with the soil bacterium Arthrobacter globiformis; and one cluster of the
0.1 Ma sample SM00-20D-480N affiliating with Propionibacterium sp., isolated from moderately acidic mine
drainage waters. The Bacteroidetes group was divided
into one cluster of three 20 Yr and 0.1 Ma basalt
sequences and one seawater sequence affiliated with
Fig. 5. Phylogenetic tree based on partial 16S rRNA gene sequences from Archaea inhabiting environmental basalt samples (in bold, sequence names
ends with N), sediment sample, seawater samples, and reference strain sequences obtained from GenBank. Reference sequences were cut to the same
length as the sample sequences. The scale bar corresponds to 0.1 change per nucleotide, and GenBank accession numbers are in parenthesis.
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K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
Flavobacterium sp., isolated from the northern Baltic
Sea, and one cluster of three seawater sequences affiliated
with Polaribacter irgensii, previously found in Arctic
and Antarctic sea ice. One sequence from the 0.1 Ma
basalt belonged to e-proteobacteria, and showed highest
phylogenetic affiliation to a sequence recently recovered
from a study by Huber et al. [11] on bacterial diversity in
a subsurface habitat following a deep-sea volcanic eruption. The d-proteobacteria sequences from this study
were mainly found in the seawater samples, but also
one sequence from the 0.1 Ma basalt was retrieved.
All d-proteobacteria seawater sequences were affiliated
to a sequence from a study from the Arctic Ocean,
whereas the sequence from the basalt was most similar
to a psychrophilic sulfate reducing isolate from marine
Arctic sediments. Sequences affiliated to plastids and
Verrucomicrobia were only retrieved from seawater
samples.
The bacterial DNA sequences retrieved from bottom
seawater (16 sequences), surface seawater (3 sequences),
and sediment (1 sequence) differed from those retrieved
from basalt samples (Fig. 4). No sequences retrieved from
seawater were more than 89% similar to sequences from
basalt communities.
3.3. Phylogenetic diversity of Archaea
Sequencing of archaeal 16S rRNA gene DGGE band
fragments were carried out on the 0.1 Ma basalt samples
(SM99-6R, SM00-2D, -17D, -18D, -20D, -54D), the sediment sample SM00-2DS, and the bottom seawater samples SM00-50SW, -65SW, SM01-8SW, and -45SW. All
archaeal sequences retrieved (21 from basalt, 3 from sediment, and 12 from seawater) belonged to the non-thermophilic marine Group 1: Crenarchaeota (Fig. 5). The
archaeal DNA sequences from basalt, sediment, and
seawater samples were similar, but not identical.
Seven sequences from 0.1 Ma basalt and tree sequences retrieved from sediment clustered together with
an uncultured archaeon from the 11,000 m deep Mariana trench (D87350) and an uncultured archaeon from
a deep-sea carbonate crust. Overall the archaeal se-
Fig. 6. Phylogenetic tree of sequences from enriched bacteria (in bold italic, sequence names ends with E) compared to environmental sequences (in
bold, see Fig. 4) from basalt. Only those enrichment culture sequences displaying at least 92% similarity to environmental sequences are included.
Reference strains are from GenBank. The scale bar corresponds to 0.1 change per nucleotide, and GenBank accession numbers are in parenthesis.
OTU8E = SM00-47D, -52D; OTU9E = SM00-27D, -47D, -52D, -54D; OTU10E = SM00-27D, -52D; OTU11E = SM99-5R, -7R, SM00-7D, -18D, 22D, -27D, -52D, -54D; OTU12E = SM00-18D, -47D, -52D, -54D; OTU13E = SM99-5R, -6R.
K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
quences resembled uncultured strains previously found
in seawater [40–42], deep-sea sediments [43], and deepsea carbonate crusts [44].
3.4. Primary enrichment cultures
After 4 months of incubation, growth was observed
in 82 of the 171 enrichment cultures inoculated with basalt. In addition, growth was observed in 24 of the 35
cultures inoculated with seawater and in 14 of the 18 cultures inoculated with sediment. Enrichments with observed growth were screened using PCR-DGGE
analysis.
Reduction of ferric iron (Fe3+) to ferrous iron (Fe2+)
was observed in 4 of the 22 basalt enrichment cultures
with ‘‘Fe-TSB’’ medium (SM00-2D, -7D, -47D, and
-54D). This medium was amended with organic compounds as sources of carbon and electron donor. Enrichments SM00-7D, -47D, and -54D had a dominant band
in common, which was not seen in the SM00-2D culture.
Sequencing of this band position from SM00-7D resulted
in a sequence forming part of OTU11E and affiliating
with Shewanella frigidimarina (100% identity) and the environmental sequence SM00-52D-121N (Fig. 6). The
SM00-47D and -54D bands in this position were not
successfully sequenced, because we were unable to reamplify the excised DNA. The SM00-7D culture had
an additional dominant band, not seen in the other cultures. Sequencing analysis of this unique band (SM007D) showed that the DNA sequence also resembled the
iron-reducer Shewanella frigidimarina, but at a lower similarity (99%). Iron reduction was not detected in any of
the enrichments inoculated with seawater (eight cultures)
or sediment (five cultures), nor in any enrichment cultures
with ‘‘FePPI’’ or ‘‘Fe-reducer’’ medium, either with H2 or
organic compounds as electron donors.
223
Sulfide production, indicating the presence of sulfatereducing bacteria (SRB), was not detected in any of the
24 basalt enrichment cultures with ‘‘W20’’ medium. In
contrast, sulfide was produced in corresponding enrichments inoculated with bottom and surface seawater (6
out of 8) and sediment (2 out of 4).
Methane was produced in two out of seven basalt enrichment cultures with methanogenic media and H2 and
CO2 as energy- and carbon sources. These methane-producing cultures originated from the basalt samples
SM00-2D and -18D, but archaeal 16S rRNA gene sequences retrieved from these cultures did not match sequences from any known methanogens. Methane
production was not detected in the eight enrichment cultures inoculated with basalt and amended with acetate,
lactate, or trimethylamine, nor was it detected in corresponding enrichment cultures inoculated with bottom
and surface seawater (three cultures) or sediment (four
cultures). The archaeal DGGE profiles in the two basalt
cultures where methane production was detected differed (results not shown). In the SM00-2D DGGE pattern, only three bands were detected, whereas in the
SM00-18D pattern, seven bands were seen. One band
was present in both profiles, but sequencing of these
bands (SM00-2D-857 and –18D-853) showed that they
differed (Fig. 7).
The bacterial DNA sequences from enrichment cultures inoculated with basalt (a total of 136 sequences) affiliated with the same eight main phylogenetic groups of
the domain Bacteria that were present in the native basalt samples. The major fraction of the sequences from
enrichment cultures grouped within the c-proteobacteria
(114 sequences). Twenty-nine sequences retrieved from
basalt enrichment cultures were at least 92% similar to
environmental basalt sequences (Fig. 6). Sequences
from environmental and enriched basalt communities
that were 98–100% similar were only found within the
Fig. 7. Phylogenetic tree of archaeal partial sequences from enrichment cultures (in bold italic, sequence names ends with E) compared to
environmental sequences (in bold, see Fig. 5) from basalt. Only those enrichment culture sequences displaying at least 92% sequence similarity to
native sequences are included. Reference strains are from GenBank. The scale bar corresponds to 0.1 change per nucleotide, and GenBank accession
numbers are in parenthesis.
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K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
Firmicutes resembling Clostridium estertheticum (SM0017D-287N and SM99-2R-2E and OTU 8E) [45]. All
other sequences were less than 98% similar.
Chloroflexi, one of the most frequently retrieved phylogenetic groups from environmental samples, was only
represented by one sequence from enrichment cultures
(sequence SM00-2D-18E) resembling the five environmental sequences SM00-25D-351N, -25D-353N, and
OTU7N (containing three sequences). Three sequences,
all from enrichment cultures inoculated with basalt from
the Kolbeinsey ridge, resembled the oligotrophic bacterium Hyphomicrobium indicum and the environmental
sequence SM00-52D-119N from the Knipovich ridge
(Fig. 6). 12 sequences obtained from basalt enrichment
cultures (with ‘‘Fe-TSB’’, ‘‘Ppi’’, ‘‘Fe-red’’, ‘‘PYGV’’,
and ‘‘W20’’ media) resembled the iron reducer Shewanella frigidimarina and the environmental basalt sequence SM00-52D-121N (Fig. 6). Iron reduction was,
however, only observed in one of these cultures, as mention above. Manganese oxidation and reduction was not
measured in the ‘‘PYGV’’ cultures. Within the main
groups Actinobacteria and d-proteobacteria, the sequences retrieved from enrichment cultures did not
match any environmental sequences.
Bacterial sequences retrieved from sediment and seawater enrichment cultures (a total of 39 sequences) affiliated with six of the same phylogenetic groups retrieved
from basalt enrichment cultures: Firmicutes, Actinobacteria, Bacteroidetes, and the a-proteobacteria, c-proteobacteria, and e-proteobacteria (results not shown). Only
one sequence from a sediment enrichment was identical
to a basalt enrichment sequence (SM99-2R-2E), grouping with Clostridium estertheticum (phylum Firmicutes,
Fig. 6).
All archaeal sequences from enrichment cultures
were between 92% and 98% similar to the sequences retrieved directly from the basalt, except for one sequence
obtained from an enrichment culture (SM00-2D-857E),
which was 99% similar to an environmental sequence
(SM00-18D-837N). These sequences, although obtained
from different samples, came from the same ridge and
age group (0.1 Ma). All archaeal sequences retrieved
from basalt enrichment cultures were obtained from
methanogenic media in which methane production
was detected (Fig. 7). Archaeal sequences were not retrieved from any sediment or seawater enrichment
cultures.
4. Discussion
4.1. Bacterial diversity
DGGE analysis showed that the bacterial DNA
banding patterns obtained for the basalt samples was
complex (20–40 distinguishable bands), indicating a
relatively high diversity. This was higher than for seawater samples, which had 21–28 bands. Only the
clearly visual bands were excised and sequenced,
which gives an indication of the most abundant part
of population, but not the absolute diversity (Figs.
3,4). Several DNA bands from different profiles appeared to migrate the same distance on the gel. Sequencing, however, often showed that the sequences
were related but not identical. There are two possible
explanations for this: (1) closely related but non-identical sequences migrated to the same distance on the
gel (microheterogeneity), or (2) the bands with the
same position contained originally identical DNA
fragments which have accumulated ambiguous nucleotide positions or misreads during reamplification and
sequencing.
The diversity of sequences retrieved from basalts was
dominated by Proteobacteria (Figs. 3,4), primarily cproteobacteria. c-proteobacteria and Firmicutes were
found in most samples in all three age groups. The 18
c-proteobacterial sequences originated from 12 different
samples, whereas the 10 Firmicute sequences originated
from 10 different samples. Sequences affiliating with aproteobacteria, Chloroflexi and Actinobacteria were
also repeatedly found, but with a more inconsistent distribution, as these divisions were abundant in a few samples, but completely absent in others. Chloroflexi, which
was only found in 0.1 Ma basalts, has previously been
detected in seawater [46], non-hydrothermal subsurface
basalt [17], and sediment deposits [47]. Actinobacteria
was absent in the youngest basalts but showed an increased abundance with age, and appeared to be the
dominant group in the oldest (1 Ma) basalt. Actinobacteria are gram-positive bacteria that are well represented
by cultivated organisms, and have been reported in subsurface environments [48]. However, it is not possible to
draw any distinct conclusions regarding the timescale, as
these data reflect sequences obtained from only one microbial community from the oldest sample.
Most DNA sequences from environmental basalt did
not match precisely any sequences in the database (Figs.
4,5). One sequence (SM00-19D-300N) did not affiliate
with any known phyla, and could belong to an uncharacterized candidate division. This suggests that most of
the microorganisms in Arctic ridge basalts are previously uncharacterized. Exceptions were: (i) the sequence
SM00-20D-480N, which was identical to the V3 region
of Propionibacterium sp. WJ6, previously identified from
mine waters [49], (ii) OTU6N, which was identical to the
aquatic heterotrophic bacterium Brevundimonas sp. [50],
and (iii) SM00-22D-295N, which was identical to Hyphomonas oceanitis [51]. Both Brevundimonas and Hyphomonas are able to attach to solid surfaces and are
typical oligotrophic microorganisms. Hyphomonas are
often the first to colonize surfaces and initiate biofilm
formation [52].
K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
Some of the bacterial DNA sequences resembled
those of previously detected microorganisms associated
with sponges, corals, or vestimentiferans (Pogonophora)
(Fig. 4). These sequences were from samples collected at
different depths on all three ridges, so no correlation between depth of samples and sequences resembling
macrofauna-associated microorganisms could be
detected.
The sequences retrieved from basalt samples grouped
into nine phylogenetic main groups; Firmicutes, Chloroflexi, Actinobacteria, Bacteroidetes, a-, c-, d-, and e-proteobacteria, and Crenarchaeota (Figs. 4,5). In
comparison, previous studies of non-hydrothermal seafloor [15] and subseafloor [17] basalt reported four and
six phylogenetic main groups, respectively. These previously reported phylogenetic groups were among the nine
found in this study, except the b-proteobacteria, which
were only retrieved from the older subsurface basalt
[17]. Some sequences retrieved in the current study
matched (93–100%) sequences previously retrieved from
similar environments. Within c-proteobacteria, Chloroflexi, and Actinobacteria, sequences retrieved in the current study matched nine different reference strains
previously obtained from a similar study of subsurface
basalt from the Southeast Indian ridge (marked with *
in Fig. 4) [17]. The uncultured c-proteobacterium
ODP-155B-597 was 93% similar to SM00-54D-12N,
whereas the c-proteobacterium ODP-1162-327 was
identical to SM00-5R-460N (Fig. 4(b)). Within c- and
e-proteobacteria, our sequences matched two reference
strains previously obtained from a diffuse vent on the
Juan de Fuca ridge (marked with in Fig. 4) [11]. This
suggests that some populations might be common to
low-temperature seafloor environments.
In contrast, the sequences retrieved from basalt communities differ from those obtained from seawater, as
shown in Figs. 3 and 4. Seawater was dominated by cand d-proteobacteria. Only one sequence affiliated with
the d-proteobacteria was retrieved from basalt, and this
sequence did not resemble the sequences from seawater.
Several of the sequences retrieved from seawater were
affiliated to Pseudoalteromonas, which is a common organism found in marine environments. Seawater also
contained Verrucomicrobia and plastids, which were
not detected in basalts. Verrucomicrobia is a relatively
new division of bacteria, and this group is represented
by few isolates [48], however, culture independent methods have shown that this group of organisms is widely
distributed in the marine and terrestrial environments.
Recent studies involving a substantial molecular survey
of seawater also show different microbial diversity from
that found in basalt in the current study, where more
than 80% of the rRNA genes were affiliated to a-, band c-proteobacteria [46]. Bacterial community composition and processes in sediments also differ from the
basalt communities [3,47]. In a study on microbial
225
phylogeny in sediments, populations mainly differing
from those found in this current study was reported
[47]. Exceptions were sequences resembling Acinetobacter junii and Pseudomonas stutzeri, but these sequences
are marked as laboratory contaminants in [47]. If that
is so, the similarity has nothing to do with the environments from which the samples were retrieved. Some degree of similarity was also found between the
Chloroflexi isolate ODP1176A1H3z_8_B (accession
number AY191335) [47] and the sequences SM99-2R12N (90% similar), SM00-10D-277N (90% similar),
and SM00-10D-264N (88% similar). Other subsurface
Chloroflexi resembling sequences retrieved in the current
study includes the sequences H1.2.f (AF005747) and
H1.43.f (AF005749) from a subsurface paleosol [53],
which were 92% similar to the sequences SM00-10D280N and SM00-10D-277N, respectively. Microbial
communities from hydrothermal areas differ from those
found in this study [54]. Sequences obtained from basalt
associated hydrothermal fluids from the Juan de Fuca
ridge [11,13] differ from the sequences retrieved from
the cold seafloor basalts of the current study. Exceptions
were two sequences from diffuse vent fluids (<50 C) [11]
that were similar to the c-proteobacterial sequences affiliating with A. junii and of the only e-proteobacterial sequence retrieved in this study, respectively. Also, the 65
C hydrothermal fluid [13] differed more from the nonhydrothermal basalt of this study than the >50 C diffuse vent fluid [11], indicating that temperature is an important factor controlling the microbial community. In
low-temperature areas of the Juan de Fuca ridge, psychrophilic iron oxidizers have recently been isolated
from metalliferous deposits [14]. The two identified
groups of iron oxidizers were most closely related to
Marinobacter and Hyphomonas. Sequences affiliating
with Marinobacter and Hyphomonas were also retrieved
during the current study, but our sequences could not be
compared to those of [14] due to sequencing of different
regions of the 16S rRNA gene.
4.2. Archaeal diversity
During the amplification of archaeal DNA, visible
PCR products appeared only after the second round
of the nested PCR procedure, indicating a much lower
abundance of Archaea compared to Bacteria. Also, the
DGGE analysis showed less complex patterns, with
6–12 distinguishable bands.
All archaeal sequences obtained during this study affiliated with the marine Group 1: Crenarchaeota. None
of the sequences resembled any previously characterized
species. The lack of other archaeal groups beside the
marine group 1 Crenarchaeota may be due to the low
amount of archaeal DNA in the samples, leaving part
of the archaeal diversity under the detection limit of
the method. All archaeal sequences obtained from
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K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
environmental samples were similar, however, the
archaeal sequences were short, which will affect the
percentage similarity.
The Crenarchaeota is a well defined branch of the archaeal domain, which is obvious both from sequence
data as well as biochemical investigations. The Crenarchaeota was long considered to be only present at
high temperatures, and all cultured representatives of
the Crenarchaeota are originated from extremely hot
environments, including hydrothermal vents and geothermal springs [55]. Revealing the widespread diversity
of Crenarchaeota in non-extreme habitats is one of the
remarkable findings of culture-independent surveys.
Group 1 Crenarchaeota is the most widely distributed
and abundant form of all known Archaea, as they occupy
several different habitats and ecological niches. In a
study of Archaea in the mesopelagic zone of the Pacific
Ocean, these Crenarchaeota presented one of the oceans
single most abundant cell type [56]. Thorseth et al. [15]
also found the Group 1 Crenarchaeota to be the dominant Archaea of cold seafloor basalt environments at
the Knipovich ridge.
4.3. Physiology and microbial processes in seafloor basalt
One main objective of this study was to grow microbial strains detected in the environmental samples in order to map microbial processes involved in element
fluxes and biogeochemical cycles in this seafloor basalt
environment. In most cases, the microorganisms
detected in environmental samples were not successfully
grown with the enrichment media and conditions used in
this study. Exceptions were sequences from environmental and enriched basalt, found within the (1) Firmicutes
resembling Clostridium psychrophilum, which were 98–
100% similar to each other; and (2) Crenarchaeota,
which were 99% similar to each other, and therefore
could belong to the same species. All other sequences
from enrichment cultures were less than 98% similar to
any environmental sequence, and thus probably belonged to different species. This shows that the enriched
microorganisms comprise a minor part of the total in
situ community.
Within Chloroflexi, one of the most abundant phylogenetic groups present in the environmental samples
(Fig. 4), only one sequence could be obtained from enrichment cultures (Fig. 6). This sequence was 96% similar to environmental sequences.
Observations of iron and manganese oxyhydroxides
on the surfaces and along fractures in the basalt samples
collected, as well as observations of iron reduction in
iron-reducing bacterial (IRB) cultures and retrieval of
sequences resembling the iron reducer Shewanella, suggest that microbial catalyzed reduction of iron, and
probably also manganese, is important in non-hydrothermal seafloor basalt environments. Sequences resem-
bling those of the iron reducer Shewanella frigidimarina
were retrieved from both environmental and enriched
basalt samples (Fig. 6). Shewanella resembling sequences
were only retrieved from one of the four positive IRB
cultures, whereas DGGE pattern observations indicated
that Shewanella was also present in two more cultures.
In the fourth culture, however, no indication of Shewanella was seen either by DGGE or DNA sequencing, indicating that unknown iron-reducers were present in this
culture. In addition, sequences matching Shewanella
were retrieved from enrichments with the media ‘‘Ppi’’,
‘‘Fe-red’’ (both designed for iron reducers), ‘‘PYGV’’
(designed for manganese oxidizers), and ‘‘W20’’ (designed for sulfate reducers). Indeed, Shewanella is
known to reduce manganese and sulfate, as well as iron.
The detection of sequences resembling Shewanella in
media for manganese oxidizers, as well as the lack of observation of iron reduction in the ‘‘Ppi’’ and ‘‘Fe-red’’
media, could be due to anaerobic niches in the inoculum
or to the cultures also containing metal oxidizers. Sequences resembling Shewanella were obtained from enrichment cultures inoculated with basalt sampled from
lavas of different depths and ages, including the very
young samples from the Knipovich ridge.
Iron oxidation is a chemically spontaneous process in
the presence of oxygen at near-neutral pH, but can be
biologically catalyzed under microaerophilic conditions.
In a previous study of seafloor lavas from the Knipovich
ridge, SEM analysis revealed stalks resembling those
produced by the iron oxidizer Gallionella ferruginea in
the youngest and least altered sample [15]. Also, sequences matching G. ferruginea have been retrieved
from young basalt flows collected from shallow areas
around Jan Mayen, indicating that Gallionella is an inhabitant of Arctic ridge basalt [16]. The presence of
the iron oxidizing bacteria Gallionella and iron reducers,
such as Shewanella, in young seafloor basalts indicates
that microbial redox cycling of iron starts soon after
the formation of the lava flows and may be important
in the weathering of ocean crust basalt.
Methane production was detected in two out of seven
cultures on methanogenic media inoculated with basalt,
both from 0.1 Ma samples collected from deep sites on
the Mohns ridge. Although the primers were designed
to target all known methanogens, methanogenic Archaea were not detected in the DGGE analysis. Possible
explanations for this could be that unknown methanogens with different primer homology regions were present; or that the number of methanogens was under
the detection level for the applied method. Bidle et al.
[57] also experienced the difficulty of retrieving sequences affiliating with methanogens using 16S rRNA gene
targeting primers. With these primers they only retrieved
Group 1 Crenarchaeaota, whereas using specific primers
targeting enzymes involved in methanogenesis resulted
in sequences belonging to methanogens. In another
K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
study, lithoautotrophic methanogens and heterotrophic
IRB from subseafloor basalt collected from the northern
flank of the Southeast Indian ridge were successfully enriched [17]. This indicates that both methanogens and
IRB are present in non-hydrothermal ocean crust basalt,
and that lithoautotrophic methanogens could be important primary producers in this environment. It has been
suggested that the deep biosphere has a hydrogen and
carbon dioxide dependent primary production [1,58].
The hydrogen source for methanogenesis in the seafloor
basalt studied here is unknown, and could be either a result of inorganic (fluid–rock interactions) or microbial
processes. Geochemically produced hydrogen is, however, only reported to be produced at high-temperature
fluid–rock reactions associated with volcanic eruptions
and serpentinization [59,60].
Sulfate reducing bacteria were not detected in basalt
samples, either in DNA analysis of environmental
samples or in enrichment studies. Neither was any sulfide minerals detected in the basalts by petrographical
examination, indicating that sulfate reduction is not a
dominant process. We were only able to grow a minor
part of the total microbial diversity in environmental
basalt, so there could be sulfate-reducing bacteria in
the uncultivable fraction. Sulfate reduction was, however, common in sediment and seawater enrichments.
The failure of detecting SRB in basalts thus supports
the view that basalt samples were not heavily contaminated with microorganisms from seawater or
sediment.
4.4. Evaluation of possible contamination
As the basalt samples were transported through the
seawater column, indigenous seawater microbes could
easily attach to the basalt. In order to evaluate this potential contamination, surface seawater samples as well
as bottom seawater samples were included in the analysis. The phylogenetic analysis showed that the microbial
community in the seawater samples differed from that of
the basalts. The two phylogenetic groups Verrucomicrobia and plastids derived from photosynthetic organisms
in the seawater were absent in the basalt samples. Also
the fraction of the d-proteobacteria was much higher
in the seawater samples. The quick handling of samples
and the use of methods only analyzing the dominant
part of the populations may explain the lack of extensive
seawater contaminants, which the rock samples were exposed to during dredging.
As only one sediment sample was analyzed, little can
be concluded about possible sediment contamination.
One sequence from an enrichment culture inoculated
with sediment was identical to environmental sequences
retrieved from basalt. This sequence similarity between
strains from basalt and sediment could, however, be
due to the habitats being in close contact and forming
227
a continuous habitat, rather than contamination. Most
sequences retrieved during this study were unrelated to
previously characterized bacteria from sediment and
seawater, and are good candidates for indigenous seafloor basalt inhabiting bacteria.
Another source of contamination is amplification of
contaminant DNA instead of, or in addition to, DNA
extracted from the samples. As the basalts contain low
amount of biomass and DNA, they are susceptible to
amplification of contaminant DNA. Yet another possible source of contamination is the laboratory water supply system. Even though blanks were routinely used for
DNA amplification during this study, some candidates
that have been reported as typical laboratory contaminants were found. This applies especially to sequences
affiliating with the c-proteobacteria Acinetobacter junii
(6 sequences) and Pseudomonas stutzeri (1 sequence)
(Fig. 4(b)). A. junii and P. stutzeri are common constituents of laboratory water supply [61–63] and have repeatedly been found to contaminate low-biomass
samples [2,61]. We can therefore not exclude the possibility that some of the c-proteobacterial sequences might
be derived from laboratory contaminants.
5. Conclusions
The aim of this study was to describe the microbial
diversity in seafloor basalts and to identify possible microbial groups unique to these environments. By culturing approaches, we wanted to gain an improved
understanding of the physiology of the organisms present in deep sea basalts and also determine if there is a
correlation between the diversity of microorganisms retrieved from these basalt samples and geochemical characteristics of the environments. The most frequently
retrieved sequences from the basalt communities affiliated
with the Chloroflexi, Firmicutes, Actinobacteria, and
the c- and a-proteobacteria, suggesting that members of
these phylogenetic groups dominate this habitat. Our results show that distinct microorganisms, different from
those observed in deep seawater, inhabit seafloor basalt.
Some of the phylotypes retrieved during this study were
closely related to marine bacteria, especially within the
c-proteobacteria and Marine Group 1: Crenarchaeota.
Other sequences showed less than 90% sequence similarity to any previously retrieved microorganisms or DNA
sequences, especially within the Chloroflexi. The original
source habitat for the basalt microbial community is
probably seawater, as the bacteria could not have originated from the extremely hot molten lava. However,
none of the sequences retrieved from seawater during
this study were more than 89% similar to sequences
from basalt communities. Also, the phylogenetic and
physiological diversity of the non-hydrothermal seafloor
basalt microorganisms differ from those previously
228
K. Lysnes et al. / FEMS Microbiology Ecology 50 (2004) 213–230
found at hydrothermal regions [11–13,54], seawater [46],
and sediments [3,47]. Detection of DNA sequences similar to those of previous microbial surveys of seafloor
and subseafloor basalt indicates that non-hydrothermal
ocean crust in general could be inhabited by related microbial populations. Parts of the cultivable fraction of
microorganisms participate in the reduction of iron
and in the production of methane. Evidence of oxidation and reduction of iron in very young basalts indicate
that biologically catalyzed cycling of iron initiates in the
oceanic crust relatively quickly after its formation. Reduction of sulfate was not detected in basalt cultures,
but was common in enrichments inoculated with sediment and bottom and surface seawater. The majority
of bacterial sequences retrieved from basalt samples
and enrichment cultures showed no close relation to cultured relatives. Further work to explore these unique microorganisms is thus urgently needed.
Acknowledgements
This work was supported by the Norwegian Research
Council (NFR) through the ‘‘SUBMAR’’ program
(128418/431). We thank Svein Norland for providing
the software used for DGGE gel analysis. We also thank
two anonymous reviewers for helpful and constructive
comments on the manuscript.
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