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The island of Surtsey was formed in 1963–1967 on the offshore Icelandic volcanic rift zone. It offers a unique opportunity to study the subsurface biosphere in newly formed oceanic crust and an associated hydrothermal-seawater system, whose maximum temperature is currently above 120°C at about 100m below surface. Here, we present new insights into the diversity, distribution, and abundance of microorganisms in the subsurface of the island, 50years after its creation. Samples, including basaltic tuff drill cores and associated fluids acquired at successive depths as well as surface fumes from fumaroles, were collected during expedition 5059 of the International Continental Scientific Drilling Program specifically designed to collect microbiological samples. Results of this microbial survey are investigated with 16S rRNA gene amplicon sequencing and scanning electron microscopy. To distinguish endemic microbial taxa of subsurface rocks from potential contaminants present in the drilling fluid, we use both methodological and computational strategies. Our 16S rRNA gene analysis results expose diverse and distinct microbial communities in the drill cores and the borehole fluid samples, which harbor thermophiles in high abundance. Whereas some taxonomic lineages detected across these habitats remain uncharacterized (e.g., Acetothermiia, Ammonifexales), our results highlight potential residents of the subsurface that could be identified at lower taxonomic rank such as Thermaerobacter, BRH-c8a (Desulfallas-Sporotomaculum), Thioalkalimicrobium, and Sulfurospirillum. Microscopy images reveal possible biotic structures attached to the basaltic substrate. Finally, microbial colonization of the newly formed basaltic crust and the metabolic potential are discussed on the basis of the data.
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Frontiers in Microbiology | www.frontiersin.org 1 September 2021 | Volume 12 | Article 728977
ORIGINAL RESEARCH
published: 29 September 2021
doi: 10.3389/fmicb.2021.728977
Edited by:
Mark Alexander Lever,
ETH Zürich, Switzerland
Reviewed by:
Gustavo Antonio Ramírez,
University of North Carolina at Chapel
Hill, UnitedStates
Florentin Constancias,
ETH Zürich, Switzerland
*Correspondence:
Viggó Thor Marteinsson
viggo@matis.is
Specialty section:
This article was submitted to
Extreme Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 22 June 2021
Accepted: 30 August 2021
Published: 29 September 2021
Citation:
Bergsten P, Vannier P, Klonowski AM,
Knobloch S, Gudmundsson MT,
Jackson MD and
Marteinsson VT (2021) Basalt-Hosted
Microbial Communities in the
Subsurface of the Young Volcanic
Island of Surtsey, Iceland.
Front. Microbiol. 12:728977.
doi: 10.3389/fmicb.2021.728977
Basalt-Hosted Microbial
Communities in the Subsurface of
the Young Volcanic Island of Surtsey,
Iceland
PaulineBergsten
1,2, PaulineVannier
1, AlexandraMaríaKlonowski
1, StephenKnobloch
1,
MagnúsTumiGudmundsson
3, MarieDoloresJackson
4 and ViggóThorMarteinsson
1,5*
1 Exploration & Utilization of Genetic Resources, Matís, Reykjavík, Iceland, 2 Faculty of Life and Environmental Sciences,
University of Iceland, Reykjavík, Iceland, 3 Nordvulk, Institute of Earth Sciences, University of Iceland, Reykjavík, Iceland,
4 Department of Geology and Geophysics, University of Utah, Salt Lake City, UT, United States, 5 Faculty of Food Science and
Nutrition, University of Iceland, Reykjavík, Iceland
The island of Surtsey was formed in 1963–1967 on the offshore Icelandic volcanic rift
zone. It offers a unique opportunity to study the subsurface biosphere in newly formed
oceanic crust and an associated hydrothermal-seawater system, whose maximum
temperature is currently above 120°C at about 100 m below surface. Here, wepresent
new insights into the diversity, distribution, and abundance of microorganisms in the
subsurface of the island, 50 years after its creation. Samples, including basaltic tuff drill
cores and associated uids acquired at successive depths as well as surface fumes from
fumaroles, were collected during expedition 5059 of the International Continental Scientic
Drilling Program specically designed to collect microbiological samples. Results of this
microbial survey are investigated with 16S rRNA gene amplicon sequencing and scanning
electron microscopy. To distinguish endemic microbial taxa of subsurface rocks from
potential contaminants present in the drilling uid, weuse both methodological and
computational strategies. Our 16S rRNA gene analysis results expose diverse and distinct
microbial communities in the drill cores and the borehole uid samples, which harbor
thermophiles in high abundance. Whereas some taxonomic lineages detected across
these habitats remain uncharacterized (e.g., Acetothermiia, Ammonifexales), our results
highlight potential residents of the subsurface that could beidentied at lower taxonomic
rank such as Thermaerobacter, BRH-c8a (Desulfallas-Sporotomaculum),
Thioalkalimicrobium, and Sulfurospirillum. Microscopy images reveal possible biotic
structures attached to the basaltic substrate. Finally, microbial colonization of the newly
formed basaltic crust and the metabolic potential are discussed on the basis of the data.
Keywords: 16S rRNA gene amplicon sequencing, bacterial and archaeal communities, microbial diversity, extreme
environment, subsurface, oceanic basaltic crust, Iceland
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 2 September 2021 | Volume 12 | Article 728977
INTRODUCTION
e subsurface biosphere, dened as an ecosystem encompassing
regions beneath soils and sediments, occupies roughly twice
the volume of the oceans and holds about 15% of the total
biomass on Earth (Pedersen, 2000; Heberling et al., 2010;
Bar-On et al., 2018; Orcutt et al., 2019). Recent estimates
suggest that these zones (i.e., continental subsurface, subseaoor
sediments, and oceanic crust) contain ~70% of all prokaryotic
cells and possibly more than 80% of all bacterial and archaeal
species (Magnabosco et al., 2018). is large biome has only
recently become the focus of research studies (Gold, 1992),
and most microbial surveys have, to date, focused on subseaoor
sediments (Parkes et al., 2000; D’Hondt et al., 2004; Inagaki
et al., 2015). e deep biosphere hosted in the basaltic ocean
crust, on the contrary, has been understudied because of various
challenges: the relative inaccessibility of the samples (drilling
operation oen required), the low biomass (high risk of external
contamination), the presence of minerals which generates DNA
binding and inhibitions (Direito etal., 2012; Lever et al., 2015),
and the diculties in handling the inevitable microbial
contamination from the surface (Baross et al., 2004; Sheik
et al., 2018; Ramírez et al., 2019).
Over the past decades, many microorganisms have been
discovered within Earths igneous oceanic crust despite extreme
conditions, such as oligotrophy, temperature gradients, active
circulation, and limited available space within the rock, which
were once thought unsuitable to supporting life (Cowen etal.,
2003; Nakagawa et al., 2006; Orcutt et al., 2011b; Jungbluth
et al., 2013, 2014; Lever et al., 2013; Baquiran et al., 2016).
Studies of ridge-ank systems have demonstrated that crustal
aquifers harbor aerobic mesophiles and anaerobic thermophiles,
involved in hydrogen, nitrogen, carbon, and sulfur cycling
(Cowen et al., 2003; Nakagawa et al., 2006; Smith et al., 2011;
Lever et al., 2013; Orcutt et al., 2013; Robador et al., 2015;
Jungbluth et al., 2016). e comparison of biomes in very
young (<3 Ma) and old (80 Ma) oceanic crust indicates that
microbial diversity increases with the age of the basalt and
the community compositions converge toward similar proles
over time (Lee et al., 2015). e colonization of rocks and
the succession of microbial communities mainly depend on
the temperature and reduction-oxidation conditions (Baquiran
etal., 2016; Ramírez etal., 2019). While microbial communities
hosted by young basalt (<10 Ma) have been detected (Konhauser
et al., 2002; Templeton et al., 2005), our knowledge of the
pioneering communities inhabiting newly erupted oceanic basalt
(<100 years) is extremely limited.
A recent drilling operation has addressed this gap in knowledge
through the study of the volcanic island, Surtsey, located on
the southern oshore extension of the Icelandic volcanic ri
zone (Figure 1). e island is the visible part of a volcano
formed by underwater and basaltic eruptions from the seaoor
between 1963 and 1967 (Þórarinsson, 1965; Jakobsson and
Moore, 1982; Jakobsson et al., 2009). Since the initiation of
eruptive activity, the entire area has been accessible only to
scientic investigations. Surtsey is the site of long-term
longitudinal studies, which have provided a unique record of
pioneering species of plants and animals colonizing the surface
of the basaltic deposits (Baldursson and Ingadóttir, 2007;
Magnússon etal., 2014). In 1979, a 181 m core (SE-01) drilled
through the eastern sector of the Surtur vent (Figure1), probed
the hydrothermal system in the subaerial and the submarine
deposits – above and below coastal sea level, respectively
(Jakobsson and Moore, 1982, 1986; Jackson et al., 2019a). In
2017, three new cored boreholes (SE-02a, SE-02b and SE-03)
were acquired through the International Continental Scientic
Drilling Program (ICDP) 5,059 expedition, SUSTAIN drilling
operation (Jackson et al., 2015, 2019b; Weisenberger et al.,
2019). Annual monitoring of temperatures in the 1979 borehole,
SE-01, indicates that the hydrothermal system has cooled down
over the years. e maximal temperature in 1980 was 141.3°C
at 100 m depth, and it has decreased gradually to 124.6°C in
2017 (Figure 1; Jakobsson and Moore, 1986; Ólafsson and
Jakobsson, 2009; Marteinsson etal., 2015; Jackson etal., 2019b).
e current temperatures exceed a presumed upper limit for
functional microbial life (122°C; Prieur and Marteinsson, 1998;
Kashe and Lovley, 2003; Takai etal., 2008). e highly porous
subaerial and submarine tephra deposits were largely transformed
to palagonitized lapilli tu by 1979, described in studies of
the SE-01 drill core (Jakobsson and Moore, 1982; Jakobsson
and Moore, 1986). Progressive alteration was recorded in samples
from a parallel, time-lapse drill core acquired in 2017, SE-02b
(Figure 1; Prause et al., 2020). A recent study of geothermal
water chemistries in the 1979 and 2017 Surtsey boreholes
indicates depletion in boron, magnesium, iron, carbon dioxide,
and sulfate concentrations, suggesting that the uid compositions
in the subsurface deposits are controlled by seawater-basalt
interactions and temperature (Kleine etal., 2020). is further
suggests that uid-rock interactions in the submarine Surtsey
basaltic deposits behave similarly to those interactions in basaltic
oceanic crust, where the chemical composition of rocks and
uids changes and introduces organic matter and oxygen into
the system (Furnes and Staudigel, 1999; Edwards et al., 2005;
Orcutt et al., 2011a). e Surtsey volcano geothermal system
represents an exceptional natural laboratory for studying uid-
rock-microbe interactions at temperatures approaching the
presumed thermal limit for functional life on Earth. Its boreholes
can be viewed as windows opened from the land surface that
allow the study of subsurface processes at high temperature
associated with the basaltic oceanic crust.
Several studies have described the volcanic structure of
Surtsey and the geochemical, chemical, and mineralogical
changes in the altered basaltic deposits (Jakobsson and Moore,
1982; Jakobsson and Moore, 1986; Schipper etal., 2016; McPhie
et al., 2020; Moore and Jackson, 2020; Prause et al., 2020).
Little is known, however, about the microorganisms inhabiting
the subsurface and their metabolic potential. A pioneering
study by Marteinsson et al. (2015) detected archaea of the
Archaeoglobus genus at 145 m depth (80°C) and one taxon
from the Methanobacteriales order in the SE-01 borehole at
depths to 170 m (55°C; Marteinsson etal., 2015). Furthermore,
recent studies of 1979 and 2017 drill core samples show abundant
microtubules in basaltic glass that resemble endolithic
microborings (Jackson et al., 2019a; Jackson, 2020); these are
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 3 September 2021 | Volume 12 | Article 728977
thought to indicate microbial microboring into glass (Fisk
et al., 2003; Staudigel et al., 2008; Walton, 2008; Mcloughlin
et al., 2010). ese investigations suggest that Surtsey’s basalt
and uids could reveal new information about the microbial
communities associated with the young hydrothermal-seawater
system and the newly formed oceanic basaltic crust.
is work focuses on the following questions: (i) how can
we distinguish the true residents of the subsurface from the
marine microbial taxa derived from the drilling uid? (ii)
which microorganisms inhabit Surtsey’s basalt and uids? (iii)
does life persist in the subsurface zones crossing the presumed
thermal limit for life? and (iv) can wedetect actual microbial
structures or biotic signatures in the basaltic cores only 50 years
aer eruptions terminated? Weused high-throughput sequencing
(16S rRNA gene amplicon sequence analysis) and scanning
electron microscopy (SEM) to address these questions and
enable an in-depth characterization of the basalt-hosted microbial
communities within the volcanic system. Microbial communities
from drill core samples collected at successive depths from
the subaerial and submarine basaltic tephra deposits were
compared to the communities in borehole uid samples associated
with the active hydrothermal system and in fumarole samples
from the surface of the island and seawater samples collected
several kilometers oshore. In this study, it was of crucial
importance to collect drilling uids, sequence control samples
representing potential sources of contamination, and implement
computational methods to distinguish the endemic microbial
taxa of the subsurface rocks from the microorganisms found
in the drilling uid and other potential contaminants. Here,
we report our strategy to identify and minimize contaminants
in the data sets; we describe the archaeal and bacterial taxa
that are candidate residents of the oceanic crust; we discuss
the microbial colonization and dissemination from the
surrounding ecosystems, and we explore microbial metabolic
FIGURE1 | Location of the study area of the south coast of Iceland. Map of the Surtsey volcanic island within the Westman Islands (Vestmannaeyjar) archipelago
and location of sampling sites: 1979 borehole SE-01 and 2017 borehole SE-02b (green star), drilling uid (blue line), and fumaroles (orange stars). Schematic cross
section of Surtur crater showing the geological setting of the subsurface deposits and the location of the time-lapse cored boreholes (see Jackson etal., 2019b) and
a zone of seawater inow.
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 4 September 2021 | Volume 12 | Article 728977
potentials. e results provide unique data on subsurface
microbial life in one of the newest and most pristine oceanic
basalt environments on Earth.
MATERIALS AND METHODS
Site Description
e volcanic island of Surtsey is located in the Vestmannaeyjar
archipelago, approximately 35 km from the south coast of Iceland
(63°18'10.8'N; 20°36'16.9'W; Figure 1) within the southern
oshore extension of Iceland’s Eastern Volcanic Zone. e
ocean depth was 130 m below sea level prior to eruption. Aer
3.5 years of submarine, explosive, and eusive lava-producing
eruptions of basalts from the seaoor in 1963–1967, Surtsey
had a subaerial area of 2.6 km2, and the highest point on the
island was 174 m above sea level. e island has now eroded
to less than 1.2 km2 and a height of 150 m above sea level
(Baldursson and Ingadóttir, 2007; Óskarsson et al., 2020).
e cores extracted from the SE-01 borehole in 1979 and
the SE-02b borehole in 2017 indicate that the subsurface of
Surtsey consists mainly of lithied tephra, mainly lapilli tu,
and minor amounts of weakly consolidated tephra and alkali
olivine basalt intrusions (Jakobsson and Moore, 1982; McPhie
et al., 2020; Figure 1). e principal authigenic minerals in
the lapilli tu are smectitic clay mineral (nontronite and
clinochlore), analcite, phillipsite, Al-tobermorite, and anhydrite
(Jakobsson and Moore, 1986; Jackson et al., 2019a; Prause
etal., 2020). Alteration through palagonitization and production
of authigenic cementitious minerals has progressed during the
past 38 years, from 1979 to 2017 (Jackson, 2020; Prause etal.,
2020). In situ subsurface uid temperatures have been measured
annually since 1980 in SE-01, usually at 1-m intervals from
the surface to the bottom of the borehole (e.g., Jakobsson and
Moore, 1986; Marteinsson et al., 2015). Geochemical analyses
and pH measurements of borehole uids and pore water
extracted from the 2017 drill cores are described by Kleine
et al. (2020).
Drilling Operation and Drilling Fluid
One of the principal objectives of the 2017 Surtsey Underwater
volcanic System for ermophiles, Alteration processes and
Innovative concretes (SUSTAIN) drilling project at Surtsey
volcano, sponsored in part by the ICDP, was to investigate
microbial diversity within the basaltic tephra. All possible
precautions were made to avoid microbial contamination (Jackson
et al., 2019b). Water from the sea was pumped to the drill
site and used as drilling uid since no fresh water is available
on the island (Figure 1). e conventional methods used to
track contamination during drilling operations, such as the
addition of tracer compounds in the circulating uid, could
not be applied during drilling at Surtsey due to the strict
environmental protection of the island (see discussion). To
avoid contamination, drilling uid was sterilized using two
ltration units with a pore size of 30 μm (Pentek Big Blue,
R30-BB 30 Micron cartridge lter, Lenntech, e Netherlands)
and two ultraviolet sterilization devices (AQUA4ALT from
WEDECO, Aquaculture systems, Xylem Water Solutions Herford
GmbH, Germany) with a maximum ow rate of 1.58 ls1. e
decontaminated drilling uid was stored in 1000 L containers
before pumping into the borehole. To track potential problems
with the sterilization system, 1 l of drilling uid was collected
regularly at dierent drilling depths to assess potential microbial
contaminants (Figure 1).
Sample Collection
Four types of samples were collected for molecular investigation
and comparison of microbial diversity among these sample
types: drill cores from the 2017 SE-02b borehole, borehole
uids from the 1979 SE-01 borehole, steam from surface cracks
of fumaroles, and seawater samples collected few kilometers
from the northwest coast of Surtsey (Figure 1). A description
of the samples is shown in Tabl e  1 .
Seventeen drill core samples were collected for microbial
analyses from the vertical SE-02b cored borehole that extends
to 192 m below surface and terminates in poorly consolidated
tephra a few meters above the presumed depth of the pre-eruption
seaoor (Jackson et al., 2019b). At the drill site, drill core
samples were collected from every third 3-m core run for
molecular analyses by cutting a 10-cm section at 70 cm from
the top of the core run. Immediately aer sampling, each
section was kept in the plastic core liner, taped at both ends,
wrapped in a plastic bag, kept in liquid nitrogen on site, and
at 80°C for long-term laboratory storage. Fluid samples from
SE-01 borehole were collected in 2016 and in 2017, before
drilling operations started, using a custom sampler made of
stainless steel, as described in Marteinsson et al. (2015). e
sampler was rinsed with 70% ethanol before each sampling.
Eighteen uid samples were collected from the SE-01 borehole:
18in 2016 and seven in 2017 (Tab l e 1 ). Steam from fumaroles,
located on the summit of Surtur, the eastern tephra cone
(~150 m above sea level; 63°18'15.4'N 20°36'07.7'W) and
Surtlungur, the western tephra cone (63°18'19.9'N 20°36'24.7'W)
were collected in 2017 by introducing a sterile rubber hose
into the outlet of the fumarole, with the other end connected
to a sterile plastic container. is generated 5,350 ml of condensed
water from the fumaroles over 12 h of continuous sampling.
Four liters of seawater samples were collected 25 km oshore
during the drilling operation (63°28'58.8'N; 20°54'7.2'W). All
water samples (drilling uid, borehole uid, fumarole, and
seawater samples) were immediately ltrated through 0.22-μm
Sterivex lters (Merck Millipore). e lters were stored in
liquid nitrogen on site and at 80°C for long-term storage
in the laboratory.
DNA Extraction
DNA Extraction From Rock Samples
A modified PowerMax® Soil DNA Isolation Kit protocol
(MO BIO Laboratories, Inc.) was applied to extract the
DNA from the drill core samples. Small fragments of tuff
from the interior of the 17 frozen core samples from SE-02b
were broken aseptically. After 2 min pre-cooling on ice,
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 5 September 2021 | Volume 12 | Article 728977
TABLE1 | Sample description table: locations, sampling date, depth, temperature, and DNA concentration.
Sample ID Sample types
and categories
Sample
site
Sampling
date
Collection
depth (m b.s.)
Collection
Temp (°C)
Sample
amount
DNA
concentration
NanoDrop (ng/μl)
260/280 DNA
concentration
QuBit dsDNA
(ng/μl)
Sur161 Borehole uid SE-01 6/9/2016 164 56.56 500 ml 16.14 1.41 0.16
Sur162 Borehole uid SE-01 6/9/2016 164 56.56 300 ml 15.48 1.35 0.30
Sur163 Borehole uid SE-01 6/9/2016 166 54.24 500 ml 26.65 1.43 Too low
Sur164 Borehole uid SE-01 6/9/2016 162 58.88 400 ml 8.33 1.29 Too low
Sur165 Borehole uid SE-01 6/9/2016 70 109.17 250 ml 56.12 1.56 0.09
Sur166 Borehole uid SE-01 6/9/2016 100 124.62 500 ml 34.07 1.58 0.15
Sur167 Borehole uid SE-01 6/9/2016 100 124.62 250 ml 38.24 1.50 0.81
Sur168 Borehole uid SE-01 6/9/2016 120 115.78 550 ml 97.37 1.61 0.56
Sur169 Borehole uid SE-01 6/9/2016 160 61.27 500 ml 17.59 1.05 0.13
Sur1610 Borehole uid SE-01 6/9/2016 160 61.27 150 ml 12.31 1.21 0.11
Sur16Mix Borehole uid SE-01 6/9/2016 mix n.a. 1,400 ml 52.23 1.14 0.15
Sur171 Borehole uid SE-01 8/3/2017 58 87.75 5,000 ml 48.80 1.49 n.a.
Sur172 Borehole uid SE-01 8/3/2017 120 115.78 1,820 ml 38.25 1.46 n.a.
Sur173 Borehole uid SE-01 8/3/2017 mix n.a. 980 ml 35.38 1.49 n.a.
Sur174 Borehole uid SE-01 8/3/2017 mix n.a. 805 ml 23.07 1.53 n.a.
Sur175 Borehole uid SE-01 8/3/2017 150 74.22 5,000 ml 52.80 1.45 n.a.
Sur176 Borehole uid SE-01 8/8/2017 160 61.27 5,000 ml 35.95 1.49 n.a.
Sur177 Borehole uid SE-01 8/8/2017 mix n.a. 850 ml 16.59 1.58 n.a.
C4 Drill core (DC_1) SE-02b 8/22/2017 23*20.00 15 g 181.61 1.42 0.17
C9 Drill core (DC_1) SE-02b 8/22/2017 35*36.00 15 g 196.47 1.44 0.14
C13 Drill core (DC_2) SE-02b 8/22/2017 44*59.00 15 g 202.15 1.33 0.16
C17 Drill core (DC_2) SE-02b 8/23/2017 55*82.50 15 g 137.89 1.43 0.11
C22 Drill core (DC_2) SE-02b 8/23/2017 65 101.50 15 g 115.60 1.46 0.41
C27 Drill core (DC_3) SE-02b 8/23/2017 78 114.00 15 g 204.83 1.42 0.11
C33 Drill core (DC_3) SE-02b 8/24/2017 93 123.00 15 g 202.74 1.38 0.09
C36 Drill core (DC_3) SE-02b 8/24/2017 102 124.00 15 g 202.32 1.41 0.16
C39 Drill core (DC_3) SE-02b 8/24/2017 111 121.50 15 g 197.72 1.35 0.15
C42 Drill core (DC_3) SE-02b 8/24/2017 120 116.00 15 g 57.15 1.43 0.17
C45 Drill core (DC_3) SE-02b 8/24/2017 130 107.00 15 g 171.05 1.40 0.15
C49 Drill core (DC_3) SE-02b 8/25/2017 139 97.00 15 g 168.91 1.41 0.11
C52 Drill core (DC_4) SE-02b 8/25/2017 148 84.00 15 g 180.71 1.44 0.10
C55 Drill core (DC_4) SE-02b 8/25/2017 157 64.00 15 g 97.89 1.44 0.06
C59 Drill core (DC_4) SE-02b 8/25/2017 166 55.00 15 g 148.14 1.43 0.10
C62 Drill core (DC_4) SE-02b 8/25/2017 175 44.50 15 g 178.08 1.42 0.13
C65 Drill core (DC_4) SE-02b 8/25/2017 181 37.00 15 g 203.00 1.40 0.13
Fum_1 Fumarole 63°18'19.9'N
20°36'24.7'W
8/4/2017 0 82.30 350 ml 19.05 1.88 Too low
Fum_2 Fumarole 63°18'15.4'N
20°36'07.7'W
8/4/2017 0 85.60 5,000 ml 8.39 2.33 Too low
SW_10 Seawater 63°28'58.8'N;
20°54'7.2'W
8/18/2017 10 (m b.s.l.) 12 1,000 ml 330.37 2.00 n.a.
SW_20 Seawater 63°28'58.8'N;
20°54'7.2'W
8/18/2017 20 (m b.s.l.) 11.97 1,000 ml 607.05 1.58 n.a.
SW_30 Seawater 63°28'58.8'N;
20°54'7.2'W
8/18/2017 30 (m b.s.l.) 10.81 1,000 ml 449.85 1.85 n.a.
SW_50 Seawater 63°28'58.8'N;
20°54'7.2'W
8/18/2017 50 (m b.s.l.) 9.9 1,000 ml 175.98 1.81 n.a.
1B0ZC Drilling uid 63°18'30.7'N
20°36'21.0'W
8/9/2017 1 (m b.s.l.) 10.00 1,000 ml 220.69 1.98 n.a.
1B3ZC Drilling uid 63°18'30.7'N
20°36'21.0'W
8/10/2017 1 (m b.s.l.) 10.00 1,000 ml 92.74 1.91 n.a.
1B25ZC Drilling uid 63°18'30.7'N
20°36'21.0'W
8/12/2017 1 (m b.s.l.) 10.00 1,000 ml 103.01 2.03 n.a.
149ZC Drilling uid 63°18'30.7'N
20°36'21.0'W
8/16/2017 1 (m b.s.l.) 10.00 1,000 ml 129.22 1.90 n.a.
1C17ZC Drilling uid 63°18'30.7'N
20°36'21.0'W
8/23/2017 1 (m b.s.l.) 10.00 1,000 ml 118.26 1.97 n.a.
1C39ZC Drilling uid 63°18'30.7'N
20°36'21.0'W
8/24/2017 1 (m b.s.l.) 10.00 1,000 ml 96.39 1.91 n.a.
(Continued)
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 6 September 2021 | Volume 12 | Article 728977
15 g were cryo-ground at an impact rate of 8 cycles per
second for 1 min using a 6,700 Freezer/Mill cryogenic grinder
(SPEX). Phosphate-ethanol solution (1 M phosphate buffer,
15% ethanol, pH 8.0; Direito et al., 2012) and proteinase
K (20 mg/ml) were added to the lysis solution provided by
the kit. Samples were vortexed twice for 30 s with a 1 min
cooling step in between instead of the bead-beating step,
followed by incubations at 55°C for 1 h and at 80°C for
40 min. Subsequent DNA isolation was carried out according
to the standard PowerMax® Soil DNA isolation protocol.
DNA precipitation was done overnight with isopropanol (0.7
volume) and glycogen (20 mg/ml). After a washing step with
70% ethanol, DNA was resuspended in Tris buffer (10 mM,
pH 8) and quantified using both NanoDrop® ND-1000
UV-Vis Spectrophotometer (Thermo Fisher Scientific) and
a Qubit fluorometer and high-sensitivity dsDNA reagents
(Invitrogen) before being stored at 20°C. Attempts to
estimate the microbial biomass within the drill core samples
using fluorescence microscopy were unsuccessful because
of the non-biological background signals that occurred during
recognition of cells for counting. Two controls were carried
out to test for contamination during DNA extraction of
the core samples. One used sterile water (instead of the
basalt powder) to test for contaminants derived from the
kit and reagents. The other used 15 g of basalt powder from
a drill core that had been treated with 70% ethanol and
heated at 180°C for 24 h. Both controls resulted in
amplifications with primers targeting bacterial and archaeal
16S rRNA genes, which were then sequenced.
DNA Extraction From Fluid Samples
DNA was extracted from Sterivex filters containing biomass
from borehole water, condensed fumarole water and drilling
fluid samples following a modified protocol by Neufeld etal.
(2007). Sucrose EDTA Tris buffer (SET buffer: 40 mM
ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl
pH 9, and 0.75 M sucrose) and 20 mg/ml lysozyme solution
were added to the Sterivex filters. Filters were incubated
at 37°C for 30 min. After the addition of 10% (w/v) sodium
dodecyl sulfate (SDS) and proteinase K (20 mg/ml), the filters
were incubated with rotation at 55°C for 2 h. Lysates were
collected into syringes, while the filters were rinsed twice
with SET buffer and the rinsed buffer was combined to
the lysate. One volume of phenol:chloroform:isoamyl alcohol
(PCI: 25:24:1, pH 8) was added, and the aqueous phase
was transferred to a new tube after a 15-min centrifugation
at 10,000 × g at 4°C. The phenol was removed from the
aqueous phase by adding 1 volume of chloroform. The
cleaned aqueous phase was transferred to a new tube after
a 5-min centrifugation at 10,000 × g at 4°C, and 0.7 volume
of cold isopropanol was added. After inverting the tube
several times, the sample was incubated for 15 min at room
temperature and then overnight at 20°C. After a 20-min
centrifugation at 16,000 × g at 4°C, the DNA pellet was
washed twice with 75% (v/v) ethanol, dried for 5 min using
a SpeedVac and 15 min at room temperature, and finally
resuspended in sterile Tris-HCl buffer (10 mM, pH 8). DNA
was quantified using a NanoDrop® ND-1000 UV-Vis
Spectrophotometer (Thermo Fisher Scientific) and Qubit
fluorometer (Invitrogen, Quant-iT dsDNA HS) and stored
at 20°C. One negative extraction control was carried out
for each round of extractions by rinsing a sterile Sterivex
filter with sterile laboratory-grade water.
Partial 16S rRNA Gene Amplication and
Tag Sequencing
Illumina MiSeq paired-end (2 × 300 base pair) tag sequencing
was carried out using the Earth Microbiome Project universal
primers 515f (5'-GTG CCA GCM GCC GCG GTA A-3') and
806r (5'-GGA CTA CHV GGG TWT CTA AT-3'), which
amplify the V4 region of the bacterial and archaeal 16S rRNA
genes (Caporaso et al., 2012). Since this primer pair does not
show a high coverage for Archaea (Parada etal., 2016), specic
archaeal primer sets were used with a nested PCR approach.
e rst-round PCR was performed to amplify the V3-V5
region of archaeal 16S rRNA gene with the primer set Parch340F
(5'- CCC TAY GGG GYG CAS CAG -3'; Øvreås et al., 1997)
and Arch958VR (5'- YCC GGC GTT GAV TCC AAT T -3';
Klindworth etal., 2013). en, a second round was performed
on the rst PCR product to amplify the V3 region of archaeal
16S rRNA gene with the primer set Arch349F (5'-
GYGCASCAGKCGMGAAW -3'; Takai and Horikoshi, 2000)
and Parch519R (5'- TTACCGCGGCKGCTG -3'; Øvreås
et al., 1997).
Sample ID Sample types
and categories
Sample
site
Sampling
date
Collection
depth (m b.s.)
Collection
Temp (°C)
Sample
amount
DNA
concentration
NanoDrop (ng/μl)
260/280 DNA
concentration
QuBit dsDNA
(ng/μl)
1C51ZC Drilling uid 63°18'30.7'N
20°36'21.0'W
8/25/2017 1 (m b.s.l.) 10.00 1,000 ml 252.97 2.05 n.a.
1C59ZC Drilling uid 63°18'30.7'N
20°36'21.0'W
8/24/2017 1 (m b.s.l.) 10.00 1,000 ml 152.45 2.04 n.a.
Cw Control n.a. n.a. n.a. n.a. n.a. 27.11 1.74 Too low
Cr Control n.a. n.a. n.a. n.a. 15 g 25.05 1.69 Too low
*Drill core samples from the subaerial tuff cone, located above the sea level. Too low: for detection with Qubit uorometer and high-sensitivity dsDNA reagents, <0.5ng/ml. n.a.: not
available. m b.s.l.: meter below sea level. m b.s.: meter below surface.
TABLE1 | Continued
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 7 September 2021 | Volume 12 | Article 728977
All PCR reactions were carried out in 25 μl reactions with
20 μl of Q5® High-Fidelity PCR Master Mix (New England
Biolabs, MA, United States) following the manufacturer’s
amplication protocol and 5 μl of DNA at 10 ng/μl (NanoDrop
quantication). e second round of the nested PCR used 5 μl
of amplied DNA from the rst round. Bovine serum albumin
was added to the master mix at a nal concentration of 0.5 ng/
μl for rock samples that could not beamplied. ermal cycling
consisted of an initial denaturation step at 98°C for 30 s,
followed by 30 cycles of denaturation at 98°C for 10 s, annealing
at 52°C (using the bacterial primer set) or at 55°C (using the
archaeal primer sets) for 30 s, and elongation at 72°C for 60 s.
Final elongation was set at 72°C for 2 min. Amplication
products were visualized on 1% (w/v) agarose gels.
Sequencing libraries were generated using the “16S
Metagenomic Sequencing Library Preparation guide” from
Illumina and barcodes from the Illumina Nextera®XT DNA
Sample Preparation Kit (8 cycles for index PCR). e libraries
were assessed on a Qubit Fluorometer (Invitrogen, Quant-iT
dsDNA HS) and a Bioanalyzer system (Agilent Technologies).
Aer normalization and quantication, the nal pooled library
was loaded on a MiSeq Desktop sequencer (Illumina) and
sequenced with V3 chemistry and 2 × 300 cycles across two
sequencing runs. Raw sequences have been deposited in the
European Nucleotide Archive (ENA) at EMBL-EBI under
accession number ERP126178.
16S rRNA Gene Amplicon Sequence
Analysis
Bioinformatic analysis was conducted in RStudio running R
version 4.0.2 (R Core Team, 2019; Rstudio, 2020). Sequence
variants were inferred using the R Package DADA2 (Callahan
et al., 2016) version 1.4, available at https://benjjneb.github.io/
dada2/tutorial.html. e following trimming parameters were
used: trimLe = 10, maxN = 0, maxEE = c(2,5), truncQ = 2, and
truncLen = c(230,215) for the universal primer set and
truncLen = c(115,110) for the archaeal primer sets. Aer quality
screening and trimming, forward and reverse reads were merged
to remove chimeric variants and singletons and to identify
amplicon sequence variants (ASV). Non-target-length sequences
were removed, and only amplicons of 270–275 bp length were
kept with the universal primer set and amplicons of 130–165 bp
for the archaeal primer sets. Data sets from three MiSeq sequencing
runs (universal primer set) were processed separately using the
same pipeline and same parameters and merged into a unique
ASV table using the function “mergeSequenceTables.” e
taxonomy was assigned using the function “assignTaxonomy”
(minimum bootstrap condence at 50) and the SILVA SSU
database release 138 (Quast et al., 2013).
In total, all 51 samples were successfully sequenced using
the universal primer set, including extraction controls and
drilling uid samples. e R package Decontam (version 1.10.0;
Davis et al., 20181) was used to identify contaminant ASVs
in the universal primer data set. Weidentied 160 contaminant
1
https://github.com/benjjneb/decontam
ASVs (Supplementary Table S1) using the Decontam prevalence
method (threshold value of 0.5), based on the prevalence
comparison of each sequence in true samples and negative
controls (Supplementary Figure S1). In addition, sequences
that were identied as chloroplast at the order level, mitochondria
at the family level, Eukaryote at the kingdom level, and those
that could not beidentied at the kingdom level were subtracted
from the data set prior to analysis, as well as putative contaminants
identied by taxonomic aliation at the genus level by a study
on common contaminants from the Census of Deep Life data
set (Sheik et al., 2018). Supplementary Table S2 gives a list
of 95 potentially contaminant genera removed from the analyses.
A total of 588,510 sequences were removed from the samples
by the abovementioned procedures (Details are available in
Supplementary Table S3).
From the archaeal nested PCR, 17 samples were analyzed.
Sequences identied at the kingdom level as Eukaryote, Bacteria,
or not assigned were removed from libraries prior to analysis,
as well as sequences detected in the DNA extraction blanks
(Supplementary Table S4, 26 ASVs). Using the archaeal primer
sets, the number of sequences removed from the samples was
74,315 (Details available in Supplementary Table S3).
Microbial Community Analysis
Microbial community analysis (α and β diversity, community
composition, and statistical analysis) was conducted in R (R
Core Team, 2019) with the Phyloseq (McMurdie and Holmes,
2013) and Vegan (Oksanen et al., 2007) packages. ANOVA
and Tukey’s HSD (Honestly Signicant Dierence) test were
conducted to evaluate the dierences in α diversity values.
For β diversity assessment, the data were normalized using
“rarefy_even_depth” function prior to performing a non-metric
multidimensional scaling (NMDS) ordination on Bray-Curtis
dissimilarities. e signicance of sample type variable was
assess using permutational multivariate analysis of variance
(PERMANOVA) using distance matrices and multilevel pairwise
comparison (Martinez Arbizu, 2020). e command envt
was used to investigate the correlation between the community
structure and environmental variables (depth and temperature).
Finally, DESeq2 was used to identify ASVs signicantly dierent
among sample types (F, fumarole; BF, borehole uid; DC,
drill core; SW, seawater samples) and categories of drill cores
(DC_1, DC_2, DC_3 and DC_4; Love et al., 2014). Details
of the data analyses can be found in the Supplementary
Material. Predictive functional analyses of the prokaryotic
communities were performed using Phylogenetic Investigation
of Communities by Reconstruction of Unobserved States 2
(PICRUSt2) on both data sets, obtained with the universal
primer set and the archaeal primer sets, separately (Douglas
et al., 2020). From the universal data set, a total of 2,333
PICRUSt2 predicted KEGG orthologs (enzymes) were collapsed
into 425 MetaCyc pathways, while 828 predicted KEGG
orthologs and 124 MetaCyc pathways were obtained from
the archaeal data set. Only few MetaCyc pathways were selected
to represent sulfur, nitrogen, methane, and carbon metabolism
(Supplementary Figures S8, S9).
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 8 September 2021 | Volume 12 | Article 728977
Microscopy
For SEM images, drill core samples were crushed in a sterile
mortar to obtain rock grains <0.5 mm diameter, which were
dehydrated by four 10-min wash steps in increasing
concentrations of ethanol (30, 50, 80, and 100%). Aer drying,
samples were placed on carbon conductive tabs (PELCO Tabs,
9 mm) and gold-coated. SEM used a Zeiss Auriga 40 Focused
Ion Beam Field Emission Scanning Electron Microscope coupled
with an energy dispersive X-ray spectroscopy analyzer (EDX)
at the Institute de Physique du Globe de Paris (University
Sorbonne Paris Cité, Paris, France) using two types of secondary
electron detectors: In-Lens and SESI, and a backscattered electron
detector: EsB. e acceleration voltage (EHT) ranged from 5
to 15 kV.
RESULTS
DNA Yields From Rocks
Based on Qubit quantication, DNA concentration extracted
from 15 g of each drill core sample yields from 0.2 to 1.36 ng.
g1 of rocks with an average of 0.48 ng.g1. However, no
signicant correlations in DNA yield were apparent with
sampling depth or in situ temperature (Supplementary
Figure S2).
Special Considerations for the Drilling
Operation and the Drilling Fluid
e island of Surtsey is subjected to strict environmental
protection and is a UNESCO World Heritage site (Baldursson
and Ingadóttir, 2007). For this reason (and other reasons
mentioned later in the study), the use of tracer compounds
to track contamination could not be considered during the
design of the 2017 SUSTAIN drilling operation. Instead, a
dierent approach was used to assess contamination. Filtered
and UV-sterilized water pumped from the sea was used as
drilling uid (see above). Aliquots of those uids were then
collected to track microbial DNA potentially contaminating
the core. DNA extracted from 1 l of each drilling uid sample
yields ~96 to 252 ng/μL of DNA (Tab l e  1 ).
e 16s rRNA gene sequencing of the drilling uid samples
thus showed, unfortunately, that the sterilization system failed
(Supplementary Table S3, Supplementary Figure S3).
Consequently, marine microbial taxa from the drilling uid
were introduced into the subsurface. e failure can possibly
be explained by the clogging of lters and an overly high
ow rate for the UV system.
As the contamination of the samples was evidently pervasive,
the data were evaluated with caution. A NMDS ordination
plot conducted using Bray-Curtis dissimilarity metrics (stress
value = 0.124) revealed that some of the drill core samples (e.g.,
C45, C52, C65) showed a microbial community composition
similar to the drilling uid and seawater samples
(Supplementary Figure S4). Whereas dierences in microbial
community structure between drill core and drilling uid and
between drill core and seawater samples proved to besignicant
by a Tukey HSD test (p adjusted values of 0.01 and 0.0008,
respectively), a strategy was adopted to distinguish endemic
microbial taxa of Surtsey subsurface rocks from the marine
residents introduced to the subsurface by the drilling uid.
e distributions of individual ASVs were evaluated by a simple
overlap approach using a Venn diagram to compare ASVs
shared among the seawater samples, drilling uid, drill core
from the subaerial deposits, and from the submarine deposits
(Figure 2).
In the subaerial tephra deposits, located above coastal
sea level, meteoric water was presented before drilling. In
the zone of tidal flux at ~58 m b.s., temperatures up to
100°C could operate as a natural autoclave or biological
barrier, preventing transfer of live microorganisms between
the subaerial and submarine basaltic deposits (Ólafsson and
Jakobsson, 2009; Marteinsson etal., 2015). Hence considering
that no marine taxa should be detected in the subaerial
deposit, all ASVs shared between the subaerial samples (C4,
C9, C13, and C17; 23–55 m b.s.; 20–82.5°C), the drilling
fluid, and the seawater samples (Figure 2:
93 + 84 + 14 + 11 + 16 + 14 ASVs, Supplementary Table S6)
were considered as potential marine contaminants; these
sequences were removed from the data set. In a second
iteration, ASVs shared only between the drill core samples
located in the submarine deposits and the drilling fluid
(Figure 2, Supplementary Table S6, 128 ASVs) were also
considered as marine contaminants and were removed from
the data set. Natural infiltration of cool seawater occurs
in the subsurface of Surtsey at 144–155 m b.s (Jakobsson
and Moore, 1986; Jackson etal., 2019b; Kleine etal., 2020).
This suggests that marine taxa detected in the submarine
deposit could derive from the inflow of seawater that infiltrate
the subsurface. Therefore, ASVs shared between the submarine
drill core, seawater samples, and the drilling fluid were
retained (Figure2, 61 ASVs). The total number of sequences
removed from the drill core samples by the abovementioned
procedures was 138,913 (Supplementary Table S3), which
represents on average 47% of the reads per drill core samples.
The subsequent analyses were performed on the
decontaminated data set, excluding the drilling fluid samples.
Microbial Community Structure Among the
Sample Types
Sequencing provided enough reads to capture the total richness
of the samples as all libraries reached saturation in rarefaction
curves (Supplementary Figure S5). e 41 samples were
catalogued by sample type: 18 samples from borehole uids,
17 samples from drill cores, 2 samples from fumaroles, and
4 seawater samples (Tabl e  1 , Supplementary Table S3).
Amplication of the partial 16S rRNA gene using the universal
primer set was successful for all samples, while only 17 samples
could be amplied using the archaeal primer sets.
We obtained a total of 455,545 and 848,053 high-quality
sequences using universal and archaeal 16S rRNA primer sets,
respectively (Supplementary Table S3). A total of 4,222 ASVs
ranging from 41 (Sur168) to 317 (SW50) ASVs per sample
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 9 September 2021 | Volume 12 | Article 728977
were obtained using the universal primer set, whereas a total
of 157 ASVs ranging between 3 (Sur4a) and 41 (Sur1a) ASVs
per sample were obtained using the archaeal primer sets.
α Diversity
Analysis of variance on species richness showed signicant
dierences between sample types (ANOVA, F value = 9.082,
Pr(>F) = 0.000123). A Tukey HSD test highlighted signicant
dierences among them between seawater samples and borehole
uid (<0.001), seawater samples and drill core (0.0339), and
between drill core and borehole uid (0.0184). Shannon diversity
also diered signicantly among them (ANOVA, F value = 5.998,
Pr(>F) = 0.00195), and Tukey’s HSD test revealed signicant
dierences between seawater samples and borehole uid (0.00323)
and between seawater samples and drill core (0.02570; Figure3A,
Supplementary Table S7). e SE-01 borehole uids displayed
a signicantly lower observed diversity than the drill cores,
but Shannon diversity was not signicantly dierent, indicating
that the evenness of the species present in these sample types
was comparable. Observed diversity and evenness were
signicantly higher in the seawater samples than in drill core
and borehole uid samples.
β Diversity
e resulting NMDS ordination conducted using Bray-Curtis
dissimilarity metrics revealed distinct microbial communities
specic to each sample types, which clustered individually
(Figure 3B). e stress value equaled 0.18. is was further
conrmed by a PERMANOVA analysis showing that sample
types diered signicantly [Pr(>F) = 0.001, r2 = 274], and a multilevel
pairwise comparison showed signicant dierences among drill
cores and borehole uids (adjusted p value = 0.0184), seawater
samples and borehole uids (<0.001), and between seawater
samples and drill cores (0.0338). is indicates that the samples
within a given sample type show microbial communities that
are more similar to one another than to samples from a dierent
sample type. e cluster of SE-01 borehole uid samples appears
wider than the drill core cluster, indicating more dierences in
microbial community (Figure 3B).
Community Composition
At the phylum level, some taxa were common across the four
sample types (Figure3C). e phylum Proteobacteria dominated
all sample types, for example, whereas its relative proportion
varied greatly among them. Within Proteobacteria,
Alphaproteobacteria was the most abundant class followed by
Gammaproteobacteria. Fumarole samples showed a higher
relative proportion of Cyanobacteria (15.3%), Deinococcota
(12.6%), Acidobacteriota (7.84%), and Chloroexi (4.52%) than
borehole uids and drill cores. Borehole uids displayed the
highest occurrence of the bacterial phyla Acetothermia (5.56%)
and Patescibacteria (2.26%), as well as the archaeal phyla
ermoplasmatota (5.60%) and Euryarchaeota (3.13%), in
comparison with the other sample types. e class
FIGURE2 | Contamination assessment by an overlap approach between drill cores from the subaerial and submarine deposits, drilling uid, and seawater
samples. Five drilling uid samples (in grey): 1B49ZC, 1C17ZC, 1C39ZC, 1C51ZC, 1C59ZC, 4 sea samples (in blue): SW_10, SW_20, SW_30, SW_50, 4 samples
from the subaerial deposit (in yellow): C4, C9, C13 and C17, and 13 samples from the submarine deposit (in light brown): C22, C27, C33, C36, C39, C42, C45,
C49, C52, C55, C59, C62, C65. Hatched area: 360 ASVs considered as contaminants.
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 10 September 2021 | Volume 12 | Article 728977
Desulfotomaculia represented more than 5% of the borehole
uid community (data not shown). Nevertheless, due to the
compositional nature of the data, lineage-specic enrichments
necessitate further investigation, using for example a qPCR
approach (Jian et al., 2020). e drill core samples show the
highest sequence variants (2,242 ASVs), followed by the borehole
uids (1,415 ASVs), seawater samples (494 ASVs), and fumarole
samples (367 ASVs). Most ASVs were not shared between the
sample types (Figure 3D).
Microbial Community Composition and
Structure of the Rock Samples and
Inuence of Depth and Temperature
e bacterial sequences from the drill core samples are classied
into 35 phyla, with the ten phyla Proteobacteria, Bacteroidota,
Actinobacteriota, Firmicutes, Cyanobacteria, Deinococcota,
Planctomycetota, Verrucomicrobiota, Acidobacteriota, and
Chloroexi, comprising more than 95% of the sequences
(Figure4). e 29 ASVs classied as Archaea using the universal
primer set fall into the phyla ermoplasmatota, Crenarchaeota,
Nanoarchaeota, and Iainarchaeota.
Archaeal sequences obtained by nested PCR from 10 of
the drill cores were more diverse. e 88 unique ASVs
include the phyla Crenarchaeota (mainly Nitrososphaeria),
ermoplasmatota (mainly ermoplasmata), Euryarchaeota,
Halobacterota, Hydrothermarchaeota, Nanoarchaeota, and
unassigned Archaea (Figure 4). Remarkably, the sample C55
(157 m b.s.; 64°C) below the submarine inow zone is dominated
by the archaeal genus Methanobacterium, while the sample
C65 (181 m b.s.; 37°C) in weakly consolidated tephra near the
pre-eruption seaoor shows the highest abundance of the genus
ermococcus (Figure 4).
Canonical correspondence analysis (CCA) ordination
(ANOVA, p = 0.018) and envt analyses demonstrate that both
depth and in situ temperature are signicantly correlated with
microbial community structure of the drill core samples
(Supplementary Figure S6). However, it is unclear which
A
CD
B
FIGURE3 | Overview of the microbial community diversity, structure, and composition of the samples types: borehole uid, drill core, fumarole, and seawater
samples. (A) α diversity indices: observed diversity and Shannon’s diversity indexes. Signif. codes: 0.001 “**“0.01 “*”. (B) β diversity assessed with non-metric
multidimensional scaling ordination using Bray-Curtis dissimilarity metrics of microbial community within sample types. The stress value equaled 0.18. (C) Mean
relative abundance of the 14 most abundant phyla. Proteobacteria are divided into classes. (D) Venn diagram showing the number of shared and unique ASVs
among the sample types.
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 11 September 2021 | Volume 12 | Article 728977
variable has the most inuence on the microbial community
structure, since both are linked. Observed and Shannon diversity
indices showed no signicant dierence between the four
categories of drill core samples (data not shown).
To identify the ASVs causing the dissimilarity of community
structure in the CCA ordination plot, the drill core
samples are grouped in four categories as follows
(Supplementary Figure S6): (DC_1) drill core samples from
the subaerial deposits (23–35 m b.s.; 20–36°C in 2017; n = 2,
C4, C9) (DC_2) drill core samples from the zone of daily
intertidal uctuations at coastal sea level (44–65 m b.s.;
59–101.5°C; n = 3, C13, C17, C22), (DC_3) drill core samples
from the submarine deposits near the hydrothermal temperature
maximum (78–139 m b.s.; 97–124°C in 2017; n = 7, C27, C33,
C36, C39, C42, C45, C49), and (DC_4) drill core samples
from the submarine deposits, below the zone of seawater inow
(157–181 m b.s.; 37–84°C in 2017; n = 5, C52, C55, C65,
C62, C59).
Differential Abundance Analyses
To identify dierences in ASVs abundance between the four
sample types (F, fumarole; BF, borehole uid; DC, drill core;
SW, seawater samples), we performed dierential abundance
analyses (two-by-two comparisons) using separately the universal
primer and the archaeal primer data sets (Supplementary
Table S5). Additionally, the same analyses were performed for
the four categories of drill core samples to highlight the ASVs
contributing to the dissimilarity in the CCA plot (Supplementary
Figure S6). Due to the compositional features of the data, the
identication of dierentially abundant taxa between the dierent
groups of samples must be assessed carefully, since it is based
on a count-based method. Being aware of the limitations of
this approach, a total of 95 and 26 unique ASVs were proved
to be signicantly represented using the universal primer data
set (grouping into 59 taxonomic bins) and the archaeal primer
sets (7 taxonomic bins), respectively (Figure5), in accordance
with the log fold change of the mean normalized read counts
(p < 0.01).
Compared to the seawater samples, the drill cores showed
overrepresented ASVs assigned to the taxa Caldisericum,
Galbitalea, Geobacillus, Glaciecola, Oceaniserpentilla, Oleispira,
Piscinibacter, Pseudoarthrobacter, Psychromonas, Reyranella,
Sulfurospirillum, ermaerobacter, ermus, unassigned bacteria
from the Sphingomonadaceae family, and unassigned
ermoplasmata. ASVs assigned to Aliivibrio and
Pseudoalteromonas were signicantly underrepresented in the
drill cores compared to the seawater samples. Although the
latter genera are generally detected in seawater, they were
FIGURE4 | Vertical distribution of the microbial communities at the phylum or class level detected in the 17 drill core samples from the subsurface of the Surtsey
volcano using universal primer set and archaeal primer sets.
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 12 September 2021 | Volume 12 | Article 728977
signicantly overrepresented in the borehole uids collected
before drilling, indicating that marine microorganisms (or their
DNA) naturally inltrate the subsurface basaltic deposits. Other
overrepresented ASVs in the borehole uids, as compared to
the drill core samples, included unassigned Acetothermiia,
BRH-c8a (Desulfallas-Sporotomaculum), Ruegeria, Rhodobaca,
unassigned RBG-16-55-12 (Actinobacteriota), Conexibacter,
Desulfosporosinus, Desulfatiglans, unassigned Ammonifexales,
and Ammonicaceae, among others (Supplementary Table S5).
Archaeal taxa that proved to be overrepresented in the
borehole uids included unassigned ermoplasmata,
Hydrothermarchaeales, Syntrophoarchaeaceae, and the genus
ermococcus. Using the archaeal primer sets, unassigned
ermoplasmatota and Bathyarchaeia were added to the list
of overrepresented archaeal taxa detected using the universal
primer set (Figure 5, Supplementary Table S5).
Comparing the four categories of drill cores, ASVs
overrepresented in the drill core samples from the subaerial
tu cone (DC_1; 23–35 m b.s.; 20–36°C in 2017) were assigned
to the genera ermaerobacter, ioalkalimicrobium,
Salinarimonas, Marinobacter, Nioella, Ectothiorhodospira,
Polaribacter, and unassigned Limnochordaceae (Figure5). No
ASVs were signicantly overrepresented in drill core samples
from the zone of daily intertidal uctuations (DC_2; 44–65 m
b.s.; 59–101.5°C in 2017) compared with the other categories
of drill core samples. Although one ASV assigned to Geobacillus
is signicantly overrepresented in the drill core samples from
the hydrothermal temperature maximum (DC_3; 78–139 m
b.s.; 97–124°C in 2017), this is similar to the drill core samples
located below the zone of seawater inow (DC_4; 157–181 m
b.s.; 37–84°C in 2017). ese samples shared signicant ASVs
with the seawater samples, including Flavicella, NS4 marine
group from the Flavobacteriaceae family, Synechococcus CC9902,
Planktomarina, and Sulfitobacter. e presence of these taxa
further suggests an inltration of marine microorganisms
associated with the inltration of cool seawater at 145–155 m
depth, if we presume that no drilling uid contamination
remains.
Possible Biotic Structures Revealed by
SEM Images
To determine whether the detected DNA sequences could
be derived from planktonic cells in interstitial pore uids or,
alternatively, attached to surfaces of the lapilli tu and to
investigate their organization on the basaltic substrate, SEM
studies were undertaken on six drill core samples, diering
in depth (C4, C9, C22, C49, C55, and C65).
All six lapilli tu samples are porous with high water
absorption (Jackson etal., 2019b). Vesicles with spherical shapes
10–80 μm in diameter occur in all samples, and platy clay
mineral structures, with ower petal morphologies, indicate
alteration of the original volcanic glass (Jakobsson and Moore,
1986; Figure 6B). e distribution of microstructures varies
with sampling depth; here, wefocus on instructive samples at
32 m b.s (C9) and 65 m b.s (C22). Some vesicle surfaces are
covered by a net of thin laments with an approximate diameter
of 10 nm. ese features are closely intertwined with one other
and could possibly correspond to extracellular polymeric
substances-like (EPS) structures (orseth et al., 2001, 2003;
Sudek etal., 2017), perhaps representing a “relic” of past biolm
activity (Figures 6A,B). In other vesicles, spheroidal elements
correspond to putative microbial cells 2 μm in diameter
(Figure 6C) as previously described (orseth et al., 2001,
2003). Furthermore, these vesicles are covered by brillar-like
microstructures (Figure 6D), that appear texturally dierent
from the EPS-like structures (Figure 6B). ey occur as nets
of oriented bers with ramications; some seem to beattached
FIGURE5 | Relative abundance of taxonomic bins from all signicant ASVs
identied by differential abundance analyses using both universal and
archaeal primer data sets. Sample types: F, fumarole (n = 2); BF, borehole uid
(n = 18); DC, drill core (n = 17); SW, seawater samples (n = 4). Categories of drill
cores: DC_1, drill cores from the subaerial deposit (n = 2); DC_2, just above
and at a zone of daily intertidal uctuations at coastal sea level (n = 3); DC_3,
in the submarine deposit at the hydrothermal temperature maximum (n = 7);
DC_4, in the submarine deposit below the zone of seawater inow (n = 5).
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
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to the spheroidal structures. Energy dispersive X-ray analysis
on the spheroidal elements does not detect carbon or phosphate
but, instead, detects magnesium, aluminum, and iron. ey
therefore seem to have an inorganic origin.
Wrinkled dome structures are observed in many vesicles
at 65 m b.s (Figure 6E) with a diameter size of 10–20 μm. A
network of intercrossed laments covers the surface of the
dome or mound (Figure6F). eir wrinkled appearance could
beexplained by the sample preparation for SEM images, which
includes gradual dehydration. EDX analyses investigate the
chemical signature of the wrinkled dome structures
(Supplementary Figure S7). e superposition of the spectra
corresponding to the adjacent clay mineral(s)
(Supplementary Figure S7, red area) and the wrinkled dome
structures (Supplementary Figure S7, blue area) shows
enrichment in carbon, oxygen, sodium, phosphate, and calcium
in the crinkled ridge of the mound. ese could possibly
indicate elements associated with the cells and biomass of
a biolm.
DISCUSSION
Microbial colonization by pioneer communities can occur
within months on freshly deposited erupted volcanic rocks
in geothermally active environments (e.g., Konhauser et al.,
2002; Kelly et al., 2014). e Surtsey geothermal system
oers a highly unusual site to study rapid microbial colonization
in newly formed oceanic crust at <100 years and > 50°C.
Results of the analysis described in Figures 3, 4, and 6
suggest that diverse microbial communities have developed
in the Surtsey deposits only 50 years aer the eruptions
ended. Communities attached to the lapilli tu of drill core
samples are signicantly dierent from communities in
associated uids (Figures 3A,B). Similar observations have
previously been reported in crustal environments (Ramírez
etal., 2019). e Surtsey data suggest that fumaroles, borehole
uids, drill cores, and seawater samples harbor signicantly
distinct microbial communities (Figures3B,C). Yet, taxa are
shared between these sample types (Figure 3D), suggesting
possible transfer of microorganisms among these habitats.
e hot fumarole samples from the surface of Surtsey at
82.3 and 85.6°C harbor microbial communities that are more
similar to those of the drill core samples located above sea
level in the subaerial deposits (e.g., samples C4, C9, 23–35 m
b.s.; Figure2B). is suggests that some species could disperse
through the subaerial tu cone above sea level up to the
surface through fumarole activity (Tin etal., 2011). Excluding
all the drilling uid ASVs contaminants, microbial communities
detected in the seawater samples are closer in community
structure to the drill core samples from the zone of seawater
inow (sample C52, 148 m b.s., Figure2B) and to the samples
in proximity to the seaoor (sample C65, 181 m b.s.,
Figure 2B). is suggests that microorganisms from the
seawater surrounding the island (i) inltrate the submarine
tephra deposits, following the seawater inow at about 148 m
b.s (Jakobsson and Moore, 1986; Jackson etal., 2019b; Kleine
et al., 2020), and (ii) occupy the deeper zone of poorly
consolidated tu through indirect inltration of the seawater
and its circulation via the seaoor. e ability of some species
to expand across ecosystems and adapt to new environmental
conditions (Sriswasdi et al., 2017) has possibly driven the
subsurface colonization of the Surtsey volcano. Species that
cannot survive might serve as supply of fermentable organic
molecules for the subsurface heterotrophic microorganisms
(Li et al., 2020).
Assessment of contamination by Drilling
Fluid
e problem of subsurface sample contamination during
drilling operations is well-known (Barton etal., 2006; Lever
et al., 2006; Kie, 2010; Santelli et al., 2010; Sheik et al.,
2018) since drilling operations require the use of circulating
uid that inevitably inltrates into the drill core. As a result,
the recovery of uncontaminated rock is nearly impossible
(Friese et al., 2017; Kallmeyer, 2017). Conventional methods
to assess contamination include the addition of tracer
compounds to the drilling uid. ese include uorescent
dyes (Pellizzari et al., 2013), peruorocarbon tracers (PFT;
Lever et al., 2006; Inagaki et al., 2016; Orcutt et al., 2017),
and microsphere tracers (Kallmeyer et al., 2006; Yanagawa
etal., 2013). e use of these tracer compounds is prohibited
on Surtsey due to the protected environmental status of the
island (Baldursson and Ingadóttir, 2007). Furthermore, their
performance in the Surtsey system would have been quite
problematic. Although uorescent dyes have the advantages
of high sensitivity for detection, low cost, and ease of use
(Pellizzari et al., 2013), they are unstable at low pH (Zhu
et al., 2005) and are also susceptible to degradation in the
presence of light (Diehl and Horchak-Morris, 1987).
Measurements of Surtsey subsurface borehole uids extracted
from SE-01 in 2016 showed pH values decreasing to 5 at
some depths (Kleine et al., 2020). Water tanks containing
the drilling uid were exposed to sunlight during the long
Icelandic summer days. e detection of PFTs must
beperformed immediately on fresh cores because of the high
volatility of the PFTs and requires elaborate equipment (Lever
et al., 2006) whose transport by helicopter to Surtsey would
have been very dicult. Finally, microsphere tracers decompose
under high-temperature conditions (Yanagawa et al., 2013)
and would not have performed well in the Surtsey hydrothermal
system, which currently exceeds 120°C at some depths
(Marteinsson et al., 2015; Jackson et al., 2019b). Because of
the unique circumstances of drilling on Surtsey, the ltration
and UV sterilization of the drilling uid was the most eective
strategy to manage contamination. We then mitigated this
strategy with a simple overlap approach that is commonly
used in environmental microbiology studies to identify and
remove contaminants (Sheik etal., 2018). At the ASVs level,
this approach enabled the distinction between marine
contaminants from the drilling uid and possible endemic
residents, providing a rm basis for the exploration of microbial
life in this extreme and unusual habitat (Figure 2).
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 14 September 2021 | Volume 12 | Article 728977
e DNA concentration in the drill core samples, measured
using a QuBit uorometer, ranged from 0.2–1.36 ng.g1 of basalt,
which is in good correlation with other basaltic environments
(Fisk et al., 2003; Herrera and Cockell, 2007). Surprisingly,
DNA concentration did not correlate with variations in
temperature (Supplementary Figure S2a). It does decrease with
depth (Supplementary Figure S2b), as previously observed in
terrestrial and marine subsurface habitats (Cockell etal., 2012;
Ciobanu et al., 2014; Mcmahon and Parnell, 2014), but not
signicantly. e presence of contaminant DNA in the
low-biomass basaltic core samples quite possibly distorts these
results. Indeed, low DNA concentration exacerbates issues of
external contamination (Sheik et al., 2018). When using 16S
rRNA gene sequencing to analyze low-biomass samples, the
amplication and sequencing of contaminant DNA are introduced
during extraction and library preparation steps, and the risk
of well-to-well contamination is high (Minich et al., 2019).
e low biomass and the presence of external contamination
also inuence the sequencing results by aecting the magnitude
and biological provenance of analyzed sequences. erefore,
it is of crucial importance to use negative controls and optimal
decontamination approaches. e inclusion of bacterial mock
communities as extraction and sequencing controls would have
been benecial for the study (Pollock etal., 2018). Weemphasize
FIGURE6 | Scanning electron micrographs of Surtsey tephra surfaces in drill core samples. (A,B) Platey clay mineral and EPS-like structures, 65 m b.s., sample
C22. (C,D) Vesicle surfaces in altered basaltic glass are covered by a net of brillar-like structures that appears to beattached to spheroidal elements, 32 m b.s,
sample C9. (E,F) Wrinkled dome structures, or mounds, are covered by a network of laments, 65 m b.s, sample C22.
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
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that all samples were handled with extreme care during all
steps of the project to minimize, identify and remove
contaminants using a combination of experimental and
computational methods. e lists of contaminants that have
been identied based on the presence/absence of ASVs across
samples, their relative abundances, and their taxonomic
assignment (Supplementary Tables S1, S2 and S4) should
be valuable for future microbial explorations of the oceanic
crust. In addition to the low biomass and the presence of
external contamination aecting the results, we should keep
in mind that the compositional nature of the data has limits.
Indeed, the data set reported in this study was obtained by
16S rRNA gene amplicon sequencing; thus, the data are
compositional being based on relative abundances, which sum
to a constant. erefore, the analytical approaches (e.g.,
rarefaction, normalization) and statistical methods (e.g., ANOVA)
used to study microbiome data inuence the results and can
potentially be are subjected to inated false discovery rates
(Gloor etal., 2017). is can lead to the lack of reproducibility
among microbiome studies and misinterpretations of microbial
community structures (e.g., alpha diversity).
Putative Inhabitants of the Subsurface
Many of the taxa detected in the SE-01 borehole uid samples
match DNA sequences previously identied in hot springs,
hydrothermal vents, and subsurface environments (Figure5).
For example, sequences assigned to the class Acetothermiia
closely match with DNA sequences previously found in
hydrothermal sediments (GenBank: FM868292). Members
of this class were previously detected in anaerobic digesters,
hot springs, and other deep biosphere studies (Takami et al.,
2012; Jungbluth etal., 2017; Zaitseva et al., 2017; Hao et al.,
2018; Korzhenkov et al., 2018). Sequences assigned to the
genus BRH-c8a from the family Desulfallas-Sporotomaculum
match closely with sequences retrieved from deep groundwater
(LC179584) and group with the genus Desulfotomaculum
that can be found in deep subsurface environments (Sousa
etal., 2018; Watanabe etal., 2018). Other sequences belonging
to the class Desulfotomaculia are assigned to Ammonifexales
and Ammonicaceae; these are related to DNA sequences
retrieved from petroleum reservoirs (MF470409). In addition,
sequences assigned to Desulfosporosinus closely match
sequences retrieved from coal formation waters (KC215435),
while sequences assigned to Desulfatiglans match with
sequences found in hydrothermal vents (AB294892). Among
the abundant ASVs detected in the borehole uids, an early-
branching, uncultivated actinobacterial clade identied as
RBG-16-55-12 in the SILVA database release 138 has been
previously detected in serpentinite-hosted systems (Merino
etal., 2020). Uncharacterized ermoplasmata are also detected
in serpentinite subsurface deposits (Motamedi etal., 2020),
yet numerous sequences assigned to this class from the SE-01
borehole uid samples showed less than 90% of sequence
similarity with the rst match on the NCBI Nucleotide
collection database (AB327321). ese results suggest that
all the latter taxa could beendemic in the Surtsey subsurface
deposits and perhaps could be common in other oceanic
or continental subsurface habitats, as well. Other taxa detected
in the SE-01 borehole uid samples are usually found in
seawater, including Halomonas, Pseudoalteromonas,
Sulfitobacter, Aliivibrio, and Shewanella (Figure 5). e
presence of marine microorganisms in the SE-01 borehole
uid before new drilling began in 2017 further demonstrates
that the inltration of seawater transports marine
microorganisms into the subsurface and its hydrothermal
system. ese species are also frequently detected in cool
basaltic oceanic crust (Templeton etal., 2005; Mason et al.,
2007; Zhang et al., 2016), and the question of whether or
not these species survive in the Surtsey hydrothermal system
could depend on the inuence of temperature (Edwards
et al., 2012; Baquiran et al., 2016; Ramírez et al., 2019).
Scanning electron microscopy studies of instructive drill
core samples reveal possible biotic structures whose size,
morphology, and fabrics seem to be consistent with
EPS-microcolony complexes that are attached to the basaltic
substrate (Figure 6, Supplementary Figure S7; Thorseth
et al., 2001, 2003; Nakagawa et al., 2006). Indeed, some
taxa detected in the drill core samples could represent
endemic inhabitant of the basaltic subsurface of the island,
which can bedivided into two distinct habitats: the subaerial
tuff cone and the submarine tuff deposits. In the subaerial
tuff cone (DC_1; 23–35 m b.s.; 20–36°C in 2017), the taxa
include the genera Thermaerobacter, Thioalkalimicrobium,
Salinaromonas, Marinobacter, and Ectothiorhodospira
(Figure 5). Thermaerobacter are thermophilic to extremely
thermophilic bacteria found in terrestrial and oceanic
subsurface environments (Spanevello etal., 2002; Takai etal.,
1999). Thioalkalimicrobium, Ectothiorhodospira, and
Salinarimonas are detected in alkaline and saline habitats
such as soda lakes (Sorokin et al., 2001), saline soil (Cai
et al., 2011), or salt mines (Liu etal., 2010). The presence
of Marinobacter only in the tuff deposits above sea level
is curious since it is a common lineage found in marine
basaltic habitats, including ridge-flank systems and seamount
(Templeton et al., 2005; Zhang et al., 2016). Nevertheless,
the presence of these taxa in the subaerial deposits suggests
adaptation of the microbial communities to more extreme
environmental conditions, such as high temperature, high
salt concentration produced by NaCl saturation from seawater
evaporation or from alkaline pH produced by basaltic glass
dissolution at low fluid-rock ratios (Kleine et al., 2020).
No hyperthermophilic species were significantly enriched
in the drill core samples from the hydrothermal temperature
maximum (DC_3; 78–139 m b.s.; 97–124°C in 2017 and
100–141°C in 1979), yet two thermophilic species Thermus
and Geobacillus from the Thermoleovorans group were
detected (Figure 5). Based on these results and the taxa
detected in the SE-01 borehole fluids, it seems that microbial
life may persist in subsurface deposits that have experienced
temperatures >120°C, the presumed temperature for functional
microbial life. The hydrothermal zone at about 100 m b.s.
could act as a dispersal barrier that provides an obstacle
to the transfer of live cells from the zone of tidal flux and
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 16 September 2021 | Volume 12 | Article 728977
upper submarine deposits to the deeper submarine deposits
(e.g., samples C27; 78 m b.s. and C52; 148 m b.s.). Furthermore,
no significant difference exists between the observed microbial
communities from the lowermost submarine deposits below
the zone of cool seawater inflow (DC_4; 157–181 m b.s.;
37–84°C in 2017) and the other submarine deposits
(Supplementary Figure S6). Shared taxa mainly
include mesophilic Proteobacteria such as unassigned
Sphingomonadaceae, Glaciecola, Arenicella, Oceaniserpentilla,
and Piscinibacter that are commonly found in marine habitats.
In addition, Candidatus Nitrosopumilus and unassigned
Thermoplasmata from the Marine Group II were detected
in great abundance (Figure5). Those taxa have been previously
detected in marine basalts and deep seawater circulation
through oceanic crust (Mason et al., 2007; Singer et al.,
2015; Suzuki et al., 2020; Bergo et al., 2021). Although the
deep ocean is typically enriched in archaeal cells, other
marine environments usually show a dominance of bacterial
lineages (Karner et al., 2001). This has been previously
observed in the basaltic crust. For example,
Gammaproteobacteria and Alphaproteobacteria are detected
in great abundance in the seafloor basaltic glass of the East
Pacific Rise (Santelli etal., 2008, 2009), the Arctic spreading
ridges (Lysnes etal., 2004), altered basalts from the Hawaiian
Loihi Seamount (Templeton etal., 2005; Santelli etal., 2008;
Jacobson Meyers et al., 2014), and the Mid-Atlantic Ridge
(Rathsack etal., 2009; Mason etal., 2010). Deltaproteobacteria,
Firmicutes, Gammaproteobacteria, and Bacteroidetes are also
detected in great abundance in the Juan de Fuca Ridge
flank and the Costa Rica Rift (Nigro et al., 2012;
Jungbluth et al., 2013, 2014). Our results support the
dominance of bacterial lineages in marine basalts.
While most taxa could be identied at a low taxonomic
rank, many others were not assigned to a known genus,
family, or even order. For example, many sequences detected
in this study fell into unknown clades, including Acetothermiia,
Ammonifexales, Bacilli, RBG-16-55-12 from the phylum
Actinobacteriota, Sphingomonadaceae, Limnochordaceae, and
Saccharimonadales, among others. Likewise, many archaeal
sequences could not be identied further than the phylum
level, including unassigned ermoplasmatota and
Halobacterota, or the class level, including unassigned
ermoplasmata and Bathyarchaeia. Hence, the subsurface
biosphere of Surtsey lapilli tu and tephra, as well as uids,
could have high potential for discoveries of new
microbial clades.
FIGURE7 | Overview of the putative metabolisms and microbial dispersion scenario of the subsurface of Surtsey volcano.
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 17 September 2021 | Volume 12 | Article 728977
Metabolic Potential
e metabolic potential of the endemic subsurface microbial
communities can bediscussed with some degree of certainty,
while remaining mindful of the diculties inherent to
identications based on 16S rRNA gene sequence analysis.
To support our hypotheses, predictive functional analyses
were performed using PICRUSt2. A few MetaCyc pathways
were selected to represent the functional potential of the
bacterial and archaeal communities for carbon, sulfur, nitrogen,
and methane metabolism (Supplementary Figures S8, S9).
Many genera reported in this study belong to taxonomic
clades with known metabolisms that are involved in both
heterotrophy and chemolithoautotrophy (Figure 7). e
presence of putative sulfate-reducing bacteria strongly suggests
a potential for active sulfate reduction, including
Desulfosporosinus, Desulfatiglans, and the Desulfotomaculia
class, among others. In addition, sulfur oxidizers were detected,
such as the genera ioalkalimicrobium, Sulfurospirillum,
Sulfurimonas, Ectothiorhodospira, and Sulfurihydrogenibium.
e observation of these bacterial taxa possibly involved in
sulfate reduction and sulfur oxidation suggests an active sulfur
cycle in the subsurface of Surtsey (Supplementary Figure S8),
as has previously been reported in similar ecosystems (Bach
and Edwards, 2003; Lever et al., 2013; Suzuki et al., 2020).
is is further reinforced by the detection of archaea possibly
involved in the sulfur cycle, such as ermococcus, Pyrococcus,
and Archaeoglobus. Also, characterized members of the
ermoplasmatales are typically involved in sulfur cycling
(Barton et al., 2014; Arce-Rodríguez et al., 2019). ese taxa
coincide with deposits that contain sulfate minerals, principally
anhydrite and gypsum (Jakobsson and Moore, 1986; Kleine
et al., 2020; Prause et al., 2020).
In addition, the genus Methanobacterium from the order
Methanobacteriales dominates the archaeal sequences of drill
core sample C55, at 157 m depth (Figure 3). The MetaCyc
pathways detected in this sample using the archaeal data
set were mainly involved in methane metabolism (e.g.,
methanogenesis from H2 and CO2, coenzyme B biosynthesis,
coenzyme M biosynthesis I; Supplementary Figure S9). One
taxon from the same order of Methanobacteriales was
previously reported to dominate the SE-01 borehole fluid
microbial communities sampled in 2009 at similar depth
(Marteinsson etal., 2015). Methanobacterium spp. grows by
reducing carbon dioxide to methane and uses molecular
hydrogen as the electron donor (Kern et al., 2015). Hence,
these taxa could play an important role as primary producers
in this ecosystem, at least at certain depths. The possible
occurrence of an active methane cycle is supported by the
presence of other methanogens (e.g., Methanosarcinia
(Syntrophoarchaeaceae), Methanomassiliicoccales
(Methanothermus, Methanoregula), as well as methanotrophic
(e.g., Methylocella, Methylacidiphilaceae), and methylotrophic
(e.g., Hansschlegelia, Methylopila, Methylophilaceae (OM43
clade), Methylophaga) bacteria, despite their relatively low
abundances. The observation of genera such as Geobacter,
Rhodoferax, Marinobacter, Shewanella, and Ferruginibacter,
as well as Hydrogenophilus and Hydrogenophaga, could indicate
that iron and hydrogen are electron donors within the
ecosystem, as previously reported for other subsurface habitats
(Bach and Edwards, 2003; Bach, 2016; Zhang etal., 2016).
In addition, putative ammonium-oxidizing archaea belonging
to the Marine Group II and Candidatus Nitrosopumilus
suggest an ability to transform nitrogen compounds as
previously reported in the oceanic crust (Daae etal., 2013;
Orcutt et al., 2015; Jørgensen and Zhao, 2016; Zhao
et al., 2020).
CONCLUSIONS
e 1979 and 2017 drilling projects at Surtsey volcano provide
a rare opportunity to explore the subsurface microbial diversity
of a very young basaltic island associated with an active
hydrothermal-seawater system in newly formed oceanic crust.
A cored borehole dedicated to microbiology research in the
2017 drilling operation probes a low biomass but highly diverse
habitat that hosts bacterial and archaeal clades, including
extremophiles, that have been previously detected in other
terrestrial and marine environments. Many clades, however,
fall into as-yet-unknown lineages. e 16 s rRNA gene amplicon
sequencing data provide insights into diverse sources of microbial
colonization in newly formed oceanic crust, with potential
dissemination from the deep subsurface, surrounding seawater,
and surface ecosystems. e island of Surtsey may thus
be regarded as a porous, sponge-like basaltic structure that
absorbs cells from the surrounding environments and selects
microorganisms that can adapt to the extreme environmental
conditions that exist within the volcano. e data also provide
a baseline for long-term observations of the microbial
communities inhabiting the subsurface of Surtsey and their
temporal succession amid a changing hydrothermal environment,
in which poorly consolidated tephra lithify to form well-
consolidated lapilli tu. Further research, including metagenomic
sequencing, will increase our knowledge of the metabolism
and function of the microbiome in this very young
basaltic environment.
DATA AVAILABILITY STATEMENT
e data sets (accession number ERP126178) for this study can
befound in the European Nucleotide Archive (ENA) at EMBL-EBI
(https://www.ebi.ac.uk/ena/browser/view/PRJEB42339).
AUTHOR CONTRIBUTIONS
VM (PI of the microbiology part of drilling operation), MJ,
and MG conceived the study. PB, AK, VM, and members of
the SUSTAIN onsite, science teams conducted eld operations
and sampling. PB and PV processed the samples and conducted
the molecular biology experiments. PB, PV, and SK performed
the data analysis. PB wrote the original dra. All authors took
part in writing the manuscript.
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 18 September 2021 | Volume 12 | Article 728977
FUNDING
Sample collection and sample processing were funded with a
grant of excellence from the Icelandic Science fund, ICF-RANNÍS
IceSUSTAIN (163083-051). e drilling operation was funded
with International Continental Scientic Drilling Program (ICDP)
through the SUSTAIN project and the IceSUSTAIN grant. A
doctoral student grant from RANNÍS (206582-051) was
attributed to PB.
CODE AVAILABILITY STATEMENT
e pipeline used to analyze the data set reported in this
manuscript can be found at https://benjjneb.github.io/dada2/
tutorial.html. e code used to identify contaminants can befound
at https://benjjneb.github.io/decontam/vignettes/decontam_intro.
html, and the code used to build the gures can be found in
the supplementary material under the section code.
ACKNOWLEDGMENTS
The authors would like to thank all members of the SUSTAIN
onsite and science teams, and the Surtsey Research Society
for their contributions to the drilling project and sampling,
especially B. Kleine, T. Weisenberger, S. Prause, C.F. Gorny,
and S. Couper. The Icelandic Science fund, ICF-RANNÍS
Jules Verne fund (scientific and technical cooperation between
Iceland and France) contributed additional funds. The authors
thank E. Gérard, A. Lecoeuvre, B. Ménez, and S. Borensztajn
for their contributions to sample preparation and SEM
images.
SUPPLEMENTARY MATERIAL
e Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2021.728977/
full#supplementary-material
REFERENCES
Arce-Rodríguez, A., Puente-Sánchez, F., Avendaño, R., Martínez-Cruz, M., de
Moor, J. M., Pieper, D. H., et al. (2019). ermoplasmatales and sulfur-
oxidizing bacteria dominate the microbial community at the surface water
of a CO2-rich hydrothermal spring located in Tenorio volcano National
Park, Costa Rica. Extremophiles 23, 177–187. doi: 10.1007/s00792-018-
01072-6
Bach, W. (2016). Some compositional and kinetic controls on the bioenergetic
landscapes in oceanic basement. Front. Microbiol. 7:107. doi: 10.3389/
fmicb.2016.00107
Bach, W., and Edwards, K. J. (2003). Iron and sulde oxidation within the
basaltic ocean crust: implications for chemolithoautotrophic microbial biomass
production. Geochim. C osm ochi m. Acta 67, 3871–3887. doi: 10.1016/
S0016-7037(03)00304-1
Baldursson, S., and Ingadóttir, Á. (2007). Nomination of Surtsey for the UNESCO
world heritage list. Icelandic Inst. Nat. Hist. Reykjavik Available at: https://
whc.unesco.org/uploads/nominations/1267.pdf
Baquiran, J. P. M., Ramírez, G. A., Haddad, A. G., Toner, B. M., Hulme, S.,
Wheat, C. G., et al. (2016). Temperature and redox eect on mineral
colonization in Juan de Fuca Ridge Flank subsurface crustal uids. Front.
Microbiol. 7:396. doi: 10.3389/fmicb.2016.00396
Bar-On, Y. M., Phillips, R., and Milo, R. (2018). e biomass distribution on
earth. Proc. Natl. Acad . Sci. U. S. A. 115, 6506–6511. doi: 10.1073/
pnas.1711842115
Baross, J. A., Wilcock, W. S. D., Kelley, D. S., Delong, E. F., and Cary, S. C.
(2004). e subsurface biosphere at mid-ocean ridges: issues and challenges.
Geophys. Monogr. Ser. doi: 10.1029/GM144
Barton, L. L., Fardeau, M. L., and Fauque, G. D. (2014). Hydrogen sulde: A
toxic gas produced by dissimilatory sulfate and sulfur reduction and consumed
by microbial oxidation. Met. Ions Life Sci. 14, 237–277. doi: 10.1007/978-
94-017-9269-1_10
Barton, H. A., Taylor, N. M., Lubbers, B. R., and Pemberton, A. C. (2006).
DNA extraction from low-biomass carbonate rock: An improved method
with reduced contamination and the low-biomass contaminant database.
J. Microbiol. Methods 66, 21–31. doi: 10.1016/j.mimet.2005.10.005
Bergo, N. M., Bendia, A. G., Ferreira, J. C. N., Murton, B. J., Brandini, F. P.,
and Pellizari, V. H. (2021). Microbial diversity of deep-sea ferromanganese
crust eld in the Rio Grande Rise, Southwestern Atlantic Ocean. Microb.
Ecol. 82, 344–355. doi: 10.1007/s00248-020-01670-y
Cai, M., Wang, L., Cai, H., Li, Y., Wang, Y. N., Tang, Y. Q., et al. (2011).
Salinarimonas ramus sp. nov. and Tessaracoccus oleiagri sp. nov., isolated
from a crude oil-contaminated saline soil. Int. J. Syst. Evol. Microbio l. 61,
1767–1775. doi: 10.1099/ijs.0.025932-0
Callahan, B. J., McMurdie, P. J., Rosen, M. J., Han, A. W., Johnson, A. J. A.,
and Holmes, S. P. (2016). DADA2: high-resolution sample inference from
Illumina amplicon data. Nat. Methods 13, 581–583. doi: 10.1038/nmeth.3869
Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Huntley, J.,
Fierer, N., et al. (2012). Ultra-high-throughput microbial community analysis
on the Illumina HiSeq and MiSeq platforms. ISME J. 6, 1621–1624. doi:
10.1038/ismej.2012.8
Ciobanu, M. C., Burgaud, G., Dufresne, A., Breuker, A., Rédou, V., Maamar, S. B.,
et al. (2014). Microorganisms persist at record depths in the subseaoor
of the Canterbury Basin. ISME J. 8, 1370–1380. doi: 10.1038/ismej.2013.250
Cockell, C. S., Voytek, M. A., Gronstal, A. L., Finster, K., Kirshtein, J. D.,
Howard, K., et al. (2012). Impact disruption and recovery of the deep
subsurface biosphere. Astrobiology 12, 231–246. doi: 10.1089/ast.2011.0722
Cowen, J. P., Giovannoni, S. J., Kenig, F., Johnson, H. P., Buttereld, D.,
Rappé, M. S., et al. (2003). Fluids from aging ocean crust that support
microbial life. Science 299, 120–123. doi: 10.1126/science.1075653
D’Hondt, S., Jørgensen, B. B., Miller, D. J., Batzke, A., Blake, R., Cragg, B. A.,
et al. (2004). Distributions of microbial activities in deep subseaoor sediments.
Science 306, 2216–2221. doi: 10.1126/science.1101155
Daae, F. L., Økland, I., Dahle, H., Jørgensen, S. L., orseth, I. H., and
Pedersen, R. B. (2013). Microbial life associated with low-temperature alteration
of ultramac rocks in the Leka ophiolite complex. Geobiology 11, 318–339.
doi: 10.1111/gbi.12035
Davis, N. M., Proctor, D. M., Holmes, S. P., Relman, D. A., and Callahan, B. J.
(2018). Simple statistical identication and removal of contaminant sequences
in marker-gene and metagenomics data. Microbiome 6:226. doi: 10.1186/
s40168-018-0605-2
Diehl, H., and Horchak-Morris, N. (1987). Studies on uorescein-V e
absorbance of uorescein in the ultraviolet, as a function of pH. Talanta
34, 739–741. doi: 10.1016/0039-9140(87)80232-1
Direito, S. O. L., Marees, A., and Röling, W. F. M. (2012). Sensitive life detection
strategies for low-biomass environments: optimizing extraction of nucleic
acids adsorbing to terrestrial and Mars analogue minerals. FEMS Microbiol.
Ecol. 81, 111–123. doi: 10.1111/j.1574-6941.2012.01325.x
Douglas, G. M., Maei, V. J., Zaneveld, J. R., Yurgel, S. N., Brown, J. R.,
Taylor, C. M., et al. (2020). PICRUSt2 for prediction of metagenome functions.
Nat. Biotechnol. 38, 685–688. doi: 10.1038/s41587-020-0548-6
Edwards, K. J., Bach, W., and McCollom, T. M. (2005). Geomicrobiology in
oceanography: microbe-mineral interactions at and below the seaoor. Tr end s
Microbiol. 13, 449–456. doi: 10.1016/j.tim.2005.07.005
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 19 September 2021 | Volume 12 | Article 728977
Edwards, K. J., Fisher, A. T., and Wheat, C. G. (2012). e deep subsurface
biosphere in igneous ocean crust: frontier habitats for microbiological
exploration. Front. Microbiol. 3:8. doi: 10.3389/fmicb.2012.00008
Fisk, M. R., Storrie-Lombardi, M. C., Douglas, S., Popa, R., McDonald, G.,
and Di Meo-Savoie, C. (2003). Evidence of biological activity in Hawaiian
subsurface basalts. Geochem. Geophys. Geosyst, 12:1103. doi:
10.1029/2002GC000387
Friese, A., Kallmeyer, J., Kitte, J. A., Martínez, I. M., Bijaksana, S., and Wagner, D.
(2017). A simple and inexpensive technique for assessing contamination
during drilling operations. Limnol. Oceanogr. Methods 15, 200–211. doi:
10.1002/lom3.10159
Furnes, H., and Staudigel, H. (1999). Biological mediation in ocean crust
alteration: how deep is the deep biosphere? Earth Planet. Sci. Lett. 166,
97–103. doi: 10.1016/S0012-821X(99)00005-9
Gloor, G. B., Macklaim, J. M., Pawlowsky-Glahn, V., and Egozcue, J. J. (2017).
Microbiome datasets are compositional: And this is not optional. Front.
Microbiol. 8:2224. doi: 10.3389/fmicb.2017.02224
Gold, T. (1992). e deep, hot biosphere. Proc. Natl. Acad . Sci. U. S. A. 89,
6045–6049. doi: 10.1073/pnas.89.13.6045
Hao, L., McIlroy, S. J., Kirkegaard, R. H., Karst, S. M., Fernando, W. E. Y.,
Aslan, H., et al. (2018). Novel prosthecate bacteria from the candidate
phylum Acetothermia. ISME J. 12, 2225–2237. doi: 10.1038/s41396-018-0187-9
Heberling, C., Lowell, R. P., Liu, L., and Fisk, M. R. (2010). Extent of the
microbial biosphere in the oceanic crust. Geochem. Geophys. Geosyst. 8,
1525–2027. doi: 10.1029/2009GC002968
Herrera, A., and Cockell, C. (2007). Exploring microbial diversity in volcanic
environments: A review of methods in DNA extraction. J. Microbiol. Method.
70, 1–12. doi: 10.1016/j.mimet.2007.04.005
Inagaki, F., Hinrichs, K. U., Kubo, Y., Bihan, M., Bowden, S. A., Bowles, M.,
et al. (2016). IODP expedition 337: deep coalbed biosphere o Shimokita
– microbial processes and hydrocarbon system associated with deeply buried
coalbed in the ocean. Sci. Drill. 21, 17–28. doi: 10.5194/sd-21-17-2016
Inagaki, F., Hinrichs, K. U., Kubo, Y., Bowles, M. W., Heuer, V. B., Hong, W. L.,
et al. (2015). Exploring deep microbial life in coal-bearing sediment down to
~2.5km below the ocean oor. Science 349, 420–424. doi: 10.1126/science.aaa6882
Jackson, M. D. (2020). Petrograpic and material observations of basaltic lapilli
tu, 1979 and 2017 Surtsey drill cores, Iceland. Surtsey Res. 14, 47–62. doi:
10.33112/surtsey.14.4
Jackson, M. D., Couper, S., Stan, C. V., Ivarsson, M., Czabaj, M. W., Tamura, N.,
et al. (2019a). Authigenic mineral texture in submarine 1979 basalt drill
Core, Surtsey volcano, Iceland. Geochem. Geophys. Geosyst. 20, 3751–3773.
doi: 10.1029/2019GC008304
Jackson, M. D., Gudmundsson, M. T., Bach, W., Cappelletti, P., Coleman, N. J.,
Ivarsson, M., et al. (2015). Time-lapse characterization of hydrothermal
seawater and microbial interactions with basaltic tephra at Surtsey volcano.
Sci. Drill. 20, 51–58. doi: 10.5194/sd-20-51-2015
Jackson, M. D., Gudmundsson, M. T., Weisenberger, T. B., Michael Rhodes, J.,
Stefánsson, A., Kleine, B. I., et al. (2019b). SUSTAIN drilling at Surtsey
volcano, Iceland, tracks hydrothermal and microbiological interactions in
basalt 50 years aer eruption. Sci. Drill. 25, 35–46. doi: 10.5194/sd-25-
35-2019
Jacobson Meyers, M. E., Sylvan, J. B., and Edwards, K. J. (2014). Extracellular
enzyme activity and microbial diversity measured on seaoor exposed basalts
from Loihi seamount indicate the importance of basalts to global biogeochemical
cycling. Appl. Environ. Microbiol. 80, 4854–4864. doi: 10.1128/AEM.01038-14
Jakobsson, S. P., and Moore, J. G. (1982). e Surtsey research drilling project
of 1979. Surtsey Res. 9, 76–93.
Jakobsson, S. P., and Moore, J. G. (1986). Hydrothermal minerals and alteration
rates at Surtsey volcano, Iceland. Geol. Soc . Am. Bull. 97, 648–659. doi:
10.1130/0016-7606(1986)97<648:HMAARA>2.0.CO;2
Jakobsson, S. P., ors, K., Vésteinsson, Á. T., and Ásbjörnsdóttir, L. (2009).
Some aspects of the seaoor morphology at Surtsey volcano: e new
multibeam bathymetric survey of 2007. Surtsey Res. 12,9–20.
Jian, C., Luukkonen, P., Yki-Järvinen, H., Salonen, A., and Korpela, K. (2020).
Quantitative PCR provides a simple and accessible method for quantitative
microbiota proling. PLoS One 15:e0227285. doi: 10.1371/journal.pone.0227285
Jørgensen, S. L., and Zhao, R. (2016). Microbial inventory of deeply buried
oceanic crust from a young ridge ank. Front. Microbiol. 7:820. doi: 10.3389/
fmicb.2016.00820
Jungbluth, S. P., Bowers, R. M., Lin, H. T., Cowen, J. P., and Rappé, M. S.
(2016). Novel microbial assemblages inhabiting crustal uids within mid-
ocean ridge ank subsurface basalt. ISME J. 10, 2033–2047. doi: 10.1038/
ismej.2015.248
Jungbluth, S. P., del Rio, T. G., Tringe, S. G., Stepanauskas, R., and Rappé, M. S.
(2017). Genomic comparisons of a bacterial lineage that inhabits both marine
and terrestrial deep subsurface systems. PeerJ 5:e3134. doi: 10.7717/peerj.3134
Jungbluth, S. P., Grote, J., Lin, H. T., Cowen, J. P., and Rappé, M. S. (2013).
Microbial diversity within basement uids of the sediment-buried Juan de
Fuca Ridge ank. ISME J. 7, 161–172. doi: 10.1038/ismej.2012.73
Jungbluth, S. P., Lin, H. T., Cowen, J. P., Glazer, B. T., and Rappé, M. S.
(2014). Phylogenetic diversity of microorganisms in subseaoor crustal uids
from holes 1025C and 1026B along the Juan de Fuca Ridge ank. Front.
Microbiol. 5:119. doi: 10.3389/fmicb.2014.00119
Kallmeyer, J. (2017). Contamination control for scientic drilling operations.
Adv. Appl. Microbiol. 98, 61–91. doi: 10.1016/bs.aambs.2016.09.003
Kallmeyer, J., Mangelsdorf, K., Cragg, B., and Horseld, B. (2006). Techniques
for contamination assessment during drilling for terrestrial subsurface
sediments. Geomicrobiol J. 23, 227–239. doi: 10.1080/01490450600724258
Karner, M. B., Delong, E. F., and Karl, D. M. (2001). Archaeal dominance in
the mesopelagic zone of the Pacic Ocean. Nature 409, 507–510. doi:
10.1038/35054051
Kashe, K., and Lovley, D. R. (2003). Extending the upper temperature limit
for life. Science 301:934. doi: 10.1126/science.1086823
Kelly, L. C., Cockell, C. S., orsteinsson, T., Marteinsson, V., and Stevenson, J.
(2014). Pioneer microbial communities of the Fimmvörðuháls lava ow,
Eyjaallajökull, Iceland. Microb. Ecol. 68, 504–518. doi: 10.1007/s00248-014-0432-3
Kern, T., Linge, M., and Rother, M. (2015). Methanobacterium Aggregans sp.
nov., a hydrogenotrophic methanogenic archaeon isolated from an anaerobic
digester. Int. J. Syst . Evol . Microbiol. 65, 1975–1980. doi: 10.1099/ijs.0.000210
Kie, T. L. (2010). “Sampling the deep sub-surface using drilling and coring
techniques,” in Handbook of Hydrocarbon and Lipid Microbiology. ed. K. N.
Timmis (Berlin/Heidelberg: Springer).
Kleine, B. I., Stefánsson, A., Kjartansdóttir, R., Prause, S., Weisenberger, T. B.,
Reynolds, H. I., et al. (2020). e Surtsey volcano geothermal system: An
analogue for seawater-oceanic crust interaction with implications for the
elemental budget of the oceanic crust. Chem. Geol. 550:119702. doi: 10.1016/j.
chemgeo.2020.119702
Klindworth, A., Pruesse, E., Schweer, T., Peplies, J., Quast, C., Horn, M., et al.
(2013). Evaluation of general 16S ribosomal RNA gene PCR primers for
classical and next-generation sequencing-based diversity studies. Nucleic Acids
Res. 41:e1. doi: 10.1093/nar/gks808
Konhauser, K. O., Schiman, P., and Fisher, Q. J. (2002). Microbial mediation
of authigenic clays during hydrothermal alteration of basaltic tephra, Kilauea
volcano. Geochem. Geophys. Geosyst. 3, 1–13. doi: 10.1029/2002GC000317
Korzhenkov, A. A., Teplyuk, A. V., Lebedinsky, A. V., Khvashchevskaya, A. A.,
Kopylova, Y. G., Arakchaa, K. D., et al. (2018). Members of the uncultured
taxon OP1 (“Acetothermia”) predominate in the microbial community of
an alkaline hot spring at East-Tuvinian upland. Microbiol. 87, 783–795. doi:
10.1134/S0026261718060115
Lee, M. D., Walworth, N. G., Sylvan, J. B., Edwards, K. J., and Orcutt, B. N.
(2015). Microbial communities on seaoor basalts at Dorado Outcrop reect
level of alteration and highlight global lithic clades. Front. Microbiol. 6:1470.
doi: 10.3389/fmicb.2015.01470
Lever, M. A., Alperin, M., Engelen, B., Inagaki, F., Nakagawa, S., Steinsbu, B. O.,
et al. (2006). Trends in basalt and sediment core contamination during IODP
expedition 301. Geomicrobiol J. 23, 517–530. doi: 10.1080/01490450600897245
Lever, M. A., Rogers, K. L., Lloyd, K. G., Overmann, J., Schink, B., auer, R. K.,
et al. (2015). Life under extreme energy limitation: A synthesis of laboratory-
and eld-based investigations. FEMS Microbiol. Rev. 39, 688–728. doi: 10.1093/
femsre/fuv020
Lever, M. A., Rouxel, O., Alt, J. C., Shimizu, N., Ono, S., Coggon, R. M.,
et al. (2013). Evidence for microbial carbon and sulfur cycling in deeply
buried ridge ank basalt. Science 339, 1305–1308. doi: 10.1126/science.1229240
Li, J., Mara, P., Schubotz, F., Sylvan, J. B., Burgaud, G., Klein, F., et al. (2020).
Recycling and metabolic exibility dictate life in the lower oceanic crust.
Nature 579, 250–255. doi: 10.1038/s41586-020-2075-5
Liu, J. H., Wang, Y. X., Zhang, X. X., Wang, Z. G., Chen, Y. G., Wen, M. L.,
et al. (2010). Salinarimonas rosea gen. nov., sp. nov., a new member of
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 20 September 2021 | Volume 12 | Article 728977
the α-2 subgroup of the proteobacteria. Int. J. Sy st. Ev ol. Mi crobiol . 60,
55–60. doi: 10.1099/ijs.0.006981-0
Love, M. I., Huber, W., and Anders, S. (2014). Moderated estimation of fold
change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15:550.
doi: 10.1186/s13059-014-0550-8
Lysnes, K., Torsvik, T., orseth, I.H., and Pedersen, R.B. (2004). “Microbial
populations in ocean oor basalt: Results from ODP leg 187.” in Proc. ODP,
Sci . Results. eds. R. B. Pedersen, D. M. Christie and D. J. Miller, 187.
Magnabosco, C., Lin, L. H., Dong, H., Bomberg, M., Ghiorse, W., Stan-Lotter, H.,
et al. (2018). e biomass and biodiversity of the continental subsurface.
Nat. Geosci. 11, 707–717. doi: 10.1038/s41561-018-0221-6
Magnússon, B., Magnússon, S. H., Ólafsson, E., and Sigurdsson, B. D. (2014).
Plant colonization, succession and ecosystem development on Surtsey with
reference to neighbouring islands. Biogeosciences 11, 5521–5537. doi: 10.5194/
bg-11-5521-2014
Marteinsson, V., Klonowski, A., Reynisson, E., Vannier, P., Sigurdsson, B. D.,
and Ólafsson, M. (2015). Microbial colonization in diverse surface soil types
in Surtsey and diversity analysis of its subsurface microbiota. Biogeosciences
12, 1191–1203. doi: 10.5194/bg-12-1191-2015
Martinez Arbizu, P. (2020). pairwiseAdonis: pairwise multilevel comparison
using Adonis. R Packag version 04. Available at: https://github.com/
pmartinezarbizu/pairwiseAdonis
Mason, O. U., Nakagawa, T., Rosner, M., van Nostrand, J. D., Zhou, J.,
Maruyama, A., et al. (2010). First investigation of the microbiology of the
deepest layer of ocean crust. PLoS One 5:e15399. doi: 10.1371/journal.
pone.0015399
Mason, O. U., Stingl, U., Wilhelm, L. J., Moeseneder, M. M., Di Meo-Savoie, C. A.,
Fisk, M. R., et al. (2007). e phylogeny of endolithic microbes associated
with marine basalts. Environ. Microbiol. 9, 2539–2550. doi: 10.1111/j.1462-
2920.2007.01372.x
Mcloughlin, N., Staudigel, H., Furnes, H., Eickmann, B., and Ivarsson, M.
(2010). Mechanisms of microtunneling in rock substrates: distinguishing
endolithic biosignatures from abiotic microtunnels. Geobiology 8, 245–255.
doi: 10.1111/j.1472-4669.2010.00243.x
Mcmahon, S., and Parnell, J. (2014). Weighing the deep continental biosphere.
FEMS Microbiol. Ecol. 87, 113–120. doi: 10.1111/1574-6941.12196
McMurdie, P. J., and Holmes, S. (2013). Phyloseq: An R package for reproducible
interactive analysis and graphics of microbiome census data. PLoS One
8:e61217. doi: 10.1371/journal.pone.0061217
McPhie, J., White, J. D. L., Gorny, C., Jackson, M. D., Gudmundsson, M. T.,
and Couper, S. (2020). Lithofacies from the 1963-1967 Surtsey eruption in
SUSTAIN drill cores SE-2a, SE-2b and SE-03. Surtsey Res. 14, 19–32. doi:
10.33112/surtsey.14.2
Merino, N., Kawai, M., Boyd, E. S., Colman, D. R., McGlynn, S. E., Nealson, K. H.,
et al. (2020). Single-cell genomics of novel actinobacteria with the Wood–
Ljungdahl pathway discovered in a serpentinizing system. Front. Microbiol.
11:1031. doi: 10.3389/fmicb.2020.01031
Minich, J. J., Sanders, J. G., Amir, A., Humphrey, G., Gilbert, J. A., and Knight, R.
(2019). Quantifying and understanding well-to-well contamination in
microbiome research. mSystems. 4:e00186-19. doi: 10.1128/mSystems.00186-19
Moore, J. C., and Jackson, M. D. (2020). Observations on the structure of
Surtsey. Surtsey Res. 14, 33–45. doi: 10.33112/surtsey.14.3
Motamedi, S., Orcutt, B. N., Früh-Green, G. L., Twing, K. I., Pendleton, H. L.,
and Brazelton, W. J. (2020). Microbial residents of the Atlantis massif’s
shallow serpentinite subsurface. Appl. Environ. Microbiol. 86:e00356-20. doi:
10.1128/AEM.00356-20
Nakagawa, S., Inagaki, F., Suzuki, Y., Steinsbu, B. O., Lever, M. A., Takai, K., et al.
(2006). Microbial community in black rust exposed to hot ridge ank crustal
uids. Appl. Environ. Microbiol. 72, 6789–6799. doi: 10.1128/AEM.01238-06
Neufeld, J. D., Schäfer, H., Cox, M. J., Boden, R., McDonald, I. R., and
Murrell, J. C. (2007). Stable-isotope probing implicates Methylophaga spp
and novel Gammaproteobacteria in marine methanol and methylamine
metabolism. ISME J. 1, 480–491. doi: 10.1038/ismej.2007.65
Nigro, L. M., Harris, K., Orcutt, B. N., Hyde, A., Clayton-Luce, S., Becker, K.,
et al. (2012). Microbial communities at the borehole observatory on the
Costa Rica Ri ank (ocean drilling program hole 896A). Front. Microbiol.
3:232. doi: 10.3389/fmicb.2012.00232
Oksanen, J., Blanchet, F. G., Kindt, R., Legendre, P., Minchin, P. R., O’Hara, R. B.,
et al. (2007). e vegan package. Commun . Ecol. Packag 10, 631–637.
Ólafsson, M., and Jakobsson, S. P. (2009). Chemical composition of hydrothermal
water and water-rock interactions on Surtsey volcanic island: A preliminary
report. Surtsey Res, 12, 29–38.
Orcutt, B. N., Bach, W., Becker, K., Fisher, A. T., Hentscher, M., Toner, B. M.,
et al. (2011b). Colonization of subsurface microbial observatories deployed
in young ocean crust. ISME J. 5, 692–703. doi: 10.1038/ismej.2010.157
Orcutt, B. N., Bergenthal, M., Freudenthal, T., Smith, D., Lilley, M. D.,
Schnieders, L., et al. (2017). Contamination tracer testing with seabed drills:
IODP expedition 357. Sci. Drill. 23, 39–46. doi: 10.5194/sd-23-39-2017
Orcutt, B.N., Daniel, I., and Dasgupta, R. (2019). Book deep carbon: past to
present. Cambridge: Cambridge University Press.
Orcutt, B. N., Sylvan, J. B., Knab, N. J., and Edwards, K. J. (2011a). Microbial
ecology of the Dark Ocean above, at, and below the seaoor. Microbiol.
Mol. Biol. R ev. 75, 361–422. doi: 10.1128/MMBR.00039-10
Orcutt, B. N., Sylvan, J. B., Rogers, D. R., Delaney, J., Lee, R. W., and Girguis, P. R.
(2015). Carbon xation by basalt-hosted microbial communities. Front.
Microbiol. 6:904. doi: 10.3389/fmicb.2015.00904
Orcutt, B. N., Wheat, C. G., Rouxel, O., Hulme, S., Edwards, K. J., and Bach, W.
(2013). Oxygen consumption rates in subseaoor basaltic crust derived from
a reaction transport model. Nat. Commun. 4:2539. doi: 10.1038/ncomms3539
Óskarsson, B. V., Jónasson, K., Valsson, G., and Belart, J. M. C. (2020). Erosion
and sedimentation in Surtsey island quantied from new DEMs. Surtsey
Res. 4, 63–77. doi: 10.33112/surtsey.14.5
Øvreås, L., Forney, L., Daae, F. L., and Torsvik, V. (1997). Distribution of bacterioplankton
in meromictic lake Saelenvannet, as determined by denaturing gradient gel
electrophoresis of PCR-amplied gene fragments coding for 16S rRNA. Appl.
Environ. Microbiol. 63, 3367–3373. doi: 10.1128/aem.63.9.3367-3373.1997
Parada, A. E., Needham, D. M., and Fuhrman, J. A. (2016). Every base matters:
assessing small subunit rRNA primers for marine microbiomes with mock
communities, time series and global eld samples. Environ. Microbiol. 18,
1403–1414. doi: 10.1111/1462-2920.13023
Parkes, J. R., Cragg, B. A., and Wellsbury, P. (2000). Recent studies on bacterial
populations and processes in subseaoor sediments. A review. Hydrogeol. J.
8, 11–28. doi: 10.1007/PL00010971
Pedersen, K. (2000). Exploration of deep intraterrestrial microbial life: current
perspectives. FEMS Microbiol. Lett. 185, 9–16. doi: 10.1111/j.1574-6968.2000.
tb09033.x
Pellizzari, L., Neumann, D., Alawi, M., Voigt, D., Norden, B., and Würdemann, H.
(2013). e use of tracers to assess drill-mud penetration depth into sandstone
cores during deep drilling: method development and application. Environ.
Earth Sci. 70, 3727–3738. doi: 10.1007/s12665-013-2715-2
Pollock, J., Glendinning, L., Wisedchanwet, T., and Watson, M. (2018). e
madness of microbiome: attempting to nd consensus “best practice” for
16S microbiome studies. Appl. Environ. Microbiol. 84:e02627-17. doi: 10.1128/
AEM.02627-17
Prause, S., Weisenberger, T. B., Cappelletti, P., Grimaldi, C., Rispoli, C.,
Jónasson, K., et al. (2020). Alteration progress within the Surtsey hydrothermal
system, SW Iceland – A time-lapse petrographic study of cores drilled in
1979and 2017. J. Volcanol . Geothe rm. Res. 392:106754. doi: 10.1016/j.
jvolgeores.2019.106754
Prieur, D., and Marteinsson, V.T. (1998) “Prokaryotes living under elevated
hydrostatic pressure,” in Biotechnology of Extremophiles. Advances in Biochemical
Engineering/Biotechnology. Vol 61. ed. G. Antranikian (Berlin, Heidelberg:
Springer).
Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., et al.
(2013). e SILVA ribosomal RNA gene database project: improved data
processing and web-based tools. Nucleic Acids Res. 41, D590–D596. doi:
10.1093/nar/gks1219
R Core Team (2019). R: A language and environment for statistical computing.
R Found Stat Comput. Vienna. Available at: https://www.R-project.org/
Ramírez, G. A., Garber, A. I., Lecoeuvre, A., D’angelo, T., Wheat, C. G., and
Orcutt, B. N. (2019). Ecology of subseaoor crustal biolms. Front. Microbiol.
10:1983. doi: 10.3389/fmicb.2019.01983
Rathsack, K., Stackebrandt, E., Reitner, J., and Schumann, G. (2009).
Microorganisms isolated from deep sea low-temperature inuenced oceanic
crust basalts and sediment samples collected along the Mid-Atlantic Ridge.
Geomicrobiol J. 26, 264–274. doi: 10.1080/01490450902892456
Robador, A., Jungbluth, S. P., LaRowe, D. E., Bowers, R. M., Rappé, M. S.,
Amend, J. P., et al. (2015). Activity and phylogenetic diversity of sulfate-
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 21 September 2021 | Volume 12 | Article 728977
reducing microorganisms in low-temperature subsurface uids within the
upper oceanic crust. Front. Microbiol. 5:748. doi: 10.3389/fmicb.2014.00748
Rstudio, T. (2020) RStudio: Integrated Development for R. Rstudio Team, PBC,
Boston, MA. Available at: Available at: http://www.rstudio.com/
Santelli, C. M., Banerjee, N., Bach, W., and Edwards, K. J. (2010). Tapping
the subsurface ocean crust biosphere: low biomass and drilling-related
contamination calls for improved quality controls. Geomicrobiol J. 27, 158–169.
doi: 10.1080/01490450903456780
Santelli, C. M., Edgcomb, V. P., Bach, W., and Edwards, K. J. (2009). e
diversity and abundance of bacteria inhabiting seaoor lavas positively
correlate with rock alteration. Environ. Microbiol. 11, 86–98. doi: 10.1111/j.
1462-2920.2008.01743.x
Santelli, C. M., Orcutt, B. N., Banning, E., Bach, W., Moyer, C. L., Sogin, M. L.,
et al. (2008). Abundance and diversity of microbial life in ocean crust.
Nature 453, 653–656. doi: 10.1038/nature06899
Schipper, C. I., Le Voyer, M., Moussallam, Y., White, J. D. L., ordarson, T.,
Kimura, J. I., et al. (2016). Degassing and magma mixing during the eruption
of Surtsey volcano (Iceland, 1963–1967): the signatures of a dynamic and discrete
ri propagation event. Bull. Volcanol. 78:33. doi: 10.1007/s00445-016-1025-4
Sheik, C. S., Reese, B. K., Twing, K. I., Sylvan, J. B., Grim, S. L., Schrenk, M. O.,
et al. (2018). Identication and removal of contaminant sequences from
ribosomal gene databases: lessons from the census of deep life. Front.
Microbiol. 9:840. doi: 10.3389/fmicb.2018.00840
Singer, E., Chong, L. S., Heidelberg, J. F., and Edwards, K. J. (2015). Similar
microbial communities found on two distant seaoor basalts. Front. Microbiol.
6:1409. doi: 10.3389/fmicb.2015.01409
Smith, A., Popa, R., Fisk, M., Nielsen, M., Wheat, C. G., Jannasch, H. W.,
et al. (2011). In situ enrichment of ocean crust microbes on igneous minerals
and glasses using an osmotic ow-through device. Geochem. Geophys. Geosyst.
6, 1525–2027. doi: 10.1029/2010GC003424
Sorokin, D. Y., Lysenko, A. M., Mityushina, L. L., Tourova, T. P., Jones, B. E.,
Rainey, F. A., et al. (2001). ioalkalimicrobium aerophilum gen. nov., sp.
nov. and ioalkalimicrobium sibericum sp. nov., and ioalkalivibrio versutus
gen. nov., sp. nov., ioalkalivibrio nitratis sp. nov. and ioalkalivibrio
denitricans sp. nov., novel obligately alkaliphilic. Int. J. Syst. Evol. Mi crobiol .
51, 565–580. doi: 10.1099/00207713-51-2-565
Sousa, D. Z., Visser, M., Van Gelder, A. H., Boeren, S., Pieterse, M. M.,
Pinkse, M. W. H., et al. (2018). e deep-subsurface sulfate reducer
Desulfotomaculum kuznetsovii employs two methanol-degrading pathways.
Nat. Commun. 9:239. doi: 10.1038/s41467-017-02518-9
Spanevello, M. D., Yamamoto, H., and Patel, B. K. C. (2002). ermaerobacter
subterraneus sp. nov., a novel aerobic bacterium from the Great Artesian
Basin of Australia, and emendation of the genus ermaerobacter. Int. J.
Syst. Evol. Microbiol. doi: 10.1099/ijs.0.01959-0
Sriswasdi, S., Yang, C. C., and Iwasaki, W. (2017). Generalist species drive microbial
dispersion and evolution. Nat. Commun. 8:1162. doi: 10.1038/s41467-017-01265-1
Staudigel, H., Furnes, H., McLoughlin, N., Banerjee, N. R., Connell, L. B., and
Templeton, A. (2008). 3.5 billion years of glass bioalteration: volcanic rocks as a
basis for microbial life? Earth Sci. Rev. 89, 156–176. doi: 10.1016/j.earscirev.2008.04.005
Sudek, L. A., Wanger, G., Templeton, A. S., Staudigel, H., and Tebo, B. M.
(2017). Submarine basaltic glass colonization by the heterotrophic Fe(II)-
oxidizing and siderophore-producing deep-sea bacterium pseudomonas stutzeri
VS-10: e potential role of basalt in enhancing growth. Front. Microbiol.
8:363. doi: 10.3389/fmicb.2017.00363
Suzuki, Y., Yamashita, S., Kouduka, M., Ao, Y., Mukai, H., Mitsunobu, S., et al.
(2020). Deep microbial proliferation at the basalt interface in 33.5–104 million-
year-old oceanic crust. Commun. Biol. 3:136. doi: 10.1038/s42003-020-0860-1
Takai, K., and Horikoshi, K. (2000). Rapid detection and quantication of
members of the archaeal community by quantitative PCR using uorogenic
probes. Appl. Environ. Microbiol. 66, 5066–5072. doi: 10.1128/AEM.66.
11.5066-5072.2000
Takai, K., Inoue, A., and Horikoshi, K. (1999). ermaerobacter marianensis
gen. nov., sp. nov., an aerobic extremely thermophilic marine bacterium
from the 11 000 m deep Mariana Trench. Int. J. Syst. Bacteriol. 2, 619–628.
doi: 10.1099/00207713-49-2-619
Takai, K., Nakamura, K., Toki, T., Tsunogai, U., Miyazaki, M., Miyazaki, J.,
et al. (2008). Cell proliferation at 122°C and isotopically heavy CH4 production
by a hyperthermophilic methanogen under high-pressure cultivation. Proc.
Natl. Acad. S ci. U. S. A. 105, 10949–10954. doi: 10.1073/pnas.0712334105
Takami, H., Noguchi, H., Takaki, Y., Uchiyama, I., Toyoda, A., Nishi, S., et al.
(2012). A deeply branching thermophilic bacterium with an ancient acetyl-
CoA pathway dominates a subsurface ecosystem. PLoS One 7:e30559. doi:
10.1371/journal.pone.0030559
Templeton, A. S., Staudigel, H., and Tebo, B. M. (2005). Diverse Mn(II)-oxidizing
bacteria isolated from submarine basalts at Loihi seamount. Geomicrobiol
J. 22, 127–139. doi: 10.1080/01490450590945951
Þórarinsson, S. (1965). e Surtsey eruption: course of events and the development
of the new island. Surtsey Res 1, 51–55.
orseth, I. H., Pedersen, R. B., and Christie, D. M. (2003). Microbial alteration
of 0-30-Ma seaoor and sub-seaoor basaltic glasses from the Australian
Antarctic Discordance. Earth Planet. Sci. Lett. 215, 237–247. doi: 10.1016/
S0012-821X(03)00427-8
orseth, I. H., Torsvik, T., Torsvik, V., Daae, F. L., and Pedersen, R. B. (2001).
Diversity of life in ocean oor basalt. Earth Planet. Sci. Lett. 194, 31–37.
doi: 10.1016/S0012-821X(01)00537-4
Tin, S., Bizzoco, R. W., and Kelley, S. T. (2011). Role of the terrestrial subsurface
in shaping geothermal spring microbial communities. Environ. Mi crob iol .
Rep. 3, 491–499. doi: 10.1111/j.1758-2229.2011.00248.x
Walton, A. W. (2008). Microtubules in basalt glass from Hawaii Scientic Driling
Project #2 phase 1 core and Hilina slope, Hawaii: evidence of the occurrence
and behavior of endolithic microorganisms. Geobiology 6, 351–364. doi:
10.1111/j.1472-4669.2008.00149.x
Watanabe, M., Kojima, H., and Fukui, M. (2018). Review of Desulfotomaculum
species and proposal of the genera Desulfallas gen. Nov., Desulfofundulus
gen. nov., Desulfofarcimen gen. nov. and Desulfohalotomaculum gen. nov.
Int. J. Syst. Evol. Microb iol . 68, 2891–2899. doi: 10.1099/ijsem.0.002915
Weisenberger, T. B., Gudmundsson, M. T., Jackson, M. D., Türke, A., Kleine, B. I.,
and Marshall, B. (2019). Operational report for the 2017 Surtsey underwater
volcanic system for thermophiles, alteration processes and INnovative concretes
(SUSTAIN) drilling project at Surtsey volcano, Iceland. Geo. Forschungs Zent
Rep. Available at: https://gfzpublic.gfz-potsdam.de/rest/items/item_4342891_3/
component/le_4342889/content
Yanagawa, K., Nunoura, T., McAllister, S. M., Hirai, M., Breuker, A., Brandt, L.,
et al. (2013). e rst microbiological contamination assessment by deep-
sea drilling and coring by the D/V chikyu at the Iheya North hydrothermal
eld in the Mid-Okinawa trough (IODP Expedition 331). Front. Microbiol.
4:327. doi: 10.3389/fmicb.2013.00327
Zaitseva, S., Lavrentieva, E., Radnagurueva, A., Baturina, O., Kabilov, M., and
Barkhutova, D. (2017) Distribution of Acetothermia-dominated microbial
communities in alkaline hot springs of Baikal Ri Zone. Peer. J. Preprints.
5:e3492v1. doi: 10.7287/peerj.preprints.3492v1
Zhang, X., Feng, X., and Wang, F. (2016). Diversity and metabolic potentials
of subsurface crustal microorganisms from the western ank of the Mid-
Atlantic Ridge. Front. Microbiol. 7:363. doi: 10.3389/fmicb.2016.02094
Zhao, R., Dahle, H., Ramírez, G. A., and Jørgensen, S. L. (2020). Indigenous
ammonia-oxidizing archaea in oxic subseaoor oceanic crust. mSystems.
5:e00758-19. doi: 10.1128/mSystems.00758-19
Zhu, H., Derksen, R. C., Krause, C. R., Fox, R. D., Brazee, R. D., and Ozkan, H. E.
(2005). Fluorescent intensity of dye solutions under dierent pH conditions.
J. ASTM Int. 2:12926. doi: 10.1520/JAI12926
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... The 16S rRNA amplicon datasets used for the comparison with the sequences of the isolated strains were previously published in the European Nucleotide Archive (ENA) at EMBL-EBI (accession number ERP126178) [37]. Briefly, the datasets were obtained by Illumina MiSeq paired-end (2 × 300 base pair) tag sequencing using the universal primers 515f (5 -GTG CCA GCM GCC GCG GTA A-3 ) and 806r (5 -GGA CTA CHV GGG TWT CTA AT-3 ) [50]. ...
... Briefly, the datasets were obtained by Illumina MiSeq paired-end (2 × 300 base pair) tag sequencing using the universal primers 515f (5 -GTG CCA GCM GCC GCG GTA A-3 ) and 806r (5 -GGA CTA CHV GGG TWT CTA AT-3 ) [50]. Sequence variants were inferred using the R Package DADA2 [51] version 1.4, as described elsewhere [37], and the SILVA SSU database release 138 was used for taxonomic assignation [43]. A phyloseq object [52] was constructed directly from the DADA2 outputs and the Amplicon Sequence Variant (ASV) table before any contaminant removal was used for the alignment. ...
... (5′-GTG CCA GCM GCC GCG GTA A-3′) and 806r (5′-GGA CTA CHV GGG TWT CTA AT-3′) [50]. Sequence variants were inferred using the R Package DADA2 [51] version 1.4, as described elsewhere [37], and the SILVA SSU database release 138 was used for taxonomic assignation [43]. A phyloseq object [52] was constructed directly from the DADA2 outputs and the Amplicon Sequence Variant (ASV) table before any contaminant removal was used for the alignment. ...
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Petrographic studies of thin sections from the 1979 and 2017 Surtsey drill cores provide new insights into microstructural features in basaltic lapilli tuff sampled from the principal structural and hydrothermal zones of the volcano. These describe narrow rims of fine ash on altered glass pyroclasts in thin sections of the 2017 cores, characteristics of granular and microtubular structures in the original thin sections of the 1979 core, and glass alteration in diverse environments. The narrow ash rims follow the outlines of glass pyroclasts in the subaerial tuff cone and in submarine and sub-seafloor deposits; they suggest complex eruptive and depositional processes. The tubular microstructures resemble endolithic microborings in older oceanic basalt; they suggest possible microbial activity. Tubule lengths indicate rapid growth rates, up to 30 µm in ~15 years. Comparisons of glass alteration in thin sections prepared immediately after drilling in 1979 and 2017 indicate differential time-lapse alteration processes in the structural and hydrothermal zones of the volcano. In contrast, thin sections of the 1979 core prepared after 38 years in the repository reveal labile glass alteration during archival storage. The oven-dry density of the sub-seafloor lapilli tuff decreases in 2017 samples with high porosity and water absorption and increases in 2017 samples with a compact ash matrix and lower water absorption. The petrographic descriptions and material measurements provide a foundational reference for further investigations of explosive eruption and deposition of basaltic tephra at Surtsey and the subsequent alteration of these deposits in the volcanic environment and, potentially, the curatorial environment.
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Surtsey is a young volcanic island in the offshore extension of Iceland's southeast rift zone that grew from the seafloor during explosive and effusive eruptions in 1963–1967. In 1979, a cored borehole (SE-1) was drilled to 181 m depth and in 2017 three cored boreholes (SE-2a, SE-2b and SE-3) were drilled to successively greater depths. The basaltic deposits host a low-temperature (40–141 °C) seawater-dominated geothermal system. Surtsey provides an ideal environment to study water-rock interaction processes in a young seawater geothermal system. Elemental concentrations (SiO2, B, Na, Ca, Mg, F, dissolved inorganic carbon, SO4, Cl) and isotope contents (δD, δ¹⁸O) in borehole fluids indicate that associated geothermal waters in submarine deposits originated from seawater modified by reactions with the surrounding basalt. These processes produce authigenic minerals in the basaltic lapilli tuff and a corresponding depletion of certain elements in the residual waters. Coupling of measured and modelled concentrations investigates the effect of temperature and associated abundance of authigenic minerals on chemical fluxes from and to the igneous oceanic crust during low-temperature alteration. The annual chemical fluxes calculated at 50–150 °C range from −0.01 to +0.1 × 10¹² mol yr⁻¹ for SiO2, +0.2 to +129 × 10¹² mol yr⁻¹ for Ca, −129 to −0.8 × 10¹² mol yr⁻¹ for Mg and −21 to +0.4 × 10¹² mol yr⁻¹ for SO4 where negative values indicate chemical fluxes from the ocean into the oceanic crust and positive values indicate fluxes from the oceanic crust to the oceans. These flux calculations reveal that water-rock interaction at varying water-rock ratios and temperatures produces authigenic minerals that serve as important sinks of seawater-derived SiO2, Mg and SO4. In contrast, water-rock interaction accompanied by dissolution of basaltic glass and primary crystal fragments provides a significant source of Ca. Such low-temperature alteration could effectively influence the elemental budget of the oceanic igneous crust and ocean waters. The modeling provides insights into water chemistries and chemical fluxes in low-temperature MOR recharge zones. Surtsey also provides a valuable young analogue for assessing the chemical evolution of fluid discharge over the life cycles of seamounts in ridge flank systems.
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Comparison of investigations of the 1979 and 2017 cored boreholes coupled with continued observations of the dynamic surface of Surtsey has modified our concepts of the subsurface structure of the volcano. A geometrical analysis of the 2017 vertical and inclined cores indicates that near-surface layering dips westerly, indicating that the boreholes are located inside the Surtur crater. In subaerial deposits, as well as in deep deposits below sea level and below the pre-Surtsey seafloor, there are zones of porous tuff that contain abundant pyroclasts with narrow rims of fine ash. These features, typical of near-surface deposits, could have been carried down the vent by downslumping during fluctuating explosive activity. They support the hypothesis that a broad diatreme underlies the Surtur vent. No major intrusions were encountered in the 2017 drilling except for coherent basalt in deep sub-seafloor deposits below the center of Surtur crater. The 2017 borehole temperature measurements indicate that the peak temperature in the vertical boreholes was 124 °C at 105 meters below the surface (m.b.s.) and that in the inclined hole it was 127 °C at 115 m.b.s. immediately after drilling. These peak temperatures are 72 meters apart horizontally yet closely resemble each other in shape and magnitude, suggesting a broad heat source. In addition, measurements in the inclined hole from 200 to 290 m.b.s. indicate a temperature of 60±2 °C. This is apparently residual heat from the volcanic action that created the diatreme. These facts cast doubt on the previous concept that the heat anomaly in the 1979 borehole was due to a nearby intrusion. Instead they suggest that heat would have been conducted down from the 85-meter-thick hot lava shield within the Surtur crater into a warm diatreme substrate containing original volcanic heat. As the conducted heat moved down into the water-saturated substrate it would have elevated the temperature above the boiling point curve, baked out water, and created a vapor-dominated system below sea level. Eventually loss of heat by boiling and rise of steam caused the vapor-dominated system to retreat upward. The resulting steam rose and warmed the tephra adjacent to the lava shields where it produced broad areas of palagonitized tuff.
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Surtsey was drilled in 2017 in the context of the Surtsey Underwater volcanic System for Thermophiles, Alteration processes and INnovative Concretes (SUSTAIN) project. Vertical drill holes, SE-02a and SE02b (drilled to 191.64 m), and angled drill SE-03 (drilled to 354.05 m), intersected armoured lapilli tuff and lapilli tuff generated mainly by explosive eruptions at Surtur from November 1963 to January 1964. The top ~20 m of lapilli tuff was erupted from Surtungur. Intervals of coherent basalt in SE-02b (15.7 to 17 m and <15 cm at the end) and in SE-03 (<1 m at ~60 m and ~238 m, and 10 m near the base) are probably intrusions that may have fed the small lavas erupted at Surtur ~2.5 years later. Although collared only a few m from the 1979 drill hole, neither SE-02a nor SE-02b intersected the 13-m-thick interval of basalt found in the 1979 drill hole. The 2017 drill cores are entirely lithified and variably altered, reflecting the effects of hydrothermal alteration and cement deposition on the originally fresh, unconsolidated ash and lapilli. Drill hole SE-03 was drilled on an azimuth of 264o and at 55o from horizontal, obliquely crossing the crater- and conduit-fill of Surtur. Although the exact trajectory of SE-03 is unknown (the drill hole was not surveyed), the drill hole ended at a vertical depth of ~100 m below the pre-eruption sea floor, however, sedimentary facies known to underlie the sea floor nearby were not intersected. Surtur eruptions therefore excavated the pre-eruption sea floor to a depth of several tens of m.