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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, UnitedStates
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
PaulineBergsten
1,2, PaulineVannier
1, AlexandraMaríaKlonowski
1, StephenKnobloch
1,
MagnúsTumiGudmundsson
3, MarieDoloresJackson
4 and ViggóThorMarteinsson
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, wepresent
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 Scientic
Drilling Program specically 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, weuse 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 beidentied 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, dened 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, subseaoor
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 subseaoor
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 oen required), the low biomass (high risk of external
contamination), the presence of minerals which generates DNA
binding and inhibitions (Direito etal., 2012; Lever et al., 2015),
and the diculties 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 Earth’s 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 etal.,
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 proles
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
etal., 2016; Ramírez etal., 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 oshore 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 seaoor
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
scientic 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 etal., 2014). In 1979, a 181 m core (SE-01) drilled
through the eastern sector of the Surtur vent (Figure1), 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 Scientic
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 etal., 2015; Jackson etal., 2019b).
e current temperatures exceed a presumed upper limit for
functional microbial life (122°C; Prieur and Marteinsson, 1998;
Kashe and Lovley, 2003; Takai etal., 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 etal., 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 etal., 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 etal., 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 wedetect actual microbial
structures or biotic signatures in the basaltic cores only 50 years
aer eruptions terminated? Weused 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 oshore. 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
FIGURE1 | 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 etal., 2019b) and
a zone of seawater inow.
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
oshore extension of Iceland’s Eastern Volcanic Zone. e
ocean depth was 130 m below sea level prior to eruption. Aer
3.5 years of submarine, explosive, and eusive lava-producing
eruptions of basalts from the seaoor 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 lithied 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
etal., 2020). Alteration through palagonitization and production
of authigenic cementitious minerals has progressed during the
past 38 years, from 1979 to 2017 (Jackson, 2020; Prause etal.,
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 ls−1. 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 dierent 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
seaoor (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 aer 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:
18in 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 oshore
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
TABLE1 | 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 etal.
(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 Amplication 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 etal., 2016), specic
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 etal., 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, <0.5ng/ml. n.a.: not
available. m b.s.l.: meter below sea level. m b.s.: meter below surface.
TABLE1 | 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
amplication protocol and 5 μl of DNA at 10 ng/μl (NanoDrop
quantication). e second round of the nested PCR used 5 μl
of amplied 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 beamplied. 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. Amplication
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).
Aer normalization and quantication, 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. Aer 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 condence 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. Weidentied 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 identied as chloroplast at the order level, mitochondria
at the family level, Eukaryote at the kingdom level, and those
that could not beidentied at the kingdom level were subtracted
from the data set prior to analysis, as well as putative contaminants
identied by taxonomic aliation 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 identied 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 Signicant Dierence) test were
conducted to evaluate the dierences 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 signicance 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 envt
was used to investigate the correlation between the community
structure and environmental variables (depth and temperature).
Finally, DESeq2 was used to identify ASVs signicantly dierent
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%). Aer 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 quantication, DNA concentration extracted
from 15 g of each drill core sample yields from 0.2 to 1.36 ng.
g−1 of rocks with an average of 0.48 ng.g−1. However, no
signicant 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
dierent 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 dierences in microbial
community structure between drill core and drilling uid and
between drill core and seawater samples proved to besignicant
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 etal., 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 etal., 2019b; Kleine etal., 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 (Figure2, 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).
Amplication of the partial 16S rRNA gene using the universal
primer set was successful for all samples, while only 17 samples
could be amplied 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 signicant
dierences between sample types (ANOVA, F value = 9.082,
Pr(>F) = 0.000123). A Tukey HSD test highlighted signicant
dierences 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 diered signicantly among them (ANOVA, F value = 5.998,
Pr(>F) = 0.00195), and Tukey’s HSD test revealed signicant
dierences between seawater samples and borehole uid (0.00323)
and between seawater samples and drill core (0.02570; Figure3A,
Supplementary Table S7). e SE-01 borehole uids displayed
a signicantly lower observed diversity than the drill cores,
but Shannon diversity was not signicantly dierent, indicating
that the evenness of the species present in these sample types
was comparable. Observed diversity and evenness were
signicantly 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
specic to each sample types, which clustered individually
(Figure 3B). e stress value equaled 0.18. is was further
conrmed by a PERMANOVA analysis showing that sample
types diered signicantly [Pr(>F) = 0.001, r2 = 274], and a multilevel
pairwise comparison showed signicant dierences 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 dierent
sample type. e cluster of SE-01 borehole uid samples appears
wider than the drill core cluster, indicating more dierences in
microbial community (Figure 3B).
Community Composition
At the phylum level, some taxa were common across the four
sample types (Figure3C). 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 Chloroexi (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
FIGURE2 | 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-specic 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
Inuence of Depth and Temperature
e bacterial sequences from the drill core samples are classied
into 35 phyla, with the ten phyla Proteobacteria, Bacteroidota,
Actinobacteriota, Firmicutes, Cyanobacteria, Deinococcota,
Planctomycetota, Verrucomicrobiota, Acidobacteriota, and
Chloroexi, comprising more than 95% of the sequences
(Figure4). e 29 ASVs classied 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 inow 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 seaoor shows the highest abundance of the genus
ermococcus (Figure 4).
Canonical correspondence analysis (CCA) ordination
(ANOVA, p = 0.018) and envt analyses demonstrate that both
depth and in situ temperature are signicantly correlated with
microbial community structure of the drill core samples
(Supplementary Figure S6). However, it is unclear which
A
CD
B
FIGURE3 | 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 inuence on the microbial community
structure, since both are linked. Observed and Shannon diversity
indices showed no signicant dierence 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 inow
(157–181 m b.s.; 37–84°C in 2017; n = 5, C52, C55, C65,
C62, C59).
Differential Abundance Analyses
To identify dierences in ASVs abundance between the four
sample types (F, fumarole; BF, borehole uid; DC, drill core;
SW, seawater samples), we performed dierential 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
identication of dierentially abundant taxa between the dierent
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 signicantly represented using the universal primer data
set (grouping into 59 taxonomic bins) and the archaeal primer
sets (7 taxonomic bins), respectively (Figure5), 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 signicantly underrepresented in the
drill cores compared to the seawater samples. Although the
latter genera are generally detected in seawater, they were
FIGURE4 | 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
signicantly overrepresented in the borehole uids collected
before drilling, indicating that marine microorganisms (or their
DNA) naturally inltrate 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 Ammonicaceae, 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 (Figure5). No
ASVs were signicantly 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 signicantly 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 inow (DC_4; 157–181 m
b.s.; 37–84°C in 2017). ese samples shared signicant 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 inltration of marine microorganisms
associated with the inltration 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, diering
in depth (C4, C9, C22, C49, C55, and C65).
All six lapilli tu samples are porous with high water
absorption (Jackson etal., 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, wefocus 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 etal., 2017), perhaps representing a “relic” of past biolm
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 dierent
from the EPS-like structures (Figure 6B). ey occur as nets
of oriented bers with ramications; some seem to beattached
FIGURE5 | Relative abundance of taxonomic bins from all signicant ASVs
identied 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 inow (n = 5).
Bergsten et al. Subsurface Microbial Communities of the Surtsey Volcano
Frontiers in Microbiology | www.frontiersin.org 13 September 2021 | Volume 12 | Article 728977
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 (Figure6F). eir wrinkled appearance could
beexplained 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 biolm.
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
oers 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 aer the eruptions
ended. Communities attached to the lapilli tu of drill core
samples are signicantly dierent from communities in
associated uids (Figures 3A,B). Similar observations have
previously been reported in crustal environments (Ramírez
etal., 2019). e Surtsey data suggest that fumaroles, borehole
uids, drill cores, and seawater samples harbor signicantly
distinct microbial communities (Figures3B,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.; Figure2B). is suggests that some species could disperse
through the subaerial tu cone above sea level up to the
surface through fumarole activity (Tin etal., 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
inow (sample C52, 148 m b.s., Figure2B) and to the samples
in proximity to the seaoor (sample C65, 181 m b.s.,
Figure 2B). is suggests that microorganisms from the
seawater surrounding the island (i) inltrate the submarine
tephra deposits, following the seawater inow at about 148 m
b.s (Jakobsson and Moore, 1986; Jackson etal., 2019b; Kleine
et al., 2020), and (ii) occupy the deeper zone of poorly
consolidated tu through indirect inltration of the seawater
and its circulation via the seaoor. 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 etal., 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 inltrates 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), peruorocarbon tracers (PFT;
Lever et al., 2006; Inagaki et al., 2016; Orcutt et al., 2017),
and microsphere tracers (Kallmeyer et al., 2006; Yanagawa
etal., 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
beperformed 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 dicult. 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 eective
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 etal., 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.g−1 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 etal., 2012;
Ciobanu et al., 2014; Mcmahon and Parnell, 2014), but not
signicantly. 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
amplication 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 inuence the sequencing results by aecting 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 benecial for the study (Pollock etal., 2018). Weemphasize
FIGURE6 | 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 beattached 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
Frontiers in Microbiology | www.frontiersin.org 15 September 2021 | Volume 12 | Article 728977
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 identied 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 aecting 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 inuence the results and can
potentially be are subjected to inated false discovery rates
(Gloor etal., 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 identied in hot springs,
hydrothermal vents, and subsurface environments (Figure5).
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 etal., 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
etal., 2018; Watanabe etal., 2018). Other sequences belonging
to the class Desulfotomaculia are assigned to Ammonifexales
and Ammonicaceae; 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 identied as
RBG-16-55-12 in the SILVA database release 138 has been
previously detected in serpentinite-hosted systems (Merino
etal., 2020). Uncharacterized ermoplasmata are also detected
in serpentinite subsurface deposits (Motamedi etal., 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 beendemic 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 inltration of seawater transports marine
microorganisms into the subsurface and its hydrothermal
system. ese species are also frequently detected in cool
basaltic oceanic crust (Templeton etal., 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 inuence 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 bedivided 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 etal., 2002; Takai etal.,
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 etal., 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 (Figure5). 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 etal., 2008, 2009), the Arctic spreading
ridges (Lysnes etal., 2004), altered basalts from the Hawaiian
Loihi Seamount (Templeton etal., 2005; Santelli etal., 2008;
Jacobson Meyers et al., 2014), and the Mid-Atlantic Ridge
(Rathsack etal., 2009; Mason etal., 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 identied 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 identied 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.
FIGURE7 | 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 bediscussed with some degree of certainty,
while remaining mindful of the diculties inherent to
identications 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 etal., 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 etal., 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 etal., 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
befound 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 Scientic 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 befound
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
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