Colonization of life on Surtsey has been observed systematically since the
formation of the island 50 years ago. Although the first colonisers were
prokaryotes, such as bacteria and blue–green algae, most studies have been
focused on the settlement of plants and animals but less on microbial
succession. To explore microbial colonization in diverse soils and the
influence of associated vegetation and birds on numbers of environmental
bacteria, we collected 45 samples from different soil types on the surface
of the island. Total viable bacterial counts were performed with the plate
count method
at 22, 30 and 37
Microorganisms are typically in great abundance and high diversity in common soil and their integrated activity drives nutrient cycling on the ecosystem scale. Organic matter (OM) inputs from plant production support microbial heterotrophic soil communities that also drive processes that make nutrients available in the system. This, in turn, supports plant primary productivity and basic food webs on the ground and in the subsurface (Fenchel et al., 2012; Roesch et al., 2007; Schlesinger, 1997; Whitman et al., 1998). Moreover, as soil develops, soil geochemistry and OM availability changes (Vitousek and Farrington, 1997) due to mineral-OM interactions and geochemical constraints on biological activity (Kleber et al., 2007; Sinsabaugh et al., 2008). Nutrient limitations can constrain plant and food web development, thus shaping the rate of succession of plant and animal life within the ecosystem (Odum, 1969; Walker and del Moral, 2003).
Subsequent to volcanic eruption, lava flow and ash deposition, new surfaces are created where both organismal growth and weathering processes are effectively reset. Microbial cells colonizing new volcanic deposits must be successful in either growing autotrophically, by fixing carbon (C) and N using light or inorganic energy sources for growth, e.g. Cyanobacteria and sulfate-reducing bacteria (Edwards et al., 2003; Ernst, 1908; Konhauser et al., 2002), using carbon monoxide as a C and energy source (Dunfield and King, 2004; King and Weber, 2008) or by growing heterotrophically using trace amounts of organic carbon (Cockell et al., 2009; Wu et al., 2007). Studies on the microbiota of volcanic terrains have only emerged within the past few years, revealing that such habitats are capable of harbouring significant microbial diversity, despite their extreme nature (Gomez-Alvarez et al., 2007; Kelly et al., 2010). However, completely isolated volcanic terrains, such as islands, are extremely rare. One of few such places is Surtsey, a neo-volcanic island, created by a series of volcanic eruption that started in 1963 and ended in 1967 (Þórarinsson, 1965, 1967, 1968). The eruption was thoroughly documented from the first plume of ash until the end of the lava flow in June 1967. In 1979 a 181 m deep hole was drilled to investigate the substructure of the volcano as well as the nature of the hydrothermal system (Jakobsson and Moore, 1979). Consequently, with its drill hole the island of Surtsey provides a unique laboratory for the investigation of biological establishment and succession on relatively newly deposited volcanic substrata, on the surface and in the subsurface. The first reports of life forms in Surtsey were from 1964 to 1966 (Brock, 1966; Friðriksson, 1965), when the first cyanobacteria were observed, even before the end of the eruption. Phototrophs were further investigated in 1968 (Schwabe, 1970) and in subsequent investigations in following years (Brock, 1973; Schwabe and Behre, 1972). However, despite such a remarkable habitat, very little research on microbiology has been performed since the first years of the island formation despite frequent research expeditions; the most recent report on microbes in Surtsey is only from the end of last century (Frederiksen et al., 2000). Additionally, no reports or data exist on heterotrophic growth or distribution of such bacteria in the surface soils of the island, and nothing is known about distribution of faecal bacteria or pathogens possibly brought by bird inputs of organic matter, such as faeces. Additionally, even less is known about the island's subsurface life although such life is well known in subseafloor sediments and within the deep biosphere where high number of microbes are present and active (Kallmeyer et al., 2012).
The overall aim of this study was to explore microbial colonization in different surface soil types and in the subsurface below 160 m depth in a drill hole in Surtsey. That was done by obtaining viable count and distribution of heterotrophic microbes on the island surface and by obtaining the correlation of nutrients and other environmental measurements to different soil types, and determining how that affects microbial communities in Surtsey. We also accomplished this by investigating the presence, survival and possible dissemination routes of pathogenic bacteria into such pristine environments. Finally, the existence and diversity of subsurface microbial biosphere and their possible dissemination routes was investigated.
Samples were collected during a sample expedition at Surtsey in July 2009. An about 1.0 cm thick layer was retrieved inside a frame of
The basic methodology used at the laboratory for media and culturing were NMKL methods (Nordisk Metodikkomité for Næringsmidler) and methods from the Compendium of Methods for the Microbiological Examination of Foods published by the American Public Health Association (APHA-2001). About 25 g of each surface sample was weighed and 225 mL Peptone water was mixed in before using a stomacher for blending the soil for 1 min. The supernatant of each sample was used and analysed with different methods.
An overview of the sampling site. The sites are marked with green squares for sand or pumice with bird droppings samples (SS), pink circles for pure sand or pumice and vegetated samples (SJ), yellow triangles for partly vegetated and non-vegetated area samples (SR) collected inside squares for activity measurements and purple circle for the drill hole site (SE). The sampling sites are distributed according to GPS points.
The conventional “pour-plate” method was used on plate count agar. Briefly,
1 mL of homogenate sample was used with 20 mL melted and cooled culture
medium. Incubation temperatures were at 22 and 30
Pictures of the sample types. Samples were divided into three
types: SJ samples (
A reference method based on most probable number (MPN) from NMKL (NMKL 96,
4th ed., 2009, Compendium 4th ed., 2001, chapter 8 (8.71, 8.72,
8.81)) was used to estimate total coliforms, faecal coliforms and
A reference method from NMKL (reference: NMKL 144, 3rd ed., 2005) was used
to estimate total Enterobacteriaceae in all 44 surface samples. The medium violet red bile glucose agar (VRBGA) was used (pour-plate method with overlay). Plates were
incubated for 24 h at 37
Reference methods from NMKL was used to estimate total number of pathogens
or for
The following NMKL method (NMKL 119, 3rd ed., 2007) was used for
The following NMKL method (reference: NMKL 136, 5th ed., 2010) was used for
The total amount of nitrogen (totN%) was measured on a nitrogen analyser
(Foss Tecator Kjeltec 2400 Analyzer Unit). About 3 g of soil was analysed at
420
The measurement of net ecosystem exchange (NEE,
Multivariate analysis was performed on the environmental parameters
collected in order to visualize environmental similarities between sample
sites. The parameters were temperature, total carbon, total nitrogen, water
content, total microbial count of PCA and counts of Enterobacteriaceae. Samples containing
missing values were excluded in the analysis except in six occasions were
total nitrogen values were not available. In these cases, the values were
estimated based on other similar samples in the data set. The other option
would have been to exclude these samples from the analysis. Data were
normalized with ln(
The subsurface was sampled through continuously cored drill hole SE-1
(Moore, 1982; Ólafsson and Jakobsson, 2009). The temperature
was measured along the drill hole at 1 m intervals from the surface down to
the bottom at 180 m with a borehole temperature meter. A temperature logger
(DST milli-PU logger from StarOddi, Reykjavík, Iceland) was placed for
approximately 21 h at 168 m depth in the borehole and the temperature
was recorded every 15 min with SeaStar software. Samples were collected
in an in-house created downhole water sampler made of stainless steel. The total
capacity of the sampler is about 1.3 L that was kept open (flow through) to
the sampling depth and closed with a messenger. Contamination of samples
were avoided by washing the sampler with several equivalent volumes of 70 %
ethanol before operation. Samples SB1, 2, 4, 5 and 6 were retrieved from
57, 58, 145, 168 and 170 m, respectively. Samples SB4, 5 and 6 were sampled
below the sea level (58 m). Samples were reduced by Na
Media for enrichment of chemolithotrophic and chemoorganotrophic organisms
were prepared by using 0.5 mL sample and 4.5 mL 0.2
To capture microbial cells for DNA extraction and analysis, 250 mL of sample
was filtered through a 47 mm, 0.22
Polymerase chain reaction (PCR) amplification was performed according to the protocol in Skírnisdóttir et al. (2001) with primers 9F (“5-GAGTTTGATCCTGGCTCAG-3”) and 805R (“5-GACTACCAGGGTATCTAATCC-3”) (Skírnisdóttir et al., 2001). PCR product was cloned by the TA method using a TOPO TA cloning kit (Invitrogen). Plasmid DNA from single colonies was isolated and sequenced using a BigDye Terminator Cycle Sequencing Ready Reaction Kit on an ABI sequencer (PE Applied Biosystems). Clones were sequenced using the reverse primer 805R. Cloned sequences were analysed and edited by using the program Sequencer 4.8 from ABI. A total of 41 clone sequences were grouped into operational taxonomic units (OTUs) at a threshold of 98 % sequence identity and then aligned by using ClustalW within the MEGA package, version 5.1 (Thompson et al., 1994). In order to check for species identification, sequences were searched against those deposited in GenBank, through the NCBI BLAST (Altschul et al., 1990). Neighbour-joining phylogenetic tree was constructed with MEGA 5.1 (Tamura et al., 2011) using a representative sequence from each OTU and related GenBank sequences.
Two sets of reactions targeting the v4–v6 regions of the archeal 16S rRNA
gene were performed using the VAMPS primers (Sogin et
al., 2006). First, pyrosequencing of short reads, 70–100 nt of the archeal
v6 variable region (primers 958F and 1048R; “5-AATTGGANTCAACGCCGG-3” and “5-CGRCGGCCATGCACCWC-3”) in the
16S ribosomal gene was performed with a 454 GS-FLX (Roche) on sample SB4.
Cycling conditions included an initial denaturation at 94
Total bacterial counts with the plate count agar method at
22
At total of 44 surface samples were collected around the island. An overview of the sampling site is shown in Fig. 1. Most of the samples were collected on the southern side of the island where the soil was highly variable ranging from sand to completely vegetated environment with significant interactive effects of bird association including nesting seabirds.
A good visual correlation was found between total bacterial counts with the
plate count agar method and growth on R2A media from all samples incubated
at 22
A total of 12 soil samples that showed significantly high numbers of
environmental bacteria or > 1
Soil nitrogen, carbon and moisture measurements were performed for all samples with sufficient soil quantity for analysis. Measurements of total nitrogen, carbon and water content was performed in 37 samples except in 6 samples that lack totN% measurements. Seven samples could not be measured (Table 1). Average totN% measurements were similar in SJ and SR samples, 0.01 and 0.02, respectively, but SS samples containing bird droppings were at least 60 times higher at 0.68. Average totC% was also highest in SS samples at 4.68 and SJ and SR samples were 1.17 and 2.74, respectively. Average water content in SJ, SR and SS samples were 0.34, 0.91 and 0.61, respectively.
Non-metric multidimensional scaling, Euclidean distances. Environmental parameters included in analysis: temperature, total carbon, total nitrogen, water content, total microbial count of PCA and counts of Enterobacteriaceae. SS samples (3–10) in light green (sand or pumice with bird droppings), SJ samples (1–7 and 10–15) in dark green (pure sand or pumice) and SR (1–12) samples in red (partly vegetated and non-vegetated area) and SR (15–17) in purple (vegetated).
Viable count of total coliforms, faecal coliforms,
nd: not determined,
CFU: colony-forming unit,
MPN: most probable number,
totN (% of dw): percentage of nitrogen,
totC (% of dw): percentage of carbon,
GWC: soil gravimetric water content,
In order to capture the niche similarities between sampling sites,
multivariate NMDS analysis was performed based on measurements of environmental
parameters. The analysis showed that the SS samples are separated from other
samples while the SR and SJ samples overlap. Samples SR-15–17 are well
separated from all other samples which is due to their higher load of
Enterobacteriaceae, total viable counts and higher water content compared to other sampling
sites (Fig. 4). For selected samples, more environmental data were recorded
(NEE, Re., GPP, PAR, Ts05, Ts10, Cov.) and were used as a base for another
sub-NMDS analysis which confirmed previous analysis and clustered the most
vegetated samples together (data not shown). Table 2 shows the CO
CO
The temperature was measured along the drill hole at 1 m intervals from the
surface down to the bottom at 178 m with a borehole temperature meter. The
temperature measurements are shown in Fig. 5 in relation to the depth
in the drill hole. The maximum temperature was 130
No growth could be observed after about 6 weeks of incubation in any of the
enrichments incubated at 40, 60 and 80
Temperatures at 5 m intervals along the drill hole, from the surface down to the bottom at 178 m. The circles show the depth and temperature of the SB samples.
Very small pellets of undetermined biomass were obtained from all SB samples, and DNA concentration was extremely low. PCR amplification products were achieved from SB4, SB5 and SB6 with both universal bacterial and archaeal primers. Library construction was successful with clones containing bacterial 16S rRNA genes that were amplified in samples SB5 and SB6 and with archaeal genes in sample SB6.
Three approaches were used to assess the bacterial and archeal taxa composition
in the samples: partial sequencing of cloned 16S rRNA fragments,
pyrosequencing of short fragment of the v6 region and pyrosequencing of a
longer fragment of v4–v6 region. Clone libraries of the 40 archaeal 16S rRNA
genes (500 bp) in sample SB6 showed high homology (99 %) to uncultured
subsurface archaea-related sequences (Genbank accession DQ354739.1) from
subsurface water of the Kalahari Shield, South Africa by BLAST method. All
the clones were dominated by this one sequence except two clones which
showed high homology to uncultured subsurface archaea-related sequences,
DQ988142 and AB301979.1, from methane cycling in subsurface marine sediments
and from hydrothermal sediments at the Yonaguni Knoll IV hydrothermal
field in the southern Okinawa Trough, respectively. Clone libraries of the
bacterial 16S rRNA genes in sample SB5 and SB6 and their closest known
relatives are presented in neighbour-joining tree of sequences which is
shown in Fig. 6. The SB4 v6 library consists mostly or 94.5 % of a
single taxon affiliated with genus
Neighbour-joining tree of sequences from the 16S rRNA clone libraries, databases showing phylogenetic relationships. The scale bar represents the expected percentage of substitutions per nucleotide position, and a marine Crenarchaeon was used as outgroup. The cluster in uncultured delta Proteobacterium clone ANOX-077 represents 11 clones with 99 % sequence similarity (5 SB6 and 10 SB5 from the borehole). The cluster in uncultured bacterium clone MD08f7 11 clones with 99 % sequence similarity (7 SB6 and 5 SB5 from the borehole).
Sequencing results of the 16S rRNA gene with a next-generation
sequencing method.
Before sampling, surface samples were classified into three types
according to their visual appearance in the field: pumice soil with bird
droppings (10 SS samples), pure pumice soil (15 SJ samples) and mixed
(19 SR samples). The SR samples were soil that were totally or partly
vegetated or pure pumice. They were all collected inside of a defined area
used for activity measurements of soil (Magnússon et
al., 2014; Sigurdsson and Magnússon, 2010). Ecosystem respiration (Re) was
measured inside these zones in order to investigate soil properties and
surface cover of vascular plants. These zones were distributed among the
juvenile communities of the island, inside and outside a seagull colony
established on the island (Sigurdsson, 2009). As shown with an overview
of the sampling sites on the island of Surtsey (Fig. 1), most of the surface
samples were collected on the southern side of the island, in the same area as
seagull (
Interestingly, the results in this study showed relatively little variance
among soil types. The controls or pure pumice samples showed little growth
by any culturing method tested as expected but soils with some vegetation
and bird droppings revealed also low cell counts apart from aerobic bacteria
growing at 30
Our classification of sample types by using multivariate NMDS analysis based on our results is in agreement with the visual classification of sample types to a certain extent. The analysis showed that all the SS samples were clearly separated from the other samples while the other two types of soil samples, SR and SJ, were gathered into one big group that could be divided into two smaller sub groups and one small group completely separate. This unique group (SR) contained samples that were highly vegetated. The vegetated samples, i.e. SR-14 to SR-19, were distinct and different from all other samples due to a higher load of Enterobacteriaceae, total viable counts, higher percentage of carbon, nitrogen and water content compared to other sampling sites (Fig. 4, Table 1). Moreover, by taking into account data only from samples (all SR samples) collected for ecosystem respiration (Re), they could be divided mainly into two groups reflecting the soil properties or vegetation, inside and outside the seagull colony. SR-16, 15, and 19 were clustered inside the main seagull colony on the southern part of the island were SR-7, 11, 5 and 9 are clustered just beside the main seagull colony or south-east part of the island, while the two most dissimilar samples SR-3 and SR-1 were collected far away from the seagull colony, on the northern part of the island.
Access to the deep biosphere in a remote neo-volcanic island is extremely
unique. We were able for the first time to collect hot subsurface samples
deep in the centre of a volcanic island, created by a series of volcanic
eruption only 42 years after the eruption break. Additionally, as reported for
geothermal boreholes in Reykjavík, the surface of the drill hole in
Surtsey can be regarded as a window to the deep subsurface biosphere of the
island (Marteinsson et al., 2001a). This window has been
open for 30 years before our sampling in 2009 as the borehole was finished
in August 1979 (Jakobsson and Moore, 1982). The purpose of the drill
hole was to obtain a core for studying the structure of the island and the
hydrothermal alteration of the tephra formed during the Surtsey eruption
(Ólafsson and Jakobsson, 2009). The drill site is located on the
edge of the Surtur tephra crater at 58 m above sea level with a total depth
of 181 m. Several temperature measurements have been taken along the depth
of the drill hole since the drilling and it appears that the hole has cooled
since 1980 (Ólafsson and Jakobsson, 2009). Our temperature
measurements along the drill hole at 1 m intervals from the surface down to
the bottom at 180 m showed drastic temperature changes compared to
previous measurements. Our highest temperature measurement was
126.5
We have explored for the first time microbial colonization in diverse
surface soils and the influence of associate vegetation and birds on viable
counts of environmental bacteria at the surface of Surtsey. The number
of faecal bacteria correlated to the higher total number of environmental
bacteria and type of soil but no pathogenic microbes were detected in any
sample tested. We were able for the first time to collect hot subsurface
samples deep in the centre of this volcanic island and record the
temperature for 21 h at 168 m depth. Both uncultivated bacteria and
archaea were found in the subsurface samples collected below 145 m. The
microbial community at 54
The authors thank Á. R. Rúnarsson and S. Magnússun for their technical contribution, and the scientific team of the Surtsey expedition in 2009, especially B. Magnússon and E. S. Hansen. We would also like to thank S. Jakobsson for helpful discussions and Náttúrufræðistofnun Íslands for the topographic map of Surtsey. Special thanks are to S. Guðbjörnsson for providing a temperature logger from Star Oddi. Many thanks are also due to the Matís staff working in the division of analysis and consulting. This work was partly financed by preliminary grant from The Icelandic Centre for Research (RANNIS) and the European Union program MaCuMBA (grant agreement 311945). Edited by: B. Magnússon