RESEARCH ARTICLE
Evidence and characteristics of a diverse and metabolically
active microbial community in deep subsurface clay
borehole water
Katinka Wouters, Hugo Moors, Patrick Boven & Natalie Leys
Expert Group for Molecular and Cellular Biology, Institute of Environment, Health and Safety, Belgian Nuclear Research Centre SCK•CEN, Mol,
Belgium
Correspondence: Natalie Leys, Expert group
for Molecular and Cellular Biology, Institute
of Environment, Health and Safety, Belgian
Nuclear Research Centre SCK•CEN,
Boeretang 200, 2400 Mol, Belgium.
Tel.: +32 14 33 27 26;
fax: +32 14 33 35 31;
e-mail: [email protected]
Received 6 March 2013; revised 14 June
2013; accepted 20 June 2013.
Final version published online 29 July 2013.
DOI: 10.1111/1574-6941.12171
MICROBIOLOGY ECOLOGY
Editor: Tillmann Lueders
Keywords
geomicrobiology; Boom Clay; metagenomics;
ATP; cultivation; borehole water.
Abstract
The Boom Clay in Belgium is investigated in the context of geological nuclear
waste disposal, making use of the High Activity Disposal Experimental Site
(HADES) underground research facility. This facility, located in the Boom Clay
at a depth of 225 m below the surface, offers a unique access to a microbial
community in an environment, of which all geological and geochemical characteristics are being thoroughly studied. This study presents the first elaborate
description of a microbial community in water samples retrieved from a Boom
Clay piezometer (borehole water). Using an integrated approach of microscopy,
metagenomics, activity screening and cultivation, the presence and activity of
this community are disclosed. Despite the presumed low-energy environment,
microscopy and molecular analyses show a large bacterial diversity and richness, tending to correlate positively with the organic matter content of the
environment. Among 10 borehole water samples, a core bacterial community
comprising seven bacterial phyla is defined, including both aerobic and anaerobic genera with a range of metabolic preferences. In addition, a corresponding
large fraction of this community is found cultivable and active. In conclusion,
this study shows the possibility of a microbial community of relative complexity
to persist in subsurface Boom Clay borehole water.
Introduction
Several countries are investigating the possibilities for
long-term geological disposal of radioactive waste in geological clay layers, granite rock or salt formations (StroesGascoyne & West, 1997; Pedersen, 1999; Fredrickson
et al., 2004; Wang & Francis, 2005; Itavaara et al., 2011).
In Belgium, the Boom Clay at 185–287 m depth beneath
the SCK•CEN facilities in Mol is investigated for this purpose. Boom Clay is a marine siliciclastic sediment deposited during the Rupelian period (c. 30 million years ago),
dominated by quartz and clay minerals. The HADES
underground facility of SCK•CEN in this clay layer not
only is an indispensable facility for nuclear waste disposal
investigations as such, but also provides unique access to
this ancient conserved ecological niche. Petrophysical and
hydraulic parameters of Boom Clay were characterized
earlier, as well as mineralogical and geochemical properties
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
(Baekelandt et al., 2001). Although Boom Clay is composed of a heterogeneous accumulation of strata with
different concentrations of ions, carbon etc., a reference
composition of Boom Clay pore water has been derived
earlier by modelling and spatial calibration efforts. This
reference composition was mainly based on borehole water
sampled from different layers within Boom Clay (De Craen
et al., 2004).
Beside the apparent physical and chemical challenges of
such long-term geological waste disposal, biogeochemical
activity of indigenous and introduced microorganisms
could compromise disposal safety (Stroes-Gascoyne &
West, 1997; Haveman & Pedersen, 2002; Fredrickson et al.,
2004; Horn et al., 2004; Wang & Francis, 2005; Fredrickson
& Balkwill, 2006; Nedelkova et al., 2007; Stroes-Gascoyne
et al., 2007; Meleshyn, 2011). First, the allochthonous
material used for waste disposal (metal, concrete) can
become deteriorated by microbial activity, especially in the
FEMS Microbiol Ecol 86 (2013) 458–473
459
Diverse and active deep subsurface microbial community
present reducing conditions. The material can become corroded, cracked or affected by production of gaseous microbial metabolites. Second, the geochemistry of the clay can
be influenced by the introduced or reactivated microbial
activity. The speciation, migration and transport of radionuclides can be affected by microbiologically induced
changes in their dissolution and complexion chemistry.
Assessment of the microbial population and its activity in
such geological formations selected for possible long-term
waste disposal is therefore of utmost relevance for future
radioactive waste management.
Despite some preliminary previous efforts, clay-associated microbial communities at depth still remain largely
unexplored (Boivin-Jahns et al., 1996; Mauclaire et al.,
2007; Stroes-Gascoyne et al., 2011). Most reviews and studies on the deep subsurface do not address clay environments (Horn et al., 2004; Wang & Francis, 2005;
Fredrickson & Balkwill, 2006; Gadd, 2007, 2010; Colwell &
D’Hondt, 2013). Nowadays, microbiologists are able to
explore subsurface ecosystems to a greater detail by combining traditional microbiology tools such as cultivation
with newer molecular tools in the popular field of metagenomics. The challenge at this point is not merely to detect
microbial life in the deep subsurface, but to find out how
microbial communities are able to persist in these environments with only very limited access to energy sources.
Indeed, Boom Clay is considered a low-energy environment with small pore sizes (average < 60 nm) and low
hydraulic conductivity (Baekelandt et al., 2001), limiting
active or passive transport of cells and nutrients.
In this study, the diversity and current metabolic activity
of a microbial community in Boom Clay borehole water
were comprehensively described. The primary aim of this
microbiological study was to sample and characterize these
communities in borehole water in order to determine a
common and dominant core bacterial community (CBC),
despite the Boom Clay heterogeneity. As such CBC would
find applications in laboratory experimental set-ups, this
aim included the indication of preferred sites to sample
borehole water to serve as CBC model inoculum. Second,
the in situ activity and some general metabolic pathways of
members of these communities were addressed to assess
their survival and proliferation rates. These analyses are
preparatory for further in-depth research on the community’s metabolic network and on the expected fate of the
microbial community under nuclear waste repository
conditions. The experimental strategy to address both aims
included sampling of the borehole water collected from
different Boom Clay stratigraphic layers independently.
An integrated approach, using molecular and cultivationbased analyses, was applied to assess the abundance, diversity, activity and metabolic properties of the bacterial
communities present.
FEMS Microbiol Ecol 86 (2013) 458–473
Materials and methods
Study site and sample collection
The Boom Clay formation was accessed through the underground research facility HADES of SCK•CEN (Mol, Belgium), which is a sophisticated concrete-lined gallery at a
depth of 225 m below the surface. Borehole water was collected from different clay layers using a vertical piezometer
named TD-11E (MORPHEUS) (Fig. 1). This piezometer
was installed in May 2001 to study the variability of the
Boom Clay pore water chemical composition, allowing
sampling from 12 filters at 12 distinct stratigraphic layers
of the Boom Clay (De Craen et al., 2004). In this study, 10
of the available filters were sampled (Fig. 1, Table 1). Relevant geochemical characteristics of the piezometer water
[total organic carbon (TOC), inorganic carbon and alkalinity] were added in Table 1, as analysed according to De
Craen et al. (2004). Materials used in the construction of
this piezometer are SchumathermTM filters (PALL, 60 lm
pore size) connected to the surface by PVC tubes, nylon
(a)
(b)
(c)
(d)
–213.25 m
–218.25 m
–223.25 m
–228.25 m
F23
F20
F18
F12
F10
F9
F8
F6
F4
F2
–233.25 m
1m
Fig. 1. Schematic view of the vertical piezometer Morpheus sampling
Boom Clay borehole water, indicating depth below sealevel (a), Boom
Clay stratigraphic layers (b) (white = clayey, grey = silty, black =
including septaria, very silty), piezometer filters, as indicated by Fxx (c)
and corresponding samples in septum bottles (d), as indicated by
arrows.
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
460
W. Katinka et al.
Table 1. General properties of 10 Boom Clay borehole water samples
Depth (m)*
F2
F4
F6
F8
F9
F10
F12
F18
F20
F23
235.23
233.83
231.83
230.28
229.80
229.13
227.83
222.63
220.83
217.13
Q (mL day 1)
d
OD600
Flocs†
TOC (mg C L 1)
IC (mg C L 1)
Alkalinity (mg L 1)
54
59
59
386
66
49
81
67
57
65
13
9
9
1.5
8
13
8
9
9
8
0.025
0.046
0.093
0.163
0.116
0.084
0.054
0.057
0.049
0.117
No
Small, transparent
Small, transparent
Small and large, transparent
Small and large, brown
Small, transparent
No
No
No
Small, transparent
101.4
93.58
81.05
121.9
87.09
80.02
69.26
90.93
75.12
87.74
166.1
161.4
157.8
209.9
170.8
156.1
177.3
162.2
158.7
165.2
13.95
13.14
13.06
17.92
14.62
13.45
14.93
13.29
12.68
13.67
Fxx, Morpheus TD-11E borehole water sample; Q, discharge of borehole water; d, number of days until a sample volume of 500 mL; OD600,
optical density at a wavelength of 600 nm; IC, inorganic carbon.
*Depth below sea level (metres).
†
Presence and appearance of flocs; < 2 mm estimated diameter = small, ≥ 2 mm estimated diameter = large.
tubes and Teflon-coated stainless steel sample cylinders.
The piezometer set-up aims at limiting disturbance of the
geochemical properties of the Boom Clay surrounding the
piezometers and its microbial community, to collect representative and unmixed water samples. Prior to sampling,
the piezometer filters were allowed to discharge an amount
of c. 500 mL, in minimum 4 days (F8) to maximum
21 days (F10), with differences in flow rates being ascribed
to the Boom Clay heterogeneity. After this flushing of the
system with original borehole water, the sample cylinders
were cleaned by rinsing three times thoroughly with deionized water and sterilized by autoclaving (20 min at 2.1 bar,
121 °C). After repeating this rinsing and sterilization once
more, the cylinders were flushed for 30 min with argon
gas, passing through a 0.22-lm filter, to ensure not only a
sterile but also an anaerobic sampling environment. Subsequently, a sampling campaign was arranged to ensure the
aseptic collection of a 500-mL sample of each filter, at the
same end point despite the different natural discharge rates
of the filters. This was accomplished by connecting the
cylinders to the system simultaneously, but opening the
sampling valves at different times. The depth of origin,
discharge and sampling time of each sample are indicated
in Table 1. An extra cylinder was used as a negative control
of the current sampling campaign, passing through all the
above steps. It was filled with autoclaved, deionized water
and subsequently connected to one of the failing filter outlets (F15), however, without opening the sampling valve.
The filled cylinders were brought to the aboveground laboratory and were handled in an anaerobic glove box with a
manually controlled atmosphere of c. 99% argon and 1%
hydrogen (Ar/H 99/1). Each sample was divided into five
100-mL aliquots for immediate analysis or storage, with
the headspace being the glove box atmosphere (Ar/H 99/
1). At that time, sample colour, turbidity (OD600 nm) and
the absence/presence of flocs were noted for each sample
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
(Table 1). Of each sample, five aliquots of 10 mL were centrifuged (12 000 g, 10 min) to collect the cell pellet, followed by storage at 20 °C to await DNA extraction.
Long-term storage was performed at a temperature of
4 °C, which is lower than the average Boom Clay temperature of c. 17 °C, in an effort to reduce microbial activity
and stop ongoing biochemical processes. One aliquot of
each sample was stored at ambient temperature for a longer
period, to be used for SEM and follow-up of ATP only.
Scanning electron microscopy
Of each sample, including the sampling blank, two aliquots
of 500 lL were used for imaging by scanning electron
microscopy (SEM). One series of samples was prepared for
SEM as such, while the other series was filtered over
1.2 lm first to remove large particles and cell aggregates.
Preparation for SEM comprised a concentration, fixation,
dehydration, drying and coating step, as described in
Supporting Information. SEM analysis was performed on a
JEOL JSM-840 (Jeol Ltd) equipped with a secondary electron
and backscatter electron detector (point electronic GmbH)
at a working distance of 20 mm and a 9-kV acceleration.
On a selection of four images per filtered sample, an
estimate of the total area covered with (apparent) biomass
was calculated using IMAGEJ software, to allow a first, rough
comparison of the biological contents between samples.
DNA extraction
Three successive rounds of DNA extraction were performed on the collected and frozen cell pellets, yielding
triplicates for each sample, following a customized protocol
after Tillett & Neilan (2000) and Leuko et al. (2008), followed by a purification step. The complete protocol is
described in Supporting Information. Total DNA yields
FEMS Microbiol Ecol 86 (2013) 458–473
461
Diverse and active deep subsurface microbial community
ranged from 61 to 977 ng per mL of original sample. This
high diversity in yields was reproducible throughout the
three successive extractions and is therefore believed not to
be the result of random errors in the efficiency of our customized DNA extraction protocol. The triplicate extractions were pooled for each sample, to minimize extraction
bias in further community analysis (Feinstein et al., 2009).
It was reported that genome sizes of specialist microorganisms are on average 2 Mb, while generalist genomes are
bigger, ranging around 5 Mb (De Bruijn et al., 1998).
Assuming the microbial community in Boom Clay is a
mixture of specialists and generalists, an average genome
size of 3.5 MB was chosen, providing rough estimations
about the amount of cells DNA was extracted from. The
DNA extraction yields were quite variable between samples,
but variability was not related to any of the metadata (e.g.
TOC concentration) (data not shown).
Polymerase chain reaction
DNA was amplified by polymerase chain reaction (PCR)
using universal primers for the bacterial genes coding for
16S rRNA, either yielding 918-bp amplicons for further
automated capillary electrophoresis sequencing (Amann
et al., 1995; Muyzer et al., 1995), 507-bp amplicons for further 454 sequencing (Wu et al., 2010) or 455-bp amplicons
holding a GC clamp at the 5′ end for further denaturing
gradient gel electrophoresis (DGGE; Muyzer et al., 1993;
Marchesi et al., 1998; Klammer et al., 2008). In addition,
PCR with primers targeting relevant metabolic genes apsA
(adenosine 5′-phosphosulphate reductase a-subunit gene,
involved in sulphate reduction; Bodelier, 2011) and nirS
(nitrite reductase gene; Braker et al., 2001) was performed
on each sample and on anaerobic enrichments (see Cultivation). All information on primers and their respective PCR
protocols are provided in Table S1 and Supporting Information.
analyses. PCR of a bacterial 16S rRNA gene fragment (V1–V3
region, 507 bp; Table S1; Wu et al., 2010) and subsequent
tag-encoded pyrosequencing were performed at DNAVision (Charleroi, Belgium). Pyrosequencing was carried out
using the forward primer on a 454 Life Sciences Genome
Sequencer FLX instrument (Roche) following titanium
chemistry. Depth of the sequencing was 10 000 sequences
per sample on average. Sequences of all six samples were
pooled and preprocessed, including trimming, denoising
and chimera removal, using a MOTHUR pipeline based on
the existing standard operational procedure of Schloss
(2009) (Supporting Information). Preprocessing of the metagenomic data was performed quite stringently, discarding
33% of the sequences, thereby assuring accurate downstream results based on the most trustworthy sequences.
Using the MOTHUR software, sequences were subsequently
clustered into operational taxonomic units (OTUs; Schloss
& Westcott, 2011) at 1% genetic distance. Of each OTU, a
representative sequence was classified by BLAST against the
GreenGenes database (DeSantis et al., 2006) at minimum
90% sequence similarity.
Sequence data analysis
PCR samples were analysed by DGGE using an INGENYphorU system (INGENY International), as described in
Supporting Information. BIONUMERICS 4.10 was used for
fingerprint analysis of the DGGE profiles. Profiles were
compared based on Jaccard similarity coefficients and
UPGMA clustering. Based on the resulting dendrogram,
six representative samples were selected for next-generation sequencing (NGS) of 16S rRNA genes.
Processed metagenomic sequences of the six selected samples were subsampled towards the lowest number of
sequences in the sample pool (being 4008 sequences in
sample F23), hereby allowing unbiased comparison of samples based on their OTUs. Of each sample, the general
sequence statistics and a-diversity indices were calculated
by Mothur, targeting (1) the number of sequences before
and after preprocessing; (2) the number of OTUs; (3) the
estimated community richness (Chao1 index; Chao, 1984);
(4) the coverage of the sequencing effort compared with
the richness [(2) relative to (3)]; (5) the diversity weighed
towards the rare species (Shannon index; Shannon, 1948);
and (6) the diversity weighed towards the abundant species
(inverse Simpson index; Simpson, 1949), based on the recommendations of Hughes et al. (2001). Within the pool of
metagenomic sequences of all six samples, the most abundant OTUs were indicated, using a threshold of minimum
100 sequences (in the entire pool of sequences). In addition, OTUs that were present in all six samples were indicated (shared sequences). Pairwise b-diversity of the
samples was calculated according to the Yue-Clayton coefficient, which is based on the relative abundance of OTUs,
and therefore reflects structure dissimilarity (Yue & Clayton,
2005).
Next-generation sequencing
ATP measurement
Based on clustering of the DGGE profiles, samples F6, F8,
F9, F18, F20 and F23 were selected for further phylogenetic
As an indicator and estimate of microbial metabolic
activity, the presence of intracellular adenosine triphosphate
Denaturing gradient gel electrophoresis
FEMS Microbiol Ecol 86 (2013) 458–473
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Published by John Wiley & Sons Ltd. All rights reserved
462
(ATP) was analysed in each sample (stored at ambient
temperature) at different time points (day 0, day 11, day
56, day 131 and day 350), using the Microbial ATP Kit
HS of Biothema (Isogen Life Science, the Netherlands;
Lundin, 2000) and a Lumitester C-100 (Kikkoman), as
indicated in Supporting Information. From the luminescence data, the amount of active cells in the sample was
estimated and presented in units of equivalent active cells
(EAC), based on the estimation that most bacterial cells
contain c. 3 mM of ATP, being 2 9 10 18 mol ATP per
average size cell (Neidhardt et al., 1996).
Cultivation
One-millilitre sample aliquots were cultured in 9 mL of
different nutrient solutions. Six liquid culturing conditions
were chosen for the assessment of different subpopulations
with distinct metabolic properties in all samples, using one
medium both anaerobically and aerobically (R2A), one
medium only aerobically (LB) and two media anaerobically
(N43, S63). An Ar/H 99/1 headspace was used in the
anaerobic conditions. LB broth (Bertani 1951) and R2A
(Gibbs & Hayes, 1988) are general complex media, allowing
heterotrophic organisms to proliferate. Mineral N43 medium (described by Heylen et al. 2006, as G4M3 medium)
allows growth of a broad range of nitrate-reducing microorganisms that are able to use succinate as carbon source and
electron donor and nitrate as preferential electron acceptor.
Mineral S63 medium is customized for sulphate-reducing
microorganisms like Desulfovibrio species (Medium 63;
DSMZ GmbH, Germany) and contains sodium lactate as
carbon and electron source, FeSO4 as electron acceptor and
sodium thioglycolate as reducing agent. The microbial
community showing growth on these media after c.
1 month of incubation was estimated by most probable
number technique (MPN) in Hungate tubes (Hungate,
1969), based on a 10-fold dilution series up to 10 9 dilution, in triplicate for each sample. MPN estimates and confidence intervals were calculated according to the method
described by Jarvis et al. (2010). After the incubation period of 1 month, one of the first dilution of each anaerobic
medium and of each sample was sacrificed for DNA
extraction and subsequent PCR of nirS and apsA genes.
The remaining tubes were incubated for two more months,
but no additional growth was observed.
Strain isolation
Of each sample, both 50 and 5 lL were spread on solid
R2A medium (agar 15 g L 1) for incubation in aerobic
conditions, and 10 lL, on slant agar surfaces of N43 and
S63 medium in Hungate tubes with Ar/H 99/1 headspace
for incubation in anoxic conditions. After 1 week (R2A)
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W. Katinka et al.
or 1 month (N43 and S63) of incubation, single colonies
were picked, suspended in MgSO4 (10 mM) and subsequently spread on new agar surfaces. This procedure was
repeated until visibly pure colonies were obtained. In the
end, 34 different colony types were selected for colony
PCR of bacterial 16S rRNA genes (918 bp). Of these, 25
positive PCR products were sent for automated sequencing with forward and reverse primers (Macrogen, Seoul,
Korea), to enable identification. Classification of all 25
PCR products was obtained by BLAST of the sequencing
results against the GreenGenes database (DeSantis et al.,
2006) at minimum 95% sequence similarity (mostly
genus level). Some turned out identical, which was caused
by strains growing on more than one medium or exhibiting different colony morphologies. Seven identifications
originated from anaerobic slant agars, and 10 came from
aerobic agar plates (leaving out identical classifications on
the same medium).
Nucleotide accession numbers
16S rRNA gene sequences of the bacterial isolates were submitted to the European Nucleotide Archive, under accession numbers HF675130–HF675179. The metagenomic
project along with 16S rRNA gene sequences has been
submitted to the Sequence Read Archive, with project accession number ERP002243, sample accession numbers ERS21
5409–ERS215414, experiment accession numbers ERX20
6502–ERX206507 and run accession numbers ERR231951–
ERR231956.
Results
Scanning electron microscopy
SEM images at 1000-fold magnification clearly showed a
large number of microbial cells present in each of the borehole water samples, while only a few in the blank sample
(Fig. S1). Without filtration, SEM images showed aggregates of microbial cells and other, (a)biotic, matter,
exceeding 100 lm diameter (not shown). The filtered
samples showed more clearly the variety of individual cell
morphologies (Fig. S1). Morphologies include cocci, rods,
cork screws, vibrio-type and long filaments. In addition, a
variation in feature sizes is apparent, ranging from over
100 lm in length (filaments) to < 0.1 lm in diameter. A
substantial heterogeneity between samples is apparent,
mostly regarding the presence/absence of distinctive
morphologies like filaments, but also the overall apparent
biological load of the filter surface. IMAGE J calculations
indicated that samples F2–F10 seem to hold a relatively similar load of apparent biological matter, with area
fractions below 20% (Fig. S1 and Table 2). Samples F12–F20
FEMS Microbiol Ecol 86 (2013) 458–473
463
Diverse and active deep subsurface microbial community
seem to have a higher biological load, with F12 even reaching above 40% (Fig. S1 and Table 2). With the exception
of sample F12, the biological load appears to decrease with
depth (R2 = 0.231 with F12, R2 = 0.744, without F12). In
contrast, an inverse, less pronounced linear correlation was
found between the TOC content of a sample and the SEM
load (R2 = 0.421).
Metagenomics
DNA was successfully extracted in triplicate from each of
the 10 water samples. Assuming an average bacterial genome size of 3.5 Mbp, the yields of DNA suggest the presence of between 1.7 9 107 and 2.7 9 108 cells per mL of
Boom Clay borehole water (Table 2). However, there
seems to be no correlation between the amount of
extracted DNA and the geochemical and geophysical
properties of the sample, nor the apparent biological load
of its respective SEM images (R2 ≤ 0.2). Correlations with
other biological parameters, OD600 and ATP, remained
low (R2 = 0.46 and R2 = 0.38, respectively).
In all samples, the presence of bacteria was confirmed
by PCR amplifying a 918-bp region of the bacterial 16S
rRNA genes (data not shown). Extracts and PCRs of the
sampling blank were negative, ruling out bacterial contamination during sampling (data not shown). DGGE
analysis revealed some phylogenetic diversity (Fig. 2).
Some bands seemed to occur in most or even all samples,
suggesting a shared CBC in all Boom Clay borehole water
samples. When targeting aps and nir genes, PCR results
were positive in all samples, indicating the presence of
both sulphate and nitrate reduction properties (Table S2).
Based on UPGMA clustering and Jaccard similarity coefficients of the DGGE fingerprints, six samples were sent for
NGS. Samples were chosen based on either representativeness (F6, F9, F20, F23) or distinctiveness (F8 and F18).
By NGS, between 7786 (F23) and 18 253 (F18) reads
were obtained, of which, respectively, between 4008 and
Table 2. Biomass estimates of Boom Clay borehole water microbial communities
F2
F4
F6
F8
F9
F10
F12
F18
F20
F23
Extracted genomes*
(cells mL 1)
EAC
(cells mL 1)
Av
StDev
Av
1.9E+07
1.4E+08
7.1E+07
2.7E+08
6.0E+07
1.4E+08
1.7E+07
4.3E+07
1.5E+08
2.4E+08
1.3E+06
3.1E+06
1.2E+06
2.2E+06
8.9E+05
8.6E+05
8.2E+05
1.6E+06
2.4E+06
3.5E+06
5.2E+06
9.7E+06
7.8E+06
1.2E+07
1.5E+07
1.5E+07
8.7E+06
8.1E+06
1.1E+07
1.8E+07
Cultivable in R2A_O2
(cells mL 1)
Cultivable in N43
(cells mL 1)
SEM coverage†
(%)
StDev
MPN
95%CI
MPN
95%CI
Av
StDev
3.1E+06
5.2E+06
1.9E+06
6.5E+06
5.7E+06
1.2E+07
5.7E+06
4.6E+06
4.8E+06
3.6E+06
1.8E+07
1.9E+08
8.5E+07
1.9E+08
8.5E+07
1.9E+08
4.8E+08
4.8E+08
4.8E+08
1.9E+08
4.3E+06
1.1E+08
4.3E+07
4.3E+07
1.1E+07
4.3E+07
4.3E+07
1.1E+08
4.3E+07
1.1E+08
1.9E+08
1.9E+08
1.8E+07
4.6E+07
1.9E+08
4.6E+07
1.9E+08
1.8E+07
4.8E+08
8.5E+07
4.3E+07
4.3E+07
4.3E+06
1.1E+07
4.3E+07
1.1E+07
4.3E+07
4.3E+06
1.1E+08
1.9E+07
12.2
15.7
14.5
14.7
18.5
16.7
41.8
21.0
29.2
22.9
2.1
2.3
3.5
1.6
1.7
3.8
10.9
2.0
1.8
4.6
7.9E+07
2.0E+09
8.0E+08
8.0E+08
1.9E+08
8.0E+08
8.0E+08
2.0E+09
8.0E+08
2.0E+09
8.0E+08
8.0E+08
7.9E+07
1.9E+08
8.0E+08
1.9E+08
8.0E+08
7.9E+07
2.0E+09
3.8E+08
Fxx, Morpheus TD-11E borehole water sample; EAC, equivalent active cells; R2A_O2, aerobic R2A medium; N43, anaerobic N43 medium; SEM,
scanning electron microscopy; Av, average; StDev, standard deviation; MPN, most probable number; CI, confidence interval.
*Based on an average genome size of 3.5 Mbp.
†
Fraction of filter membrane covered by assumed biomass, as seen by SEM.
Table 3. Sequence and diversity statistics of six Boom Clay borehole water metagenomes (16S rRNA genes)
F6
F8
F9
F18
F20
F23
nseqs in raw
data set
nseqs in clean
data set
nOTUs
Chao
Coverage* (%)
Shan
InvSim
14509
16163
17050
18253
17571
7786
9861
10352
12588
12627
11711
4008
141
151
130
156
105
143
213
277
179
226
184
217
66.20
54.51
72.63
69.03
57.07
65.90
2.37
3.07
2.32
3.23
2.48
2.70
3.84
10.15
3.74
12.37
6.54
6.29
Project accession number: ERP002243; sample accession numbers: ERS215409–ERS215414; experiment accession numbers: ERX206502–
ERX206507; run accession numbers: ERR231951–ERR231956.
Fxx, Morpheus TD-11E borehole water sample; Nseqs, number of sequences; nOTUs, number of operational taxonomic units; Chao, Chao index
of community richness; Shan, Shannon index of community diversity, weighted towards rare species; InvSim, Inverse of the Simpson index of
community diversity, weighted towards abundant species.
*Amount of observed OTUs (nOTUs), relative to amount of predicted OTUs (Chao).
FEMS Microbiol Ecol 86 (2013) 458–473
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Published by John Wiley & Sons Ltd. All rights reserved
464
W. Katinka et al.
(a)
(b)
(c)
0.0
25 35 45 55 65 75 85 95
F23*
F9*
F10
F20*
F12
F8*
F6*
F2
F4
F18*
33.3
16.7
66.7
50.0
%
83.3 %
Fig. 2. DGGE profile of bacterial 16S rRNA gene fragments of 10 Boom Clay borehole water communities (b), with band-based sample
similarities presented in UPGMA clustering (a) and matrix (c), based on Jaccard coefficients. Samples selected for NGS are indicated with asterisks.
F6
F8
F9
F18
different samples, seven different phyla were abundantly
represented (> 100 seqs per OTU), namely the Proteobacteria, Actinobacteria, Chlorobi, Firmicutes, Bacteroidetes,
Chloroflexi and Spirochaetes (Fig. S2). Proteobacteria made
up of 76% of the community, with the Acidovorax genus
being highly prominent, representing 77% of the b-Proteobacteria, 47% of all Proteobacteria and 36% of the total
abundant community.
When analysing OTUs that are shared between the different samples in our search for a CBC, the overall distribution of bacterial phyla in this shared community is almost
identical to the abundant bacterial community. Six of seven
abundant phyla are represented in all samples. Only the
abundant phylum Spirochaetes does not seem to occur
in all samples, whereas the rare phylum DeinococcusThermus, with 37 sequences comes up in the CBC (Fig. 4).
The Acidovorax genus is the only representative of the
b-Proteobacteria, counting for 46% of the CBC.
F20
F8
F9
F18
F20
F23
0.0
0.2
0.4
0.6
0.8
1.0
Fig. 3. Yue-Clayton dissimilarity heat map of 16S rRNA gene
metagenomes. A darker colour indicates a higher dissimilarity of
bacterial community structure of two borehole water metagenomes
based on relative abundance of OTUs. Sample accession numbers:
ERS215409–ERS215414; experiment accession numbers: ERX206502–
ERX206507; run accession numbers: ERR231951–ERR231956.
12 627 sequences were left after preprocessing with Mothur. The Chao estimated richness of each sample varied
between 179 (F9) and 277 (F8) OTUs (Table 3). The
richness of the samples seems to correlate fairly well with
their TOC concentration (R2 = 0.79). The Shannon and
inverse Simpson index provided an insight on the OTU
distributions within the data set. Both indices indicated
the highest diversity in samples F8 and F18 and the lowest in samples F6 and F9 (Table 3). Similarity between
the six samples based on the Yue-Clayton dissimilarity
coefficient revealed that the samples F8 and F18 are least
similar to the other four samples (Fig. 3).
When pooling all sequencing data of the six samples,
classification of the OTUs indicated that among the
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Published by John Wiley & Sons Ltd. All rights reserved
ATP – measurement
To assess the physiological state of the cells observed, an
additional analysis of ATP was performed on each sample
at different time points. Equivalent of active cells (EAC)
values in the water samples ranged between 7 9 106 and
3 9 108 of EAC per mL (Fig. 5, Table 2). There seems to
be less than one log difference between the 10 different
Morpheus water samples measured at the same time point,
indicating that a high microbial activity is omnipresent in
the borehole water samples of the different clay layers. In
addition, the ATP recordings remained relatively similar
(less than one log difference) upon anaerobic storage during up to 12 months at ambient temperature (21 3 °C),
which is similar to the in situ temperature (17 1 °C). In
addition, ATP results correlated well with OD600 measurements of the samples (R2 = 0.88).
MPN cultivation
A more directed estimation of the viability of the microbial population was targeted by the cultivation-based
FEMS Microbiol Ecol 86 (2013) 458–473
465
Diverse and active deep subsurface microbial community
enrichment experiments with the MPN technique (Fig. 6).
All samples scored positive in at least one tube of each
medium. A large subpopulation of general anaerobic
Bacteroidetes
3%
DeinococcusThermus
0.001%
Chloroflexi
0.001%
Firmicutes
7%
Strain isolation
Chlorobi
9%
Purification of colonies on agar plates and anoxic slant
agars resulted in the distinction of 34 different colony
types, which were selected for colony PCR of bacterial 16S
rRNA genes. In the end, 15 distinct bacterial genera were
identified (Table 4), representing (sub)phyla a-Proteobacteria (1), b-Proteobacteria (2), c-Proteobacteria (2), Actinobacteria (5), Bacteroidetes (3) and Firmicutes (2) (Table 4).
All 15 genera are commonly found in either soils and or
water, or cover at least some environmental species.
α-Proteobacteria
24%
Actinobacteria
7%
γ-Proteobacteria
4%
β-Proteobacteria
46%
Discussion
Fig. 4. Pie charts of bacterial (sub)phyla based on classification of
representative sequences of OTUs, indicating OTUs that are present in
all six 16S rRNA gene metagenomes. A similar pie chart of the
abundant OTUs is provided as Fig. S2. Sample accession numbers:
ERS215409–ERS215414; experiment accession numbers: ERX206502–
ERX206507; run accession numbers: ERR231951–ERR231956.
Equivalent active cells (Log10 cells mL–1)
heterotrophs and anaerobic nitrate-reducing microorganisms was found (107–108 cells per mL), while a smaller
subpopulation of sulphate-reducing microorganisms was
accounted for (101–103 cells per mL). Assays on aerobic
LB medium (rich) and aerobic R2A medium (oligotrophic) also yielded high MPN counts, similar to the
results of anaerobic R2A.
Borehole water samples derived from different layers within
the Boom Clay were analysed by a complementary set of
techniques, providing a comprehensive view on a persistent
and metabolically active microbial population.
Evidence of microbial presence
Where obtaining good SEM images and other microscopy
images of microorganisms in geological clay samples in
8
Day 0
Day 11
Day 56
Day 131
Day 350
Average
7.5
7
6.5
6
5.5
5
F2
F4
F6
F8
F9
F10
F12
F18
F20
F23
Boom clay borehole sample
Fig. 5. EAC of 10 Boom Clay borehole water samples kept at room temperature, based on intracellular ATP analysis, at five time steps
throughout a year. The average amount of EAC is indicated as well, with error bars representing standard deviations.
FEMS Microbiol Ecol 86 (2013) 458–473
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Published by John Wiley & Sons Ltd. All rights reserved
Most probable number (Log cells mL–1)
466
W. Katinka et al.
10
LB_O2
R2A_O2
R2A
N43
S63
F20
F23
9
8
7
6
5
4
3
2
1
0
F2
F4
F6
F8
F9
F10
F12
Boom clay borehole sample
F18
Fig. 6. MPN estimates of cultivated metabolic subcommunities of 10 Boom Clay borehole water samples. Cultivation media are described to
more detail in the Materials and methods section. LB and R2A medium target general heterotrophic communities, while N43 and S63 more
specifically target, respectively, a heterotrophic nitrate-reducing and a sulphate-reducing community. The appendix ‘_O2’ indicates incubation in
an aerobic atmosphere.
Table 4. Classification of bacterial isolates from Boom Clay borehole water to genus or family level
Observed in sample Fxx
Classification
Phylum/Class
Medium
F2
Arthrobacter sp.
Cellulomonas sp.
Microbacterium sp.
Propionicimonas sp.
Rhodococcus sp.
Chryseobacterium sp.
Cyclobacteriaceae
Porphyromonadaceae
Clostridium sp.
Staphylococcus sp.
Rhizobium sp.
Acidovorax sp.
Delftia sp.
Pseudomonas sp.
Stenotrophomonas sp.
Actinobacteria
Actinobacteria
Actinobacteria
Actinobacteria
Actinobacteria
Bacteroidetes
Bacteroidetes
Bacteroidetes
Firmicutes
Firmicutes
a-Proteobacteria
b-Proteobacteria
b-Proteobacteria
c-Proteobacteria
c-Proteobacteria
R2A_O2
R2A
R2A, R2A_O2
R2A
R2A_O2
R2A_O2
R2A_O2
S63
S63
R2A_O2
R2A_O2
R2A_O2
R2A_O2
N43, R2A_O2, S63
R2A_O2
x
x
x
x
x
F4
x
x
F6
F8
F9
F10
F12
F18
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
F20
F23
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x
Match* (%)
100
95.17
99.51–99.88
98.39
100
99.27
97.35
94.39
95.38
99.77
99.39
99.76
99.88
97.44–100
99.77
Nucleotide accession numbers: HF675130–HF675179.
Fxx, Morpheus TD-11E borehole water sample; sp., species; _O2, aerobic medium; x, colony from this sample selected for colony PCR; x, sample
contains colony/colonies similar to selected colony x.
*Identity scores of BLAST search (GreenGenes database). Lowest and highest score are indicated in case of identical classifications of different isolates.
the concept of radioactive waste disposal has not proven
successful (Boivin-Jahns et al., 1996; Stroes-Gascoyne
et al., 2007), the present study succeeded in showing
microbial cells on SEM images of each of the 10 Boom
Clay borehole water samples.
The SEM images provided the first visual evidence and
estimate of microbial life in Boom Clay borehole water
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
samples, taking however into consideration that the samples were stored at ambient temperature under anaerobic
headspace for several months prior to SEM visualization.
The suggested decrease in estimated biological load on
SEM images with depth was not supported by the other
data and does not seem unanimously supported by literature (Breuker et al., 2011; Colwell & D’Hondt, 2013). The
FEMS Microbiol Ecol 86 (2013) 458–473
Diverse and active deep subsurface microbial community
variety in cell morphologies suggests a certain microbial
diversity. The images showed cellular aggregates and filaments, even in the filtered samples, which might seem
surprising. However, because the filtration was based on
membrane filtration with a cut-off value of log 7, the
passing through of a certain amount of long filaments or
large particles remains conceivable. The filaments might
be identified as Actinobacteria, which are clearly present
in all samples, evidenced by the metagenome analyses and
the isolate identifications. Filamentation has also been
reported as a means to react to environmental stress
(Hoffmann et al., 1995; Justice et al., 2008; Crabbe et al.,
2012) and would suit with the stringent conditions in the
Boom Clay. On the other hand, fungal or archaeal filaments might occur as well. Archaea and/or Fungi might
account for the SEM-observed heterogeneity among the
10 samples as well, because Bacteria seem quite homogeneously distributed, as indicated by the definition of an
abundant CBC by the molecular data.
The images also revealed cell-like shapes with sizes
< 0.1 lm in diameter in most samples, which can be
identified either as abiotic matter or as nanobes (Folk,
1993; Vainshtein & Kudryashova, 2000). The current
SEM visualization cannot be considered as conclusive
about the presence of such nanometre-scale organisms,
whose existence is debatable (Nealson, 1997). But as morphological plasticity has been linked before to nutrientdeprived environments (Justice et al., 2008) and more
specific as decrease in cell volume (Cusack et al., 1992;
Vainshtein & Kudryashova, 2000), the biotic nature of
nanometre-scale particles in these borehole water samples
is not considered impossible. It corresponds to the observation of a rather small diameter (0.15 lm) of cells cultivated from Opalinus Clay samples (Mauclaire et al.,
2007) and to the evidence of Actinobacteria, a phylum
that has been discussed to comprise nanobacteria (Hahn
et al., 2003).
Bacterial diversity and core community
DGGE of the extracted bacterial metagenomes showed
certain variety among samples, with a limited number of
bands present in all samples. These shared bands, which
do not necessarily represent the same genera and are not
considered exhaustive, present yet the first indication of a
common CBC. The feasibility of defining a CBC was sustained by the sequencing data, indicating a shared microbial community of 34 OTUs among the six samples.
Given its high overlap with both the pool of abundant
OTUs and the bacterial isolates, this CBC is considered to
be an apt representation of the borehole water bacterial
community. The diversity indices indicate that it is a
rather rich and diverse community, both when weighing
FEMS Microbiol Ecol 86 (2013) 458–473
467
towards the rare and the abundant OTUs. Indeed, some
studies report diverse microbial communities in subsurface environments and/or in nutrient-poor conditions
(Fredrickson & Balkwill, 2006; Barton & Jurado, 2007),
contrasting to others that rather speculate that limited
diversity and density are custom in most subsurface environments (Colwell & D’Hondt, 2013).
The Acidovorax genus accounts for 46% of the
sequences of the CBC and was isolated from all 10 samples as well. The Acidovorax genus is a very common,
mostly aerobic, soil bacterium, which was taxonomically
dissociated from the Pseudomonas lineage over two decades ago (Willems et al., 1990). It is mostly described as
a plant pathogen, a degrader of xenobiotics with associated mobile genetic elements and/or a nitrate reducer
(Hu & Young, 1998; Heylen et al., 2006; Ohtsubo et al.,
2012). It occurs as a metabolically versatile genus, particularly coinciding with the high nitrate-reducing potential
observed by MPN.
While Firmicutes were previously reported to seemingly
dominate the Boom Clay (Boivin-Jahns et al., 1996), the
present study indicates a majority of Proteobacteria in the
borehole water CBC with only 7% of Firmicutes, the latter
comprising sporulating species from the Clostridium and
Bacillus genus. While intuitively sporulation would be
expected as a survival strategy in stringent conditions like
Boom Clay, this is contradicted by Hoehler & Jørgensen
(2013). In environments of chronic low energy, sporulation should be considered as a dead-end situation rather
than an advantage on the long term. Over geological time
scales, microbial spores will still leak energy and eventually perish, because regermination comes with a too high
energy cost. Thus, although sporulation would be
expected as a response by an accidentally introduced
microbial population to a low-energy environment,
microorganisms that are adapted towards long-term survival on low basal power requirements are expected to
dominate in time, rather than sporulaters (Hoehler &
Jørgensen, 2013). The absence of sporulation in the extremophile Archaea is characteristic to this respect. In
addition, members of the genus Bacillus have been shown
to lose their sporulation abilities in anaerobic conditions
(Hoffmann et al., 1995). Consequently, there is no justification to expect either high or low fractions of Firmicutes
in Boom Clay conditions, be it in the borehole water or
the clay matrix.
In a fraction equal to the Firmicutes, mostly Actinobacteria (7%) and Chlorobi (9%) were observed as well. Breuker
et al. (2011) indeed previously reported Actinobacteria
among the most abundant Bacteria in deep terrestrial sediments, although Chlorobi seem more unusual. Among the
isolates, no Chlorobi species were found either, which is not
surprising because this phylum is mostly known for its
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
468
phototrophic genera, and incubations were performed in
the dark. Their presence in the dark subsurface does not
seem rational, but has been reported before (Rastogi et al.,
2009) and might be explained by either their marine origin
and subsequent enclosement since Boom Clay deposition,
or their introduction during piezometer installation. In
both cases they might be provided with energy by light
penetrating through the PVC and nylon tubing of the piezometer, or merely residing in a basal state.
Despite the clear similarities between the samples, some
differences need to be pointed out as well. Although in
general a clear correlation between sample microbial
diversity and TOC concentrations is lacking, F9 and F8
are found to be the samples with, respectively, lowest and
highest (1) carbon content; (2) OTU richness and (3)
OTU diversity. This is particularly remarkable because
these two samples are derived from bordering layers, with
a separating distance of only 48 cm. The layers are, however, very different in composition, with F8 having higher
porosity and permeability, as evidenced by the outlying
high debit rate (Table 1). As the microbial richness of the
samples does correlate fairly well with their TOC concentration (R2 = 0.79), it can be suggested that the availability of a carbon source has an impact on the richness and
tentatively the diversity of the bacterial population. Especially, samples with the highest diversity and highest TOC
concentration, F8 and F18, seem less similar to the other
samples. This dissimilarity is apparent from both the
OTU beta diversity analysis and the DGGE clustering.
Because the diversity of these two samples is explicitly
outlying through the inverse Simpson index (weighing
towards abundant OTUs), the higher availability of
organic carbon in these samples might be linked to an
increase in the proportional abundance of a specific group
of bacteria, which would logically be general heterotrophs.
It is therefore resolved that an abundant core bacterial
population is present in the piezometer borehole water,
which may shift in relative dominance between the different layers in response to the availability of specific nutrients. By combination of the similarity, richness and
diversity indicators of the 10 samples, sample F23 was
selected as the most representative sample for Boom Clay
borehole water microbial community. Apart from the
outlying samples F8 and F18, sample F23 has the highest
richness and Shannon diversity index (weighted towards
rare species) and is therefore considered the best model
inoculum for future (laboratory) experiments.
In addition to the CBC, however, a considerable
amount of Archaea is expected as well. Archaea were not
targeted using molecular techniques in this study, but are
likely to contribute to ATP and SEM results. Sample F23
might well be the best representative of the Boom Clay
borehole water bacterial community, but currently, there
ª 2013 Federation of European Microbiological Societies
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W. Katinka et al.
is no indication whether F23 is representative of the
archaeal community. The rare or dominant involvement
of Archaea in subsurface community seems to vary
between sites and studies (Colwell & D’Hondt, 2013), but
given their extremophilic properties, their presence and
characteristics should certainly be documented when
defining a core microbial community.
Contamination is not likely to be inflicted during the
sampling campaign of 2011, because the sampling blank
did not return any positive results throughout the entire
study. Nonetheless, the CBC should be put into perspective of its origin. During the Morpheus piezometer installation in 2001, no precautions were taken with respect to
sterile working. Introduction of foreign microbial cells
and the subsequent survival or even proliferation of both
indigenous and introduced species in the borehole water
since the piezometer installation is considered credible.
In addition, borehole water conditions as such offer an
increase in both space and water and possibly the availability of alternative substrates (e.g. nylon or PVC tubing
and light penetration) or natural organic matter continuously leaching from the clay, which turn the borehole
into a remarkable in situ bioreactor (Stroes-Gascoyne
et al., 2007) for both introduced and indigenous microorganisms, while the clay matrix as such represents a rather
restricted habitat. Lehman et al. (2001) and Lehman
(2007) discuss the significant difference between microbial
communities in subsurface matrix cores vs. those in the
corresponding waters, focusing on the substantial distinction between attached and unattached populations,
mostly regarding biomass and physiological capabilities.
As indicated by Horn et al. (2004), the mere availability
of water induced growth of an indigenous community
that was previously residing in subsurface rock. The fact
that a relatively high amount of organic matter is still
leaching from the clay into the borehole suggests that the
clay microbial community is unable to use it in the clay
matrix because of its restrictions, although it might also
be argued that most of the Boom Clay organic matter is
not bioavailable as such due to the amounts of kerogen
(Blanchart, 2011; Bruggeman & De Craen, 2011). Because
growth of both indigenous and introduced microorganisms is likely to be promoted in the borehole water, the
herein microbial community is expected to differ in composition, structure, activity and/or abundance from the
actual clay community, as already evidenced by the discrepancy between the current study and earlier reports on
Boom Clay microbial communities (Boivin-Jahns et al.,
1996; Stroes-Gascoyne et al., 2007). Aside from the clear
difference between clay- and borehole water-based studies, including the issue of introducing foreign microorganisms and materials, this discrepancy can be
additionally explained by the use of a customized DNA
FEMS Microbiol Ecol 86 (2013) 458–473
Diverse and active deep subsurface microbial community
extraction protocol and the use of cultivation media with
different specificities.
Microbial contamination and liquid conditions are
expected to occur when real galleries are to be excavated
for radioactive waste disposal in the future, at least during the operational phase. In addition, the common characteristics of identified bacterial genera and phyla,
including strict anaerobic respiration, marine origin, oligotrophy and sulphate reduction, suggest the presence of
at least a highly specialized, and perchance indigenous,
community. A substantial reflection of the original clay
community in the borehole water community is therefore
considered credible. Thus, although the observed population cannot be considered to be entirely indigenous and
undisturbed, it is surely relevant for the operational phase
of nuclear waste disposal. The disturbed community will
in this phase presumably be at its most active state and
will leave imprints on the further development of the
microbial community in the excavation damaged zone
and the engineered barrier system surrounding a disposal
gallery, which will gradually become encapsulated in the
clay matrix upon consolidation. Physiology and species
dominance are expected to differ between the original
clay and the borehole water communities (Lehman et al.,
2001), but likewise, communities in an original vs. a
reconsolidated clay matrix are expected to differ substantially as well. The state of the borehole water community
discussed in this study is considered to be transitional but
nevertheless relevant.
Metabolic activity and potential
For convenience and comparison purposes, values of ATP
were presented as an equivalent of active cells (EAC). However, when considering basal power requirements, as
defined by Hoehler & Jørgensen (2013), the actual number
of active cells could be many orders of magnitude higher.
Rather than refuge into dormancy, it is stated that cells in
stringent, confined conditions survive at very low but stable metabolic rates, with low ATP production and theoretical turnover times of thousands of years (Lomstein et al.,
2012). In any case, ATP values were surprisingly high, be it
as either an actively growing or a low-energy steady-state
community. The estimates of actual microbial activity
(ATP) in the Boom Clay are roughly only one log lower
than the highest MPN results. It was surprising to see stable
ATP readings throughout a year in all samples stored at
ambient temperature. It can be speculated that during this
period, the sampled community thrives on extracellular
polysaccharides (e.g. remains of biofilms) in a stable and
efficient manner. Of course, despite the stable ATP results,
community composition might have changed over time
after sampling. Although not all parameters in the sample
FEMS Microbiol Ecol 86 (2013) 458–473
469
aliquots mimic in situ conditions (e.g. gas phase, environmental matrix), the change imposed upon the community
by sampling is not reflected by a general decrease or
increase in microbial activity as might be expected. Therefore, the stable ATP readings, which correlate well with
OD600 values, are considered a representative reflection of
in situ conditions in the borehole water. Still, these results
contrast with previous studies on subsurface clay microbiology, presuming a microbial community that mostly
seems dead or inactive (Boivin-Jahns et al., 1996; StroesGascoyne et al., 2007). These results indicate an imperative
difference between microbial life in borehole water compared with that in the actual clay matrix. Microbial activity
in the clay matrix has however not been excluded and
remains to be addressed, because communities in the subsurface have been described to be metabolically active and/
or viable, even in the presence of radionuclides (Fredrickson et al., 2004; Akob et al., 2007). As such, microbial
activity has been evaluated to potentially affect the safety of
subsurface waste disposal, yet depending on the specific
environment (e.g. availability of nutrients; Wang & Francis,
2005).
In addition to the apparent activity of the microbial
community in the borehole water, members of all targeted
metabolic subcommunities were cultivated. The presence
of a relatively high fraction of aerobic microorganisms in
the anaerobic Boom Clay and also in vadose sediments has
been previously reported (Boivin-Jahns et al., 1996; Fredrickson et al., 2004), although the results of the present
study are higher than the estimates in the latter studies.
The results of anaerobic MPN cultivations rather contrast
to the results of Boivin-Jahns et al. (1996). However, the
highest number of anaerobic microorganisms was presently
found to be either or both oligotrophic and nitrate reducing, reflecting two subpopulations that were not addressed
in the latter study. In addition, the low estimates of sulphate-reducing microorganisms might be biased by the
need for more stringent or specific cultivation requirements for these type of microorganisms. It is also important to note that the different subpopulations are likely to
overlap; for example, nitrate-reducing microorganisms
might also be capable of reducing sulphate (Dalsgaard &
Bak, 1994). This is supported by the PCR results, which
indicate the presence of nirA and apsA genes in part of,
respectively, the sulphate and nitrate-reducing cultures, but
also in the general heterotrophic cultures.
Pure bacterial strains were also isolated from different
growth media, among which Rhodococcus, Arthrobacter
and Microbacterium (Actinobacteria). From vadose sediments contaminated with high-level nuclear waste, Fredrickson et al. (2004) has isolated indigenous Actinobacteria
strains closely affiliated to those three Boom Clay borehole
water isolates, rendering these genera probable (indigenous)
ª 2013 Federation of European Microbiological Societies
Published by John Wiley & Sons Ltd. All rights reserved
470
candidates for enduring the stresses in an actual nuclear
waste disposal environment. In contrast, at least one
genus that was pointed out as a probable contaminant in
Boom Clay samples by Boivin-Jahns et al. (1996) was
found among the isolates of the present study (Staphylococcus sp.). Members of for example the Pseudomonas
lineage are also well known to be affiliated with both an
anthropogenic (Crabbe et al., 2012) and a subsurface
environment (Saikia et al., 2012). It is therefore resolved
that the pool of isolates reflects the mixed character
(introduced and indigenous) of the Boom Clay borehole
water community.
When accepting that only 1% of all microorganisms is
cultivable (Mocali & Benedetti, 2010), a two log difference
between the highest MPN result and the DNA yield would
have been expected. Especially in such stringent conditions
like Boom Clay, a large fraction of uncultivable phylogenetic lineages would be rationally predicted (Breuker et al.,
2011). Instead, the bacterial isolates nicely cover the phyla
of the CBC, with exception of, on the one hand, the Chlorobi, which are mostly phototrophic and are therefore missed
by dark incubation, and, on the other hand, the phyla that
present < 1% of the CBC. In addition, the DNA extractions
seem to match the highest MPN results (R2A and N43)
completely. Assuming a satisfactory reliability of both DNA
extraction and cultivation methods, this match suggests
either the prevalence of rather small genomes or the inclination of the microbial community towards easy cultivation. The first has been reported in extreme environments
before (DeLong, 2000), and the latter could be explained
by the introduction of foreign microorganisms during the
piezometer installation in 2001 and/or by the piezometer
infrastructure as such. As mentioned above, the piezometer
might function as an in situ enrichment bioreactor due to
the availability of space, water and carbon sources (either
piezometer materials or natural organic matter) in the
borehole water. Regardless of the impact of contamination
and the piezometer infrastructure as such, this extant, disturbed community shows high metabolic activity and
potential in an environment that is still relatively stringent
and relevant for at least the operational phase of a waste
disposal site.
W. Katinka et al.
community in the Boom Clay borehole water samples is
viable and metabolically active in situ.
In the track of this study, a more directed search will
be conducted to answer opened questions, among others
regarding the nature of observed morphologies like nanobes and filaments and the diversity and metabolic properties of other microorganisms besides Bacteria, such as
Archaea. The overlap of this borehole water microbial
community with the solid phase clay community will be
included in future work as well, using the same integrated
approach on solid Boom Clay samples.
Regardless of its origin and enrichment, the omnipresence of a microbial community that is metabolically and
phylogenetically diverse, active and cultivable yet indicates
the possibility of a transient population of relative complexity to survive in Boom Clay conditions and to possibly interfere with future safe waste disposal. In addition,
the thorough study of such a mixed microbial community of historical and recent origin in this unique environment opens perspectives to gain insight in community
dynamics and evolutionary ecology.
Acknowledgements
The authors like to express their gratitude towards the
SCK•CEN Postdoc programme for the kindly provided
advisory and financial support. Within SCK•CEN, numerous colleagues from the Waste and Disposal Expert Group
and the HADES Underground Research Facility are kindly
thanked for their help during the sampling campaign and
metadata collection and interpretation, including Kris
Moerkens, Louis Van Ravestyn, Miroslav Honty and Mieke De Craen. Colleagues Benedict Vos, Wouter Van Renterghem and Willy Vandermeulen are appreciated for
their help with the SEM acquisition. Students Lotte Paulussen and Bet€
ul Aldemir efficiently assisted in the laboratory, which was highly appreciated. During the
bioinformatics analysis of the NGS data, the online forum
of MOTHUR users has been a gratifying source of support
and help as well (http://www.mothur.org/forum/).
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SEM, DGGE and NGS results clearly demonstrate that
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was selected for future laboratory scale experiments. In
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Data S1. Materials and methods.
Fig. S1. Scanning Electron Microscope images of ten 1.2
µm filtered Boom Clay borehole water samples and a
negative control, concentrated on a polycarbonate membrane with 0.1 µm pore size.
Fig. S2. Pie chart of bacterial (sub)phyla based on classification of representative sequences of OTUs, indicating
abundant OTUs (>100 sequences) of the pooled 16S
rRNA metagenomes from Boom Clay borehole water
samples.
Table S1. Target regions, primers, protocols and applications of Polymerase Chain Reaction (PCR).
Table S2. Presence of genes involved in nitrite reduction
(nirS) and sulphate reduction (apsA) in Boom Clay borehole water samples and anaerobic enrichment cultures.
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