detected via cultivation (Cragg et al., 1990, 1996; Barnes
et al., 1998; Parkes et al., 2000). Bacteria have been
enriched and isolated with different organic compounds
at different temperatures (Bale et al., 1997; Barnes et al.,
1998; Inagaki et al., 2003; Mikucki et al., 2003; Toffin
et al., 2004, 2005; Biddle et al., 2005; Lee et al., 2005;
Kobayashi et al., 2008; Parkes et al., 2009). Isolates
belonged to the alpha-, gamma-, deltaproteobacteria,
Firmicutes, Actinobacteria, and Bacteroidetes (D’Hondt
et al., 2004; Batzke et al., 2007). For example, a barophilic
sulfate-reducing bacterium, Desulfovibrio profundus
(Bale et al., 1997), and a methanogenic archaeum,
Methanoculleus submarinus, have been isolated from
deep marine sediments that contain methane hydrates
(Mikucki et al., 2003). Cultivation and isolation of microorganisms is still the only way to get novel organisms
from the environment; to study the properties for the
description of new species, understanding their role in
the environment; and to use them for biotechnological
applications. However, molecular methods provide a more
comprehensive picture about the diversity and abundance
of microbial communities in the environment.
In recent years, nucleic acid-based techniques (i.e.,
DNA and RNA extraction, PCR amplification, and
sequencing) have been developed to identify subsurface
microorganisms without cultivation. Most of these techniques make it possible to phylogenetically identify specific groups of microorganisms and can highlight
microbial diversity and detect new sequences. The analyses of 16S rRNA gene sequences showed that microbial
communities of deep marine sediments harbor members
of distinct, uncultured bacterial and archaeal phylogenetic
lineages (Teske, 2006a; Teske, 2006b; Biddle et al., 2008;
Teske and Sørensen, 2008; Fry et al., 2008; Durbin and
Teske, 2012; Kubo et al., 2012; Orsi et al., 2013b). Among
the Bacteria, 16S rRNA gene sequences belonging to the
JS-1 group and the Chloroflexi were frequently found
(Webster et al., 2004, 2007, 2011; Blazejak and Schippers,
2010). Typical groups for the Archaea include the Marine
Benthic Group B (MBG-B), a deeply branching phylumlevel lineage; the Miscellaneous Crenarchaeotal Group
(MCG), a frequently detected crenarchaeotal lineage with
high intragroup diversity; the South African Gold Mine
Euryarchaeotal Group (SAGMEG); and the Marine Benthic Group D (MBG-D), an euryarchaeotal group affiliated with the Thermoplasmatales (Durbin and Teske,
2012). The metabolic capabilities of these uncultivated
organisms have started to be explored using metagenomic
approaches (Biddle et al., 2008, 2011; Rinke et al., 2013;
Wasmund et al., 2014). For example, recent metagenomic
data indicate that uncultured archaea such as MCG and
MBG-D may have a role in protein remineralization in
anoxic marine sediments (Lloyd et al., 2013a). The microbial ecology of the deep biosphere has been mainly studied in organic-rich, meso- to eutrophic sediments, and
relatively few studies focused on organic carbon-lean, oligotrophic subsurface sediments (Inagaki et al., 2001;
Sørensen et al., 2004; Nunoura et al., 2009; Roussel
et al., 2009; Durbin and Teske, 2011; Breuker and
Schippers, 2013). Since oligotrophic sediments exhibit
specific archaeal diversity patterns, the organic carbon
content is obviously extremely relevant for the natural
selection of distinct Archaea (Durbin and Teske, 2012).
However, the organic carbon concentration is not a
directly proportional index of the sediment trophic state,
as substrate availability and organic carbon residence time
can vary between sediments with similar organic carbon
contents (Durbin and Teske, 2012). Consequently, Durbin
and Teske also defined other parameters such as the sedimentation rate, the penetration depth of electron acceptors
such as sulfate, and the ammonium concentration to characterize the trophic state of sediments.
Sulfate reduction and methanogenesis are relevant biogeochemical processes in deeply buried sediments (e.g.,
D’Hondt et al., 2004). However, sulfate-reducing Bacteria
or methanogenic Archaea, which are frequently found in
near-surface sediments, were rarely detected in deep sediments (Parkes et al., 2005; Biddle et al., 2006; Inagaki
et al., 2006; Teske, 2005; Teske, 2006a; Teske, 2006b;
Webster et al., 2006; Fry et al., 2008; Teske and Sørensen,
2008). In addition to 16S rRNA genes, sulfate reducers
and methanogens have been detected and quantified via
their functional genes encoding dissimilatory sulfite
reductase (dsr), adenosine 5
0 -phosphosulfate reductase
(apr), and methyl-coenzyme M reductase (mcr), respectively (Parkes et al., 2005; Schippers and Neretin, 2006;
Webster et al., 2006, 2009; Wilms et al., 2007; Engelen
et al., 2008; Nunoura et al., 2009; Blazejak and Schippers,
2011; Breuker et al., 2013; Lever, 2013; Ciobanu et al.,
2014).
As mentioned above CARD-FISH (and FISH) has been
applied for the quantification of living Bacteria and
Archaea in deeply buried subsurface sediments. Another
technique with high sensitivity is quantitative real-time
polymerase chain reaction (qPCR). It has been used frequently for the enumeration of phylogenetic 16S rRNA
genes as well as functional genes coding for enzymes particularly involved in biogeochemical processes. qPCR is
based on the online fluorescence detection of PCR products and allows the rapid detection and quantification of
gene sequences without the need for labor-intensive
post-PCR processing (Heid et al., 1996). For application,
DNA is quantitatively extracted from sediment samples,
purified, and specifically amplified with a thermocycler
using sequence-specific fluorescently labeled probes.
There are different formats of the used probes, but the
most common are sequence-specific TaqMan probes
(Heid et al., 1996) and the intercalating nonspecific SYBR
Green dye (Wittwer et al., 1997). The detection limit of the
method depends on the target of interest, sample purity,
PCR conditions, and other factors but theoretically allows
to detect a single DNA molecule (Lockey et al., 1998).
Additionally to DNA, RNA can be quantified after application of an additional reverse transcription step.
A quantification of particular prokaryotic groups (i.e.,
Bacteria and Archaea) in deep subsurface sediments has
DEEP BIOSPHERE
147
et al., 1998; Parkes et al., 2000). Bacteria have been
enriched and isolated with different organic compounds
at different temperatures (Bale et al., 1997; Barnes et al.,
1998; Inagaki et al., 2003; Mikucki et al., 2003; Toffin
et al., 2004, 2005; Biddle et al., 2005; Lee et al., 2005;
Kobayashi et al., 2008; Parkes et al., 2009). Isolates
belonged to the alpha-, gamma-, deltaproteobacteria,
Firmicutes, Actinobacteria, and Bacteroidetes (D’Hondt
et al., 2004; Batzke et al., 2007). For example, a barophilic
sulfate-reducing bacterium, Desulfovibrio profundus
(Bale et al., 1997), and a methanogenic archaeum,
Methanoculleus submarinus, have been isolated from
deep marine sediments that contain methane hydrates
(Mikucki et al., 2003). Cultivation and isolation of microorganisms is still the only way to get novel organisms
from the environment; to study the properties for the
description of new species, understanding their role in
the environment; and to use them for biotechnological
applications. However, molecular methods provide a more
comprehensive picture about the diversity and abundance
of microbial communities in the environment.
In recent years, nucleic acid-based techniques (i.e.,
DNA and RNA extraction, PCR amplification, and
sequencing) have been developed to identify subsurface
microorganisms without cultivation. Most of these techniques make it possible to phylogenetically identify specific groups of microorganisms and can highlight
microbial diversity and detect new sequences. The analyses of 16S rRNA gene sequences showed that microbial
communities of deep marine sediments harbor members
of distinct, uncultured bacterial and archaeal phylogenetic
lineages (Teske, 2006a; Teske, 2006b; Biddle et al., 2008;
Teske and Sørensen, 2008; Fry et al., 2008; Durbin and
Teske, 2012; Kubo et al., 2012; Orsi et al., 2013b). Among
the Bacteria, 16S rRNA gene sequences belonging to the
JS-1 group and the Chloroflexi were frequently found
(Webster et al., 2004, 2007, 2011; Blazejak and Schippers,
2010). Typical groups for the Archaea include the Marine
Benthic Group B (MBG-B), a deeply branching phylumlevel lineage; the Miscellaneous Crenarchaeotal Group
(MCG), a frequently detected crenarchaeotal lineage with
high intragroup diversity; the South African Gold Mine
Euryarchaeotal Group (SAGMEG); and the Marine Benthic Group D (MBG-D), an euryarchaeotal group affiliated with the Thermoplasmatales (Durbin and Teske,
2012). The metabolic capabilities of these uncultivated
organisms have started to be explored using metagenomic
approaches (Biddle et al., 2008, 2011; Rinke et al., 2013;
Wasmund et al., 2014). For example, recent metagenomic
data indicate that uncultured archaea such as MCG and
MBG-D may have a role in protein remineralization in
anoxic marine sediments (Lloyd et al., 2013a). The microbial ecology of the deep biosphere has been mainly studied in organic-rich, meso- to eutrophic sediments, and
relatively few studies focused on organic carbon-lean, oligotrophic subsurface sediments (Inagaki et al., 2001;
Sørensen et al., 2004; Nunoura et al., 2009; Roussel
et al., 2009; Durbin and Teske, 2011; Breuker and
Schippers, 2013). Since oligotrophic sediments exhibit
specific archaeal diversity patterns, the organic carbon
content is obviously extremely relevant for the natural
selection of distinct Archaea (Durbin and Teske, 2012).
However, the organic carbon concentration is not a
directly proportional index of the sediment trophic state,
as substrate availability and organic carbon residence time
can vary between sediments with similar organic carbon
contents (Durbin and Teske, 2012). Consequently, Durbin
and Teske also defined other parameters such as the sedimentation rate, the penetration depth of electron acceptors
such as sulfate, and the ammonium concentration to characterize the trophic state of sediments.
Sulfate reduction and methanogenesis are relevant biogeochemical processes in deeply buried sediments (e.g.,
D’Hondt et al., 2004). However, sulfate-reducing Bacteria
or methanogenic Archaea, which are frequently found in
near-surface sediments, were rarely detected in deep sediments (Parkes et al., 2005; Biddle et al., 2006; Inagaki
et al., 2006; Teske, 2005; Teske, 2006a; Teske, 2006b;
Webster et al., 2006; Fry et al., 2008; Teske and Sørensen,
2008). In addition to 16S rRNA genes, sulfate reducers
and methanogens have been detected and quantified via
their functional genes encoding dissimilatory sulfite
reductase (dsr), adenosine 5
0 -phosphosulfate reductase
(apr), and methyl-coenzyme M reductase (mcr), respectively (Parkes et al., 2005; Schippers and Neretin, 2006;
Webster et al., 2006, 2009; Wilms et al., 2007; Engelen
et al., 2008; Nunoura et al., 2009; Blazejak and Schippers,
2011; Breuker et al., 2013; Lever, 2013; Ciobanu et al.,
2014).
As mentioned above CARD-FISH (and FISH) has been
applied for the quantification of living Bacteria and
Archaea in deeply buried subsurface sediments. Another
technique with high sensitivity is quantitative real-time
polymerase chain reaction (qPCR). It has been used frequently for the enumeration of phylogenetic 16S rRNA
genes as well as functional genes coding for enzymes particularly involved in biogeochemical processes. qPCR is
based on the online fluorescence detection of PCR products and allows the rapid detection and quantification of
gene sequences without the need for labor-intensive
post-PCR processing (Heid et al., 1996). For application,
DNA is quantitatively extracted from sediment samples,
purified, and specifically amplified with a thermocycler
using sequence-specific fluorescently labeled probes.
There are different formats of the used probes, but the
most common are sequence-specific TaqMan probes
(Heid et al., 1996) and the intercalating nonspecific SYBR
Green dye (Wittwer et al., 1997). The detection limit of the
method depends on the target of interest, sample purity,
PCR conditions, and other factors but theoretically allows
to detect a single DNA molecule (Lockey et al., 1998).
Additionally to DNA, RNA can be quantified after application of an additional reverse transcription step.
A quantification of particular prokaryotic groups (i.e.,
Bacteria and Archaea) in deep subsurface sediments has
DEEP BIOSPHERE
147