Journal of Microbiological Methods 70 (2007) 1 12

www.elsevier.com/locate/jmicmeth

Review

Exploring microbial diversity in volcanic environments:

A review of methods in DNA extraction
Aude Herrera , Charles S. Cockell
Planetary and Space Sciences Research Institute, Open University, Walton Hall, Milton Keynes, MK7 6AA, UK
Received 9 March 2007; received in revised form 11 April 2007; accepted 11 April 2007
Available online 21 April 2007

Abstract
The last decade has been marked by a large number of studies focused on understanding the distribution of microorganisms in volcanic
environments. These studies are motivated by the desire to elucidate how the geochemically extreme conditions of such environments can influence
microbial diversity both on the surface and in the subsurface of the Earth. The exploration of microbial community diversity has generally not relied
on culture-dependent methods, but has been carried out using environmental DNA extraction. Because of the large diversity of chemically and
physically complex samples, extracting DNA from volcanic environments is technically challenging. In view of the emerging literature, and our own
experience in the optimisation of methods for DNA extraction from volcanic materials, it is timely to provide a methodological comparison. This
review highlights and discusses new insights and methods published on DNA extraction methods from volcanic samples, considering the different
volcanic environments. A description of a recent method for DNA extraction from basalt and obsidian glass rock samples from Iceland is included.
Finally, we discuss these approaches in the wider context of modern work to understand the microbial diversity of volcanic environments.
2007 Elsevier B.V. All rights reserved.
Keywords: Volcanic environments; DNA extraction; Microbial communities; Geomicrobiology; Extreme environments

Contents
1.
2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The diversity of volcanic environments . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Vesiculated or non-vesiculated volcanic rocks, including basalt . . . . . . . . .
2.2. Rocks and sediments with high metal concentrations and/or extreme pH ranges .
2.3. Carbonate rocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Environmental DNA extraction for microbial community analysis . . . . . . . . . . . .
3.1. DNA extraction from environmental samples: general considerations . . . . . .
3.2. DNA extraction from volcanic samples: limits due to chemical factors . . . . .
3.2.1. DNA-binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2. DNA-sequestration in hard mineral matrix . . . . . . . . . . . . . . .
3.2.3. DNA-degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. DNA extraction from volcanic samples: limits due to physical factors . . . . . .
3.4. DNA extraction from volcanic samples: limits due to biological contaminations .
3.4.1. Source of biological contaminations and prevention . . . . . . . . . . .
3.4.2. Checking results: controls and contaminant sequence database . . . . .

Corresponding author. Tel.: +44 1908 653170; fax: +44 1908 858022.
E-mail address: a.herrera@open.ac.uk (A. Herrera).
0167-7012/$ - see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2007.04.005

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A. Herrera, C.S. Cockell / Journal of Microbiological Methods 70 (2007) 112

3.5. DNA extraction from volcanic samples: conclusion . . . . .

4. Bacterial and archaebacterial communities in volcanic environments
5. Conclusion and future challenges . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
Volcanism may be defined, accurately but ponderously, as
the manifestation of the surface of a planet or satellite of internal
thermal processes through the emission at the surface of solid,
liquid, or gaseous products (Francis, 1993). Environments
resulting from volcanic activity are diverse, from acidic hot
springs to deep ocean basaltic habitats.
As volcanic environments are widely distributed on Earth, it is
of geomicrobiological importance to understand the diversity and
characteristics of microbial life that they harbour. Volcanic environments might be used as model systems to understand more
general patterns of the diversity and distribution of prokaryotes
through time. Such investigations improve our understanding of
how the geochemistry of different rocks can influence microbial
diversity that persists on the surface and in the subsurface of the
Earth.
Another major reason for astrobiologists to investigate volcanic environments is that such locations are considered analogous to some of the earliest environments on Earth (van
Kranendonk and Pirajno, 2004). During the early Archean
(approximately 3.5 to 3 Ga ago) land masses would have been
made predominantly of igneous rock. Consequently, exploring
the biological diversity of volcanic environments can help to
reveal what potential existed for early microbial life on Earth.
During the last 20 years, studies of microbial communities
have increasingly been carried out using environmental DNA
extraction. Such molecular methods can reveal the complexity of
microbial communities present in an environmental sample
through PCR amplification of the 16S ribosomal RNA (16S
rRNA) gene sequence and molecular phylogenetic techniques
(Cunnigham et al., 1995; Pace, 1997; Northup and Lavoie,
2001). This approach also applies to other genes used for
phylogenetic identification, e.g., 18S rRNA and these approaches can also be used to search for the presence of functional
genes.
In the case of volcanic environments, because of the large
diversity of chemically and physically different geological samples, extracting DNA from these specific environments is technically challenging. A range of complications, such as acidic pH
or soil-DNA interactions, can introduce non-negligible bias
during DNA extraction process (Henneberger et al., 2006).
Consequently, for a long time geomicrobial studies have been
limited to traditional microscopic, mineralogical and biogeochemical analyses.
In this review we define volcanic environments as those
environments in and around volcanoes and resulting from
volcanic activity. We recognise that there is some overlap with
non-volcanic environments, such as some low-biomass sedi-

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mentary environments. For example, because carbonate deposits are often found in volcanic areas (such as hydrothermal
travertines), we have included DNA extraction from carbonates
in this review, but of course carbonates are associated with nonvolcanic sedimentary environments. Thus, the purpose of this
review is to cover many of the environments associated with a
volcanic region, recognising that the boundaries are somewhat
grey. We discuss existing literature and we draw upon our own
experience to describe approaches to DNA extraction from
diverse volcanic environments. We also discuss how these
approaches are providing new insights into the microbial diversity of these environments.
2. The diversity of volcanic environments
In Fig. 1, we have shown just a few of the diversity of
habitats that are created in and around volcanoes. In this review
we will focus our discussion on habitats which have certain
characteristics that are linked to volcanic activity, and which
have previously been the focus of studies involving DNA extraction from volcanic regions. These include:
2.1. Vesiculated or non-vesiculated volcanic rocks, including
basalt
A majority of volcanic habitats for microorganisms are characterised by igneous rocks such as rhyolites, basalts, etc. Often
these rocks are erupted in well-defined sequences that are
influenced by the evolution of the magmatic chamber producing
them (Carmichael, 1964; Suh et al., 2003). Now considered a
classic study is the petrological examination of the tertiary basalt
sequence of the Thingmuli volcano in eastern Iceland, which
displays a sequence from olivinetholeiites (which contain over
100 ppm of Cr and Cu and over 10 ppm Fe) through to more
metal-poor rhyolites and between them a range of basalts
(Carmichael, 1964). As many of the basalts are rich in metals
such as iron, titanium and magnesium, co-extraction with DNA
may subsequently affect PCR, necessitating a cleaning step. This
problem will be less acute for the relatively metal poor rhyolites,
but nevertheless rhyolites can still contain high (N100 ppm) Zn
concentrations. Many of these rocks may be vesiculated if there
was outgassing during cooling, providing a large pore space for
microorganisms within them. Basalts in particular are widely
distributed in both terrestrial and marine volcanic settings and are
therefore of particular interest in characterising microbial community composition in volcanic environments (Staudigel et al.,
2006). As discussed later, a diversity of basalts from terrestrial and
particularly marine settings have been examined using cultureindependent methods.

A. Herrera, C.S. Cockell / Journal of Microbiological Methods 70 (2007) 112

Fig. 1. Simple schematic illustrating some of the diversity of habitats and environments found associated with volcanic activity.

2.2. Rocks and sediments with high metal concentrations and/

or extreme pH ranges
The high temperatures associated with geothermal activity
result in both surface and subsurface geothermal springs (hot
springs) which have previously been the focus of intense study.
They harbour extraordinary microbial diversity (Barns et al.,
1994, 1996) and on account of their supposed value in acting as
a window to the conditions on the Archean Earth, they have
been seen as a repository of information about the conditions for
the emergence of life. The high concentrations of sulphur
species often encountered in volcanic environments (Thordarson et al., 2003) means that many of them are acidic and this has
implications for the types of DNA extraction procedures used. It
is not possible to consider here all of the potential permutations
on sediment and rock physical and chemical conditions in
volcanic habitats and the influence of volcanic liquids on them
(see, for example, Pasternak and Varekamp, 1997; Varekamp
et al., 2000; Pedrozo et al., 2001). However, we attempt to
discuss some of the challenges in DNA extraction that have so
far been reported from sediment and hot spring environments.
2.3. Carbonate rocks
Carbonate rocks are not specific to volcanic environments,
but warm water calderas and volcanic caves often provide conditions conducive to calcium carbonate formation and associated
minerals (Forti, 2005). As these rocks present special problems
related to non-specific DNA binding, we include them in our
discussion as they are applicable to any volcanic rocks with high
metal concentrations.
3. Environmental DNA extraction for microbial
community analysis
3.1. DNA extraction from environmental samples: general
considerations
Culture-or molecular-based approaches are generally used to
characterise environmental microbial communities. Detailed
biochemical characterisation of cultured microorganisms provides valuable information on their potential roles in environmental processes. However, more than 95% of environmental

microbes are recalcitrant to laboratory culture, due to the selectivity of growth media and culture conditions (Alexander,
1977; Amann et al., 1995). The molecular techniques are able to
investigate a far greater proportion of prokaryotic communities,
based on environmental sample DNA extraction (Torsvik et al.,
1990). The use of PCR to amplify a gene common to all
organisms, such as the small ribosomal subunit (SSU) (Woese,
1987), is used in databases to provide a comparison with previously characterised organisms.
DNA extraction methods can be divided into two groups. In
one approach, the microbial cell fraction is first separated from
soil particles or rock and then lysed to purify DNA (Torsvik,
1980). This procedure requires time and effort, with generally
low DNA yield. Moreover, it has been shown that the cell
extraction method is biased as, e.g. methane-oxidising (Priem
et al., 1996) and ammonia oxidising (Aakra et al., 2000)
bacteria are more difficult to dislodge from soil particles
compared to the majority of soil bacteria. The other approach
includes direct cell lysis in the soil or rock after which DNA is
extracted (Bertrand et al., 2005). This direct approach is
generally superior to the first method in terms of DNA yield and
can be relatively easy (Tien et al., 1999).
Today, the direct lysis method is primarily used and a large
number of methods have been published for DNA extraction
from different natural and complex samples, including compost,
sediment and soil samples (Zhou et al., 1996; Ogram et al.,
1987; Smalla et al., 1993; Krsek and Wellington, 1999; Hurt
et al., 2001; LaMontagne et al., 2002). Most of these methods
consist of physical or chemical procedures for in situ cell
disruption such as sonication, grindingfreezingthawing, bead
beating, heating treatment and/or detergent, followed by phenol/
chloroform DNA extraction (Robe et al., 2003). Recently, a
number of commercial DNA extraction kits for soil has been
developed to optimize yield and reproducibility of extraction,
and they are less time-consuming than conventional methods
(Whitehouse and Hottel, 2007).
The direct DNA extraction method, including commercial
kits, is generally suitable for many environmental samples.
However, for a large variety of volcanic samples, several factors
related to their chemical and physical characteristics can limit the
efficiency of the direct DNA extraction procedure. Consequently, in the following paragraphs, we particularly focused on the
difficulties due to physical, chemical and contamination factors

A. Herrera, C.S. Cockell / Journal of Microbiological Methods 70 (2007) 112

affecting extraction, and we consider different solutions to overcome these difficulties.

3.2. DNA extraction from volcanic samples: limits due to
chemical factors
Three phenomena caused by the chemical composition of the
volcanic samples can limit the DNA extraction efficiency: (i)
DNA-binding to components naturally present in the sample,
(ii) DNA-sequestration in a hard mineral matrix and (iii) DNAdegradation particularly due to acidic conditions. These three
phenomena are not completely independent from each other and
we consider them here:
3.2.1. DNA-binding
Among the different factors limiting DNA extraction, the
interactions between DNA and the environmental sample can
greatly complicate the procedure. More precisely, several components present in volcanic environmental samples can bind
DNA (and cells), thereby making it difficult to extract the DNA or
decreasing the purity due to their co-extraction. Potential examples include clay surfaces that can bind DNA and high metal
concentrations. These problems have previously been reported for
soil and sediment samples, and several suggestions for modifications have been published (Rochelle et al., 1994; Ogram et al.,
1994; Zhou et al., 1996; He et al., 2005).
A typical example of this point concerns Andisol, a volcanic
ash soil. Andisol is widely distributed all over the world, especially in the circum-Pacific Ring of Fire, East Africa and the
Mediterranean. In a type of Andisol including allophone as a
clay mineral, it has been difficult to extract DNA with the use of
a traditional DNA extraction kit, such as FastaDNA Spin Kit for
soil (Q-BIOgene) (Takada Hoshino and Matsumoto, 2005). In a
recent study, Takada Hoshino and Matsumoto (2005) estimated
from microscopic observations and microbial cell counting data
that the concentration of DNA from their Andisol soil samples
was expected to be between 14 g DNA g 1 dry wt soil.
However, genomic DNA was not detected by agarose electrophoresis, and they showed that DNA adsorption to soil particles
is the main factor reducing extraction efficiency (Frostegard
et al., 1999; Takada Hoshino and Matsumoto, 2005). Consequently, to improve the DNA recovery, and decrease the
adsorption, they modified the FastaDNA Spin Kit by adding
skimmed milk or RNA as adsorption competitor (Volossiouk
et al., 1995; Frostegard et al., 1999). According to their results,
the combination of the commercial kit and skimmed milk
proved to be the most efficient in extracting PCR-suitable DNA
from a recalcitrant high clay-content environmental sample.
Another significant example of environmental particles adsorption and a solution for optimizing DNA extraction is given
by Purdy et al. (1996) in their previous work from sediment
samples. In their study, Purdy et al. (1996) described a novel
hydroxyapatite spin-column method of nucleic acid extraction
from natural silty clay-sediments with high organic content.
This very rapid method, using a binder which for many years
was used as a high-performance liquid chromatography column
matrix to bind and separate nucleic acids and proteins (Bernardi,

1965), produced pure nucleic acid that can be used successfully

in PCR and restriction digestions or also in slot blot hybridizations to radiolabelled 16S rRNA-targeted oligonucleotide
probes (Purdy et al., 1996). Although Purdy et al. did not apply
this directly to volcanic samples it offers another solution for
obtaining clean DNA.
Although several solutions, such as the procedures mentioned
above, have been described in the literature to avoid, or at least to
decrease, DNA adsorption on environmental particles, this
limitation remains particularly problematic for volcanic samples
characterized by very low biomass. Recent work by Barton et al.
on DNA extraction from low-biomass carbonate rock illustrates
this point (Barton et al., 2006) and may be applicable to the
extraction of DNA from hydrothermally-associated carbonates. In
their study, Barton et al. (2006) focused on the microbial community present in an extremely low-biomass calcium-rich cave
environment. In this case, the classical techniques to prevent nonspecific DNA-binding, based on blocking agents such as milk
powder or Denhardt's solution to coat reactive surfaces of CaCO3
and block DNA binding (Guthrie et al., 2000), are not suitable.
Indeed, in samples where the microbial communities abundance
is significantly lower than 108 cells/g of sample, such techniques
increase the risk of introducing bacterial contaminants that will
artificially distort the structure of the community being examined
(Barton et al., 2004, 2006; Chelius and Moore, 2004). To recover
extremely low amounts of DNA from calcium-rich rocks, Barton
et al. describe an improved protocol, relying on the use of the
synthetic DNA molecule poly-dIdC, to act both as blocking agent
and carrier molecule to increase the yield of DNA, and dialysis to
remove calcium inhibitors of PCR amplification (Barton et al.,
2006).
3.2.2. DNA-sequestration in hard mineral matrix
More than DNA-binding on environmental particles, such as
observed in soil or sediment samples, the chemical composition
of volcanic samples can lead in certain cases to complete DNA
and cell sequestration in a hard mineral matrix. In this case, the
first difficulty in DNA extraction, before being confronted by
DNA-binding to environmental particles, is actually to access
the cells and DNA which is trapped in the matrix.
An example of cell and DNA sequestration has recently been
reported by Wade and Garcia-Pichel (2003), working on modern
calcareous microbialites. Current DNA extraction protocols for
molecular analysis are poorly adapted for lithic or encrusted
microbial communities due mostly to the hard, usually cemented
nature of the mineral matrix. Consequently, in their study, the
authors optimized a procedure to minimally liberate microbial
cells from their mineral encasing, to allow their disruption
without preferential growth or death of particular cells, and to
chemically isolate the nucleic acid fraction by minimizing DNA
degradation. As with the work by Barton et al. (2006), this work
is applicable to hydrothermally-associated carbonate deposits in
volcanic environments, but it also yields more general insights
into extracting DNA and organisms from other rock matrices,
such as silicified rock from hydrothermal environments.
To evaluate the most efficient method, Wade and GarciaPichel (2003) compared three procedures for disruption of

A. Herrera, C.S. Cockell / Journal of Microbiological Methods 70 (2007) 112

calcium carbonate matrix and liberating the cells: (i) pulverization

using a sterile mortar and pestle, (ii) acid dissolution of the
carbonate matrix using sterile HCl solutions with decreasing pH
from 5.00 to 0.8 and (iii) chelator-mediated dissolution of the
matrix with sterile ethylenediaminetetraacetic acid (EDTA
disodium salt dihydrate) solutions of increasing concentrations
from 500 to 675 mM at pH 5 (Wade and Garcia-Pichel, 2003).
Following these three different procedures, the authors chose to
extract DNA by using a traditional commercial kit (Mobio
UltraClean Plant DNA Isolation). Their results clearly showed
that both pulverization and chelator-mediated dissolution of the
carbonate matrix are appropriate methods that yield sufficient
amounts of high-quality DNA suitable for PCR-based analyses.
However, the authors suggested that EDTA-dissolution, although
requiring long reaction times, might be preferred to preserve the
microbial architecture in the sample and allow for spatially
resolved analyses. This method should also be applicable to the
analyses of microbial communities in other mineral matrices, such
as gypsum endoliths (Wade and Garcia-Pichel, 2003).
On the other hand, HCl treatment for calcium carbonate
matrix disruption seemed not to be suitable for DNA extraction.
Indeed, although cell morphology appeared undisturbed by HCl
solutions, when examined microscopically, nucleic acids are
subject to facile hydrolysis (Jordan, 1960) and the proton
concentrations needed to drive carbonate dissolution at fast rates
likely resulted in the intracellular hydrolysis of DNA. Because of
the DNA instability under particular conditions such as acidic
pH, the problem of DNA degradation remains an important limit
to the DNA extraction procedures for many volcanic samples.
3.2.3. DNA-degradation
As previously mentioned, DNA is unstable under acidic
conditions, owing to depurination-induced degradation of DNA
(Garrett and Grisham, 1995). Furthermore, DNA-binding, and
particularly clay binding, can increase at acidic pH (Khanna and
Stotzky, 1992).
A recent work has been carried out by Henneberger et al.
(2006) to extract DNA from highly acidic soils collected from
Devil's Kitchen, a fumarolic field on Mt Hood, USA. These
soils were derived from andesitic and dacitic rocks altered by
volcanic heat and acidic, sulphur-rich hydrothermal steam and
did not contain visible microbial communities. The traditional
mechanical in situ cell lysis by bead beating, such as performed
by FastaPrep kit (QBiogene), was ineffective for these soil
samples. Therefore, the authors compared six other protocols to
extract DNA biomass from highly acidic mineral-containing
volcanic soils, and their results highlight the importance of
testing different extraction procedures when dealing with apparently intractable samples (Henneberger et al., 2006). For example, they successfully used for two of their samples the
FastaPrerp PEG/lysosyme modification recently described by
Bell et al. (2006, cited by Henneberger et al., 2006), whereas the
chemical cell lysis-xanthogenateSDS (XS) buffer previously
used by Tillett and Neilan (2000) led to successful results for
some of other samples.
This conclusion on the importance of testing several procedures has recently been confirmed by Brown and Wolfe

(2006), who carried out DNA extraction from diverse samples

(microbial mats, water and sediments) collected in acidic
hydrothermal environments of Lassen Volcanic National Park
(LVNP), USA. In this work, the authors compared a modified
CTAB method with chloroform purification (Barns et al., 1994),
and two soil kits, i.e. UltraClean (Mobio Laboratories) and
FastaDNA (MP Biomedicals). Although globally CTAB and
FastaDNA methods gave similar yields, superior to the UltraClean kit, their results showed that the extraction yield varied
greatly among samples. For example, many of the clay sediments samples yielded essentially no DNA and steam water
samples also often yielded low DNA recoveries (Brown and
Wolfe, 2006).
In a parallel study, Siering et al. (2006) described a different
procedure to extract DNA from similar acidic water and
sediments samples collected in LVNP. Contrarily to the study by
Brown and Wolfe (2006), a cell extraction step was first
performed by filtration and/or centrifugation, followed by DNA
extraction after the chemical and physical cell lysis (Siering
et al., 2006). This approach could be better adapted than the in
situ cell lysis to minimize DNA degradation in highly acidic
volcanic samples.
3.3. DNA extraction from volcanic samples: limits due to
physical factors
For many classical soil samples, DNA extraction is traditionally preceded by a physical soil particle disruption step using
silica or glass beads combined with chemical detergents. However, in the case of volcanic environments, some of them are
characterized by a hard rock structure, such as rocks with a high
glass content. The bead treatment is therefore not effective enough
to disrupt this structure and liberate cells. Splitting rocks with a
hammer and chisel and/or crushing in a flame-sterilised mortar is
more suitable (Thorseth et al., 2001; Lysnes et al., 2004), even if
this step could increase the risk of introducing exogenous
microbial contaminants (see below Section 3.4).
Following the rock crushing step, any specific DNA extraction methods could potentially be used as have been
previously reported for rock samples. A few authors, interested
in characterising the microbial community in basalt seafloor
rocks, directly performed 16S rDNA gene amplification from
cell suspensions obtained after washing and centrifuging their
crushed rock samples (Thorseth et al., 2001; Lysnes et al., 2004).
However, although this approach seems suitable to construct
16S rDNA clone libraries from volcanic rock samples, it has the
drawback of only releasing material not strongly bound to the
rock. For example, DNA from biofilms that might be strongly
adhered to the rock surface can only reliably be removed by
crushing the rock.
Consequently, to complete the present review, we tested and
compared different molecular methods to directly extract DNA
from two rock types collected in Iceland: a basalt rock (from 63
deg 20.07N, 19 deg 23.71W), where the microorganisms grow
within the vesiculated porous matrix and an obsidian rhyolitic
glass (from 64 deg 2.01 N, 19 deg 7.75W), where the microorganisms grow within interfaces and cracks within an otherwise

A. Herrera, C.S. Cockell / Journal of Microbiological Methods 70 (2007) 112

quite solid homogeneous rock (Fig. 2). We examined the rocks

by Scanning Electron Microscope (SEM) and the gross
mineralogy of the rocks was determined using Energy Dispersive Spectroscopy (EDS) (Fig. 2). In our work specifically,
we are interested in the correlation between microbial community composition and mineralogy in volcanic deserts, but
DNA extractions applied to metal rich basalts and volcanic
glasses such as the ones we describe here might be applied to

other types of volcanic rock samples. The experimental results

are not described here, rather we focus on the DNA extraction
methodology.
In the case of the basalt, the challenge of extraction involves
dealing with high iron concentrations and releasing the organisms from the vesicular material. In the case of the glass, the
challenge lies in the removal of the DNA from within a solid
glass matrix. Thus, because of the hard matrix of the rock

Fig. 2. Samples used to optimise DNA extraction from Basalt rock (B) and Obsidian glass (O). (1) Images of samples, (2) SEM images of samples textures. Vesicular
characteristics of basalt sample can be seen, (3) EDS of representative regions of the samples showing gross mineralogical characteristics, particularly metals, which
can be a problem for DNA extraction (see text for discussion).

A. Herrera, C.S. Cockell / Journal of Microbiological Methods 70 (2007) 112

samples, particularly the obsidian glass sample, we chose to

crush the rocks before DNA extraction with a different approach
than the traditional mortar. Pieces of rock (3 cm3) were placed in
a cylindrical metal container and crushed by pressing a heavy
metal plunger with a hammer into it. To avoid external contamination of the rock samples during this step, the samples were
kept in sterile bags and all the metal instruments in contact with
the rock were previously rinsed with ethanol and bactericide
solvent before using. After crushing, the rock powder was collected in sterile Petri dishes for the DNA extraction.
The molecular method described by Zhou et al. (1996) was
then tested from 5 g of crushed rocks for both samples. This
method, initially developed for soil DNA extraction, is mainly
based on chemical cell lysis by the common detergent sodium
dodecyl sulfate (SDS), which dissolves the hydrophobic material of cell membranes. This detergent is used in combination
with heat-treatment, with chelating agents such as EDTA and
cetyltrimethylammonium bromide (CTAB) (Zhou et al., 1996;
Robe et al., 2003), which can partially remove humic compounds by forming insoluble complexes with denatured proteins, polysaccharides and cell debris (Saano et al., 1995). The
DNA extraction results obtained by this method were compared
to those obtained by a traditional DNA extraction commercial
kit for soil: PowerMax Soil DNA Isolation Kit (MoBio
Laboratories), from 5 g of crushed rock sample. This commercial kit has been chosen for the high-quality of DNA extracted
and the rapidity with which it can be performed, compared to
the traditional [SDS-CTAB]-method. In addition, a comparable
kit has been previously found suitable to extract DNA from
acidic volcanic rocks (Walker et al., 2005).
Each of the DNA extraction methods was also tested with a
positive control using surface soil from the Open University
campus in Milton Keynes. The two DNA extraction methods
([SDS-CTAB]-method and Mobio kit) gave a DNA-yield very
similar from the basalt rock sample (between 0.35 and 0.55 ng/g
crushed rock; Fig. 3) and lower than the yield obtained from the
positive control sample, particularly with Mobio kit (3 g DNA/
g soil). However, although the DNA-yield was similar with the
both of the methods for the basalt rock sample, the DNA purity

Fig. 4. 16S rDNA amplification results for different environmental DNA: basalt
rock sample (B), obsidian glass sample (O) and control soil sample (C),
extracted by two different methods: 1= CTAB-method and 2= PowerMax Soil
DNA Kit (Mobio Laboratories). PCR amplifications were performed with the
TaqMaster kit (Eppendorf) according to the manufacturer's instructions. A
negative control (without DNA template) has been included in the protocol to
check the absence of contaminant during the amplification.

was significantly higher with the Mobio kit, because of a

specific step included in the method to efficiently remove
protein and humic acids. From the obsidian glass sample, only
the traditional [SDS-CTAB]-method led to a positive DNAyield (Fig. 3).
Following the DNA extraction, amplifications by PCR were
performed from each condition with 16S rDNA bacterial gene
specific primers (Bruce et al., 1992), in order to check the quality
and quantity of each DNA sample. For both samples, basalt rock
and obsidian glass, the 16S rDNA amplification was positive
only from DNA extracted by the Mobio kit (Fig. 4), even though
this method did not result in visible DNA biomass on the gel for
the obsidian sample (Fig. 3). These results suggested therefore
the successful extraction of endogenous DNA from the obsidian
sample using the Mobio kit.
In conclusion, the DNA quantity is not the only criterion with
which to select the best DNA extraction method from a complex
sample, such as volcanic rock samples. The method has also to
produce high-quality DNA, minimizing inhibitor co-extraction
(humic acids, protein, etc.), in order to be able to carry out
follow-on analyses, such as PCR amplifications. In our case, the
PowerMax Soil DNA Isolation Kit (MoBio Laboratories) seems
to satisfy these criteria, even if no extracted DNA could be
detected by gel electrophoresis for one of our samples.
3.4. DNA extraction from volcanic samples: limits due to
biological contaminations

Fig. 3. DNA extraction from basalt rock sample (B), obsidian glass sample (O)
and control soil sample (C), by two different methods: 1= CTAB-method
(Zhou et al.,1996) and 2= PowerMax Soil DNA Kit (Mobio Laboratories).

Microorganisms are ubiquitous in all environments. Consequently, one critical aspect of using DNA extraction and 16S
rRNA gene sequences for microbial community exploration
concerns the misinterpretation of the data due to the possible
introduction of contaminating cells or rDNA during experimental procedures (Tanner et al., 1998). Volcanic sample contamination could also occur during sampling, for example
introduction of contaminants during collection from desert
surfaces, although careful aseptic collection methods, using
sterile tongs, for example, can prevent these problems. However
in this review, we only focused on contaminant introduction
during molecular analysis.
Indeed, the difficulty in extracting environmental DNA absolutely free from contaminating DNA, coupled with the exquisite

A. Herrera, C.S. Cockell / Journal of Microbiological Methods 70 (2007) 112

sensitivity of PCR to amplify trace target DNA, make contamination a serious issue, particularly with low-biomass samples
(Barton et al., 2006). However, several solutions have been
suggested to validate the results when identifying microbial
communities from critical environmental samples, and we list
them here:
3.4.1. Source of biological contaminations and prevention
Biological contamination is not a specific problem to
volcanic samples per se. However, because volcanic samples
can have low biomass and involve multiple steps to release
DNA from rocks or sediment samples, it is valuable here to
review the potential problems encountered during the preparation of DNA from volcanic environments. Three priority issues
must be addressed to improve the likelihood of successful
contaminant-free DNA extraction and 16S rDNA amplification
from volcanic samples.
3.4.1.1. Contamination derived from the laboratory environment. Contamination with bacterial DNA is evident even in
laboratories dealing with whole-genomic DNA and contemporary low biomass samples (Tanner et al., 1998; Willerslev et al.,
2004). It is therefore prudent to assume that the laboratory
environment is contaminated with microbial cells and microbial
nucleic acids. The easiest approach to efficiently minimise
airborne contamination is certainly to do all sample handling in
a laminar flow hood, as specified by Wade and Garcia working
on modern calcareous microbialites (Wade and Garcia-Pichel,
2003). However, for critical samples, Willerslev et al. (2004)
strongly recommend carrying out all experiments (DNA
extraction and the PCR setup) in positive air hoods or glove
boxes in fully equipped laboratories dedicated to low template
number samples (clean laboratories). They suggest that these
laboratories should be physically separated from other molecular or microbial laboratories, with separate ventilation systems
and nightly UV-irradiation. Equipment and surfaces should be
cleaned regularly with bleach and personnel should also wear
full body suits, sterile gloves, caps and facemasks (Willerslev
et al., 2004).
3.4.1.2. Contamination derived from DNA extraction reagents
and commercial kits. Although commercial standardized extraction methods substantially improve conventional manual
methods, several works have shown the importance of considering potential pitfalls of these methods, especially the possibility of false positive results due to reagent contamination
(van der Zee et al., 2002; Evans et al., 2003; Mohammadi et al.,
2005). Contamination derived from extraction reagents can
result from a number of sources. For example, the identified
bacteria could be inhabitants of a variety of aquatic environments (Mohammadi et al., 2005) or indigenous to ultrapure
water in industrial systems (Kulakov et al., 2002). Therefore, it
is likely that this problem originates from the distilling systems
of the manufacturer (Mohammadi et al., 2005). To overcome the
problem of contaminating DNA in commercial kit reagents,
Mohammadi et al. (2005) suggest a method consisting of reagent filtration with the aid of the GenElute Plasmid Maxiprep

binding columns (Sigma-Aldrich, Germany). These silica-based

membrane columns are constituents of a kit for the isolation of
plasmid DNA from bacterial cultures. This easy-to-perform and
rapid method did not lead to any significant volume loss of the
reagents and did not hamper the sensitivity of the PCR assay.
Therefore, this purification procedure by centrifugation over
GenElute columns can be incorporated into large number of
protocols (Mohammadi et al., 2005).
3.4.1.3. Contamination derived from PCR amplification
procedure. In addition, the PCR amplification step constitutes a significant source of contamination. For example, autoclaving PCR material, such as ultrapure water, tubes and
pipettes tips, kills microbes but cleaves DNA into short,
amplifiable, fragments (~ 100 bp) (Willerslev et al., 2004). The
best way to efficiently decrease the risk of contamination
appears to be treating all reagents with ultra-filtration, tubes
and water with UV-irradiation, plastic and glassware via
baking and/or 5% sodium hypochlorite or 2.5 M HCl for 48 h
(Willerslev et al., 2004).
3.4.2. Checking results: controls and contaminant sequence
database
Several suggestions have been described to distinguish
external contaminants introduced during the molecular analyses
of critical environmental samples, such as low-biomass volcanic samples:
3.4.2.1. Experimental controls. Controls should be included
for every experimental step, such as an air control to monitor
possible contamination from the air circulating within the hood or
glove box, DNA extraction controls using DNA extractions
lacking samples (performed in ratio of 1:5) or PCR controls
(performed in a ratio of 1:1) (Willerslev et al., 2004). Negative
controls should also be included, wherein extracts lacking sample
are prepared in parallel throughout the protocols. These controls
allow traditionally used laboratory substrates that might contain
microbial DNA, potentially amplified through the polymerase
chain reaction (PCR) and contaminating molecular phylogenetic
profiles, to be screened.
3.4.2.2. Contaminant sequence database. While the number
of potential contaminants can be minimized, they cannot be
eliminated from extraction techniques. That is why, to identify
these contaminants, Barton et al. (2006) routinely carried out
negative control samples. By cloning and sequencing DNA
fragments detected in their negative control, they established a
database (Low-Biomass Contaminant (LBC) database, www.
cavescience.com/contaminants.htm), which contains the 16S
rRNA gene sequences of species that have been identified as
common laboratory contaminants. These identified contaminants
provide a reference database to allow investigators to critically
evaluate certain species identified within their phylogenetic
profile when examining such low-biomass environments. They
proposed that any sequences that demonstrated 98% similarity to
an identified contaminant should be regarded with a high level of
skepticism (Barton and Luiszer, 2005).

Table 1
Examples of DNA extraction method development from a large variety of volcanic environments during the last decade
Environmental
sample

Chemical
characteristics

Bacterial
DNA extraction method
level (cells/g)

Extracted DNA
yield and quality

Reference

Andisol
volcanic
ash soil

Clay mineral,
pH between 4.84
and 6.20, organic

109

Two lysis procedures: (bead-beating + 10 min heating 60 C) and (bead-beating + extraction buffer
phenol-chloroformisoamyl), and adding skim milk to the bead-beating to minimize DNA degradation
and absorption to soil particules
DNA extraction method: FastDNA Spin kit for soil (Q-BIOgene)

Detectable DNA
for all samples

Takada Hoshino
and Matsumoto
(2005)

Low-biomass
carbonate
rocks

Rich in calcium,
manganese or
silicates (DNA
binding cations)

105106

Crushing: flame sterilized Plattner's pestle and mortar

1314 ng DNA/l
Extraction Buffer: EGTA (strong calcium chelator)+ lysosyme+ poly-dIdC blocking agents of CaCO3 reactive surfaces
Purification: Phenol-Chloroform with poly-dIdC adding before precipitation and dialyse againts EGTA (to remove
calcium inhibitors of PCR, instead of gel purification which can be a contamination source)

Modern
calcareous
microbialites

Not given

Not given

Disruption matrix methods:

Pulverization using a sterile mortar and pestle (but destruction of spatial architecture of microbial communities)
Acid dissolution using sterile HCl solutions with increasing acidity from 5.0 to 0.8
Chelator-mediated dissolution with sterile
ethylenediaminetetraacetic acid (EDTA disodium salt
dihydrate) solutions increasing concentrations from 500 to
675 mM at pH 5
DNA extraction method: UltraClean Plant DNA Isolation Kit (MoBio Laboratories)

Pulverization:
3.5 g DNA/g
Acid dissolution:
no detectable DNA
EDTA dissolution:
7.8 g DNA/g

Acidic
pH between 1.4 Not given
hydrothermally 3.3
modified
volcanic soils

Comparison between several methods:

FastPrep (Qbiogene)
FastPrep PEG/lysosyme modification
PowerSoil DNA isolation Kit (MoBio Laboratories)
Chemical cell lysis (XanthhogenateSDS buffer)
Chemical-Enzymatic cell lysis (Phenol)
MechanicalChemical-Enzymatic cell lysis (phophate, SDS, Chloroform, bead beater)

None of the six

Henneberger et al.
extraction methods
(2006)
resulted in visible DNA
on gel electrophoresis

Acidic
hydrothermal
volcanic
environments

pH between 15.8 Not given

Comparison between several methods:

CTAB with Chloroform purification
UltraClean (MoBio Laboratories)
FastaDNA (MP Biomedicals)

CTAB= FastaDNA N
UltraClean, except for
3 sites (with DNA
amounts between
41 and 72 g/ml)

Brown and Wolfe

(2006)

Acidic volcanic
rocks

pH 1

UltraClean DNA extraction from crushed rocks (Mobio Laboratories)

3.1mg DNA/ g
crushed rock

Walker et al.
(2005)

Saline Mud
Volcano

1.8 105
pH 6.5, NaCl
dominant brines
with 98.0.
Rich in Al, Fe, Pb

FastDNA Spin kit for soil (Q-BIOgene)

Not given

Yakimov et al.
(2002)

Sediment
samples

Not given

Not given

DNARNA extraction method: Hydroxyapatite (HTP) spin column using after washing and bead-beating
samples in(normaly used to bind and separate nucleic acids and proteins). Elution in Potassium phosphate,
desalting with Sephadex column and ethanol precipitation

43.2 g DNA/g
sediment

Purdy et al.
(1996)

Hot Spring
sediments

Not given

Not given

SDS-CTAB method described by Zhou et al. (1996) and

purification via QIAgen gel extraction kit

Not given

Kanokratana
et al. (2004)

Wade and
Garcia-Pichel
(2003)

A. Herrera, C.S. Cockell / Journal of Microbiological Methods 70 (2007) 112

Not given

Barton et al.
(2006)

10

A. Herrera, C.S. Cockell / Journal of Microbiological Methods 70 (2007) 112

3.5. DNA extraction from volcanic samples: conclusion

The major goal of DNA extraction procedures is to obtain the
greatest DNA recovery, and hence the most representative DNA,
from the microbial community. Considering all of the previous
works, it is therefore apparent that any one DNA extraction
method did not suit all the volcanic samples. It is evident that
the efficiency of a DNA extraction method depends on the
properties of the volcanic environment sample studied.
Currently, the extraction methods mainly suffer from shortcomings, such as DNA sorption to environmental particles, extraction of environmental inhibitors and the loss, degradation or
damage of DNA. And all these shortcomings remain particularly
problematic for DNA extraction from volcanic environments.
However, during the last decade, greater efforts have been made to
improve the DNA extraction process from volcanic environmental samples, taking into account the type of volcanic environment
considered (Table 1), and in the near future, DNA extraction from
complex volcanic environments might be routinely performed.
4. Bacterial and archaebacterial communities in volcanic
environments the value of culture-independent methods
The purpose of this paper is to review methods. However, it
is worthwhile to make some final remarks how such methods
have advanced our understanding of microbial community diversity in volcanic environments.
Compared to the culture-dependent methods, the molecular
techniques based on environmental sample DNA extraction are
able to investigate a far greater proportion of the prokaryotic
communities. Moreover, another significant advantage of using
molecular approaches based on DNA extraction from complex
environments concerns the identification of unknown 16S
rDNA sequences, potentially related to new prokaryotic species.
This advantage is attractive for volcanic environments, which
remain largely unexplored until now. Some of these environments, including hydrothermal settings, are characterised by
extreme environmental conditions. Only few adapted organisms
are able to grow in these environments and they can therefore
offer an extraordinary source of new prokaryotic organisms. In
particular, (Kanokratana et al., 2004) have reported 80% of
unknown 16S rDNA sequences from hot spring sediments,
demonstrating the potential interest in exploring such environments. Indeed, these novel yet to be cultured bacteria represent
an unexplored and unexploited vast gene pool.
5. Conclusion and future challenges
In the last decade, molecular biology approaches based on
DNA extraction have given geomicrobiologists a powerful tool
to explore the microbial communities of rocky environments,
revealing uncharacterized members in both bacterial and archaeal domains. Consequently, considering the diversity of
potential habitats and the great genetic diversity of microbes, the
vast majority of as yet unknown microbes from volcanic
environments could well be a huge source of novel molecular
structures. In this review we have discussed some of the chal-

lenges, and solutions, to extracting DNA from a diversity of

volcanic samples. In the same way in which the cloning and
sequencing of environmental 16S rDNA fragments has lead to
the detection of unknown species, cloning the total environmental DNA or metagenome offers an access to the
diversity of life in volcanic environments without the initial
requirement to culture the microflora. The strategy is to clone the
metagenomic DNA in large pieces into a readily cultured organism
such as Escherichia coli, and screen the clones for biological
activity (Handelsman et al., 1998; Lorenz and Schleper, 2002).
Although this strategy has already been successfully developed in
previous works for discovering the chemical diversity of different
complex environments, such as soil or water (Daniel, 2004; Cottrell
et al., 2005), volcanic environments offer great promise for this
approach. Nevertheless, considering numerous efforts made to
extract high-quantity and quality DNA from a large variety of
volcanic environments, metagenomic library construction might be
the next challenge for geomicrobiologists.
Lastly, despite the utility of culture-independent techniques
based on environmental DNA analyses, there remains a general
need to cultivate microorganisms from volcanic habitats to
better understand their role in environmental processes and the
physiological and metabolic characteristics of the organisms
that inhabit these extreme environments.
Acknowledgments
We thank the Leverhulme Trust (project number F/00 269/N)
under whose support this work was accomplished. We also
thank Diane Johnson (from the Planetary and Space Sciences
and Research Institute, Open University) for her assistance to
use the SEM.
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