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Discovered in 1986, Movile Cave is an unusual cave ecosystem sustained by in situ chemoautotrophic primary production. The cave is completely isolated from the surface and the primary energy sources are hydrogen sulfide and methane released from hydrothermal fluids. Both condensation and acid corrosion processes contribute to the formation of Movile Cave. Invertebrates, many of which are endemic to Movile Cave, are isotopically lighter in both carbon and nitrogen than surface organisms, indicating that they derive nutrition from chemoautotrophic primary producers within the cave. Here we review work on the microbiology of the Movile Cave ecosystem, with particular emphasis on the functional diversity of microbes involved in sulfur, carbon and nitrogen cycling, and discuss their role in chemosynthetic primary production.
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Geomicrobiology Journal
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Microbiology of Movile Cave—A Chemolithoautotrophic
Deepak Kumaresan
, Daniela Wischer
, Jason Stephenson
, Alexandra Hillebrand-
& J. Colin Murrell
School of Environmental Sciences , University of East Anglia , Norwich , United Kingdom
School of Life Sciences , University of Warwick , Coventry , United Kingdom
Institute of Speleology ‘Emil Racoviţă , ’Bucharest , Romania
Published online: 30 Jan 2014.
To cite this article: Deepak Kumaresan , Daniela Wischer , Jason Stephenson , Alexandra Hillebrand-Voiculescu & J. Colin
Murrell (2014) Microbiology of Movile Cave—A Chemolithoautotrophic Ecosystem, Geomicrobiology Journal, 31:3, 186-193,
DOI: 10.1080/01490451.2013.839764
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Geomicrobiology Journal (2014) 31, 186–193
Taylor & Francis Group, LLC
ISSN: 0149-0451 print / 1521-0529 online
DOI: 10.1080/01490451.2013.839764
Microbiology of Movile Cave—A Chemolithoautotrophic
School of Environmental Sciences, University of East Anglia, Norwich, United Kingdom
School of Life Sciences, University of Warwick, Coventry, United Kingdom
Institute of Speleology ‘Emil R acovit¸
a, Bucharest, Romania
Received February 2013, Accepted August 2013
Discovered in 1986, Movile Cave is an unusual c ave ecosystem sustained by in situ chemoautotrophic primary production. The cave
is completely isolated from the surface and the primary energy sources are hydrogen sulfide and methane released from hydrothermal
fluids. Both condensation and acid corrosion processes contribute to the formation of Movile Cave. Invertebrates, many of which
are endemic to Movile Cave, are isotopically lighter in both carbon and nitrogen than surface organisms, indicating that they derive
nutrition from chemoautotrophic primary producers within the cave. Here we review work on the microbiology of the Movile Cave
ecosystem, with particular emphasis on the functional diversity of microbes involved in sulfur, carbon and nitrogen cycling, and
discuss their role in chemosynthetic primary production.
Keywords: chemoautotrophs, methanotrophs, Movile Cave, nitrifiers, sulfur oxidizers
Cave ecosystems are characterized by lack of light, nearly con-
stant air and water temperatures and relative humidity at near
saturation. They are considered to be challenging environ-
ments for microbes to colonize due to nutrient and energy lim-
itations. Usually the formation of cave systems results from the
seepage of meteoric surface waters into limestone structures
and the energy required for the formation of these caves is en-
tirely supplied by water, air, gravity and fauna from the surface
(Palmer 1991). Similarly, the biological communities within
these cave ecosystems are dependent on the flow of nutrients
and energy from the surface (Engel 2007; Forti et al. 2002).
A small percentage of the world’s caves are of hypogenic
origin, for med by ascending fluids. In this case, the energy
needed to dissolve the rock and support the biological com-
munities inhabiting the caves is supplied by ascending water
and gases (Forti et al. 2002). The geochemistry of hypogenic
caves differs depending on the origin of the rising waters, the
type of host rock and the temperature and composition of the
released gases. From a microbiological perspective, the most
interesting hypogenic caves are those with inputs of gases
such as hydrogen sulfide (H
S) and/or methane (CH
), which
could provide energy sources for microbial communities.
Address correspondence to Prof. J. Colin Murrell, School of En-
vironmental Sciences, University of East Anglia, Norwich NR4
7TJ, United Kingdom; Email:
Color versions of one or more of the figures in the article can be
found online at
Life sustained by chemosynthesis has been extensively stud-
ied using deep-sea hydrothermal vents as model ecosystems
(reviewed in Nakagawa and Takai 2008). Hydrothermal-vent
animals depend on the activity of microorganisms that derive
energy from the oxidation of inorganic compounds (H
S, H
or methane for the conversion of inorganic carbon from CO
into organic matter. These chemoautotrophic bacteria and ar-
chaea form the base of the food web, with heterotrophic bacte-
ria, protists and invertebrates completing the trophic structure
of the ecosystem. Although deep-sea hydrothermal vent en-
vironments can represent unique ecosystems that depend on
energy sources other than light, they are not completely inde-
pendent from inputs of photosynthetically derived nutrients
produced higher in the water column.
Movile Cave, unlike other cave ecosystems, does not receive
significant inputs of meteoric water from the surface, mainly
due to layers of impermeable clays and loess that cover the
limestone in which the cave is developed (Forti et al. 2002). In
this review, we focus on the microbiology of the Movile Cave
ecosystem, particularly on sulfur, carbon and nitrogen cycling
and the role of microbes that act as the primary producers in
this unique ecosystem. It should be noted that microbiological
studies in Movile Cave ecosystem have primarily focussed on
microbial mat floating on the water in the cave.
Movile Cave
Movile Cave ( is located near the town of
Mangalia in Romania, a few kilometers from the Black
Sea (43.825487N; 28.560677E). Geologist Christian Lascu
Downloaded by [Deepak Kumaresan] at 19:52 31 January 2014
Microbiology of Movile Cave 187
discovered the cave in 1986 when an artificial shaft, dug for
geological investigation created access to the narrow cave pas-
sages. Despite the lack of photosynthetically fixed carbon,
the cave hosts a remarkable diversity of invertebrates, such
as worms, insects, spiders and crustaceans (Sarbu 2000). Life
within the Movile Cave ecosystem is maintained entirely by
chemoautotrophy (Sarbu et al. 1996) an analogous system to
some deep-sea hydrothermal vents where chemoautotrophic
and methylotrophic bacteria make a substantial contribution
to primary production, which in t urn support a variety of
macrofauna (reviewed in Campbell 2006; Dubilier et al. 2008;
Lutz and Kennish 1993).
Paleogeographical evidence suggests that some animal
species were trapped in Movile Cave as early as 5.5 million
years ago (Falniowski et al. 2008; Sarbu and Kane 1995). Over
time, these animals have become adapted to life in the dark
and 33 out of 48 invertebrate species observed are endemic to
Movile Cave (Sarbu et al. 1996). In comparison to deep-sea
hydrothermal vents, the Movile Cave ecosystem offers easier
access to an interesting model ecosystem to study food-web
interactions, primarily driven by chemoautotrophic primary
production and microbial biomass.
Movile Cave—Formation and Features
Movile Cave is formed from two major corrosion processes:
condensation corrosion by carbon dioxide (CO
) and acid
corrosion by sulfuric acid (H
) (Sarbu and Lascu 1997).
Sulfuric acid corrosion that is active in the lower partially
submerged cave passages is a result of the oxidation of H
to H
in the presence of oxygen from the cave atmosphere
(Equation 1). Sulfuric acid then reacts with the limestone walls
of the cave, causing accelerated dissolution and leading to for-
mation of gypsum (calcium sulfate dihydrate) deposits on the
cave walls along with release of CO
(Equation 2). This type of
corrosion is highly efficient and also promotes condensation
corrosion due to the release of large quantities of CO
et al. 2002).
Condensation corrosion, a slower process compared to sul-
furic acid corrosion, affects the walls in the upper dry passages
of the cave and occurs when warm water vapour from the ther-
mal waters ascends and condenses on the colder walls and ceil-
ings in the upper cave passages. Carbon dioxide from the cave
atmosphere dissolves in the condensate to form carbonic acid
(Equation 3), which dissolves the carbonate bedrock forming
bicarbonate (Equation 4) (Sarbu and Lascu 1997). Carbon
dioxide in the cave is released from limestone dissolution, the
biological oxidation of methane and heterotrophic respiration
processes. Due to the absence of H
S in the upper level of the
cave, no effects of sulfuric acid corrosion are encountered here.
S + 2O
(sulfide oxidation) (1)
+ CaCO
+ H
+ CO
(sulfuric acid driven corrosion) (2)
+ H
(carbonic acid for mation) (3)
+ CaCO
+ Ca
(condensation corrosion) (4)
Although the upper passages of the cave (approximately 200 m
long) are completely dry (as a consequence of the lack of
water infiltration from the surface), the lower level (approxi-
mately 40 m long) is partly flooded by hydrothermal waters,
which contain substantial concentrations of H
(0.2 mM) and ammonium (NH
) (0.3 mM) (Figure 2)
(Sarbu and Lascu 1997). The air bells (air pockets, shown in
Figure 1; Sarbu et al. 1996) present in the cave create an ac-
tive redox interface on the surface of the water in the cave
where bacteria in floating microbial mats oxidize the reduced
sulfur compounds, methane and ammonium from the water
using O
from the atmosphere. Consequently, macrofauna in
Movile Cave appear only to live in proximity to the microbial
mats within these air bells, while the upper dry nonsulfidic
cave passages are devoid of macrofauna (Forti et al. 2002).
Physicochemical Conditions in Movile Cave
The water flooding the lower level of the cave is of hydrother-
mal origin and high in H
S (0.2–0.3 mM), CH
(0.02 mM) and
(0.2–0.3 mM), whereas oxidized compounds were not
detected (Sarbu 2000). The flow rate of the water is reported to
Fig. 1. Cross-section of Movile Cave (taken from the PhD thesis of Daniel Muschiol). © Dr. Walter Traunspurger, University of
Bielefeld, Germany. Reproduced by permission of Dr. Walter Traunspurger, University of Bielefeld, Germany. Permission to reuse
must be obtained from the rightsholder.
Downloaded by [Deepak Kumaresan] at 19:52 31 January 2014
188 Kumaresan et al.
Fig. 2. Schematic representation of microbial carbon, nitrogen and sulfur cycling in Movile Cave. soxB gene encodes the SoxB
component of the periplasmic thiosulfate-oxidizing Sox enzyme complex; dsrAB gene encodes for the α and β subunits of the
dissimilatory sulfite reductase enzyme; nifH encodes for the nitrogenase reductase subunit; amoA encodes for the α subunit of
ammonia monooxygenase (both bacteria and archaea); nirS and nirK encodes for a copper and a cytochrome cd1-containing nitrite
reductase enzyme, respectively. pmoA encodes the α subunit of the particulate methane monooxygenase enzyme, mmoX encodes
the α subunit of the hydroxylase of the soluble methane monooxygenase enzyme and mxaF encodes the α subunit of the methanol
dehydrogenase enzyme. mauA encodes for the small subunit of the methylamine dehydrogenase enzyme. gmaS encodes for the
gamma-glutamylmethylamide synthetase.
be 5 L per sec (Sarbu and Lascu 1997) and its physiochemical
properties are not affected by seasonal climatic changes (S.
Sarbu, personal communication, April 2011). The water is at
a constant pH of 7.4 due to the buffering capacity of the car-
bonate bedrock. Dissolved oxygen ranges between 9–16 μM
at the water surface and decreases to less than 1 μM after the
first few centimeters, with anoxic conditions encountered in
deeper water (Sarbu 2000).
The air temperature in the lower cave passage ranged from
C, and the temperature of the water is 20.9
C (Sarbu
and Lascu 1997) and the relative humidity ranges between 98
and 100% (Sarbu 2000). The air in the upper dry level of the
cave contains 20–21% O
, yet the air bells in
the lower level contain only 7–10% O
up to 2.5% CO
1–2% (v/v) methane (Sarbu 2000). Hydrogen sulfide is found
in proximity to the air-water interface but not in the upper level
(Sarbu 2000). Extensive microbial mats composed of bacteria,
fungi and protozoa float on the water surface (kept afloat by
rising methane bubbles) and also grow on the limestone walls
of the cave (Sarbu et al. 1994a).
Since the discovery of Movile Cave, studies have provided
evidence that the ecosystem is isolated from the surface. Ra-
dioactive artificial nuclides
Sr and
Cs, which were released
as a result of the 1986 Chernobyl nuclear accident, have b een
found in high concentrations in soil and in lakes surrounding
Movile Cave, as well as in the Black Sea and in sediments
of other caves but not within Movile Cave itself (Sarbu et al.
A Chemolithoautotrophic Ecosystem
In order to study chemolithoautotrophy in Movile Cave, mi-
crobial mat samples were incubated with
This resulted in incorporation of radioactive carbon into
microbial lipids, providing the first evidence for chemoau-
totrophic carbon fixation at the redox interface (Sarbu et al.
1994b; Sarbu et al. 1996). Furthermore, activity of ribulose-
1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a key
enzyme of the Calvin cycle, was observed in homogenates
of microbial mat samples, as well as in lysates of bacteria
cells cultivated from cave water, supporting the hypothesis that
chemosynthate is being produced in situ (Sarbu et al. 1994b).
Stable isotope ratio analyses of carbon and nitrogen pro-
vided conclusive evidence that Movile Cave is a self-sustained
ecosystem dependent on chemoautotrophically fixed carbon
(for detailed discussion, refer to Sarbu et al. 1996).
Sulfur Metabolism
Sulfur-oxidizing bacteria, first described by Winogradsky
(1887), are a heterogeneous group of organisms sharing the
ability to oxidize reduced inorganic sulfur compounds, and
are distributed within the domains of Bacteria and Archaea.
A comprehensive overview of the biochemistry and molecular
biology of sulfur oxidation can be found in Ghosh and
Dam (2009). The Movile Cave ecosystem contains a high
Downloaded by [Deepak Kumaresan] at 19:52 31 January 2014
Microbiology of Movile Cave 189
concentration of H
S and primary production is dominated
by sulfur oxidizing bacteria (Sarbu et al. 1996).
Sulfur-oxidizing bacteria (SOB) were the main focus of
early studies characterizing microbial diversity and activity
in Movile Cave (Rohwerder et al. 2003; Sarbu et al. 1994a;
Vlasceanu et al. 1997). Sarbu and colleagues ( 1994a), using
microscopic observations, identified Thiothrix and Beggiatoa-
like filamentous bacteria in the floating mat, and Vlasceanu
et al., (1997) isolated a Thiobacillus thioparus strain from cave
water samples and characteriz ed this bacterium at both the
physiological and molecular level. Using most probable num-
ber (MPN) enumeration, Rohwerder and colleagues (2003)
showed that SOB that could use sulfur, tetrathionate and thio-
sulfate as energy sources, existed at up to 10
colony forming
units (CFU) per g mat and that sulfate reducers capable of
reduction of sulfate to H
S were also present in the microbial
Clone libraries targeting bacterial 16S rRNA gene se-
quences in microbial mat DNA revealed sequences related
to the 16S rRNA genes of Thiobacillus (Betaproteobacte-
ria), Thiovirga, Thiothrix, Thioploca (Gammaproteobacteria)
and Sulfuricurvum (Epsilonproteobacteria) (Chen et al. 2009).
Employing an identical approach, also using Movile Cave
mat DNA, Porter et al. (2009) reported similar results on
the diversity of SOB with retrieval of additional 16S rRNA
gene sequences related to Halothiobacillus and Thiomonas
(Betaproteobacteria and Gammaproteobacteria, respectively)
and Sulfurospirillum (Epsilonproteobacteria). Clone libraries
of soxB genes (encoding the SoxB component of the
periplasmic thiosulfate-oxidizing enzyme complex) revealed
the widespread distribution of soxB sequences from Alpha-,
Beta- and Gammaproteobacteria. The retrieved sequences were
closely related to the soxB genes of Thiobacillus, Methyli-
bium petroleiphilum (both belonging to Betaproteobacteria),
Thiothrix and Halothiobacillus (65% identity) belonging to
By targeting RuBisCO, specifically the form I green type
RuBisCO gene (cbbL) sequences, Chen et al. (2009) de-
tected sequences closely related (80 85% identity) to Ru-
BisCO gene sequences of Thiobacillus denitrificans and T.
thioparus. DNA-stable isotope probing (SIP) (Dumont and
Murrell 2005; Radajewski et al. 2000) experiments using
C-labelled bicarbonate showed that SOB from the Beta-
and Gammaproteobacteria (Thiobacillus, Thiovirga, Thiothrix,
Thioploca) were particularly active in assimilating CO
et al. 2009).
Rohwerder and colleagues (2003) detected an extremely
acidophilic SOB, which is interesting considering the pH of the
water in Movile Cave is neutral. Based on a study in Frasassi
Caves (Galdenzi et al. 2008), Sarbu et al. (2002) reported pH
values of 3.8–4.5 on the surface of the microbial mats covering
the limestone walls in the remote air bells, suggesting that
not all of the sulfuric acid produced by SOB is immediately
buffered and indicating the possibility of ecological niches that
can be occupied by acidophilic bacteria in Movile Cave.
Rohwerder et al. (2003) also reported activity of faculta-
tively anaerobic SOB in Movile Cave, which were capable of
using nitrate (NO
) rather than oxygen as an alternative elec-
tron acceptor, and which were present at the same levels as
obligately aerobic SOB (10
CFU per gram dry weight of
mat), suggesting that both groups could contribute substan-
tially to the biomass produced. Chen et al. (2009) detected 16S
rRNA gene sequences related to Sulfuricurvum (Epsilonpro-
teobacteria), which can oxidize sulfur anaerobically (Kodama
and Watanabe 2004) in DNA extracted from Movile Cave
microbial mat, supporting the evidence for anaerobic sulfur
oxidation demonstrated by Rohwerder et al. (2003).
Endosymbiotic sulfur-oxidizing bacteria, living within in-
vertebrates, have been reported in various habitats, such as
deep sea hydrothermal vents and mangrove swamps (Distel
1998; Dubilier et al. 2008; Wood and Kelly 1989). Although
SOB benefit from sulfide, oxygen and CO
from the host,
the bacteria in turn supply organic compounds to the host
(Dahl and Prange 2006). Recently, Bauermeister et al. (2012)
reported the presence of Thiothrix ectosymbionts associated
with Niphargus species in Frasassi caves. It would be interest-
ing to look for the presence of symbiotic sulfur-oxidizing bac-
teria associated with higher organisms present in the Movile
Cave ecosystem.
Although less abundant than SOB, sulfate-reducing bacte-
ria (SRB) have been shown to be present in Movile Cave. They
appear to belong to a higher trophic level, using the organic
carbon released by SOB and other primary producers as the
electron donor (Rohwerder et al. 2003). Sulfate reducers in
sulfidic caves appear to fall mainly within the Deltaproteobac-
teria (Engel 2007). Sequences related to members of the family
Desulfobulbaceae have been found in Movile Cave in two in-
dependent 16S rRNA gene based clone library analyses (Chen
et al. 2009; Porter et al. 2009). Thus far, no Archaea capable
of oxidizing reduced sulfur compounds have been reported in
the Movile Cave ecosystem, or in fact from any other sulfidic
caves, probably due to the fact that most characterized Ar-
chaea that oxidize reduced sulfur compounds grow at elevated
temperatures (Chen et al. 2009; Engel 2007).
One-Carbon Metabolism—Methanotrophy
and Methylotrophy
Aerobic methanotrophy, the ability to use methane as a sole
carbon and energy source, is found in bacteria within the
phyla Proteobacteria and Verrucomicrobia. The biochemistry
and molecular biology of aerobic methane oxidation in
Bacteria has been extensively reviewed (Trotsenko and
Murrell 2008). Use of specific biomarkers targeting both
16S rRNA and key metabolic genes of methanotrophs to
infer phylogeny has also been reviewed (McDonald et al.
2008). Air bells within the Movile Cave contain methane and
oxygen and are therefore favourable environments for aerobic
methane-oxidizing bacteria.
Hutchens e t al. (2004) using DNA-SIP experiments with
, identified active methanotrophs, belonging to both
the Alpha- and Gammaproteobacteria in Movile Cave wa-
ter and microbial mat. Based on analysis of 16S rRNA and
functional genes (pmoA and mmoX, encoding the active-site
subunit of particulate methane monooxygenase and soluble
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190 Kumaresan et al.
methane monooxygenase, respectively) it was shown that
strains of Methylomonas, Methylococcus and Methylocys-
tis/Methylosinus had assimilated
. Sequences of non-
methanotrophic bacteria and an alga (Ochromonas danica,
based on 18S rRNA gene sequence analysis) were also re-
trieved from
C-labelled DNA in heavy fractions, indicating
the possibility of non-methanotrophs cross-feeding on
labelled biomass or metabolites arising from the initial con-
sumption of
by methanotrophs.
Both 16S rRNA and mxaF (encoding the active-site sub-
unit of methanol dehydrogenase) clone libraries from the
heavy DNA (
C-labelled DNA) contained sequences similar
to the extant methylotrophs Methylophilus and Hyphomicro-
bium, suggesting that these organisms had assimilated
methanol excreted by methanotrophs metabolizing
The extent of the contribution of methanotrophs to primary
production within the Movile Cave ecosystem is not known
and future research should focus on understanding their role
as primary producers. The possibility of anaerobic methane
oxidation (Knittel and Boetius 2009) occurring in Movile Cave
also warrants investigation in the future.
Other Methylotrophs
Methylotrophs utilize reduced carbon substrates that have no
carbon carbon bond (eg methanol and methylated amines),
as their sole carbon and energy source (Chistoserdova et al.
2009). Enumeration studies by Rohwerder et al. (2003) re-
vealed methylotrophs in the floating mat at up to 10
per gram dry weight of mat. It is possible that these methy-
lotrophs can f eed on methanol released by methanotrophs
during methane oxidation. Chen et al. (2009) retrieved soxB
and cbbL sequences related to those of M. petroleiphilum,a
facultative methylotroph. Recently, Kalyuhznaya et al. (2009)
reported that methylotrophs within the Methylophilaceae, par-
ticularly some species of Methylotenera, require nitrate for
growth on methanol. Investigating the role of methylotrophy-
linked to denitrification within the Movile Cave ecosystem will
yield more insights into the interactions between the carbon
cycle and the nitrogen cycle in this ecosystem.
Chen et al. (2009) reported that the obligate methy-
lated amine-utilizing methylotroph Methylotenera mobilis
was present in high numbers, while Methylophilus and
Methylovorus were also detected. Similar studies by Porter
et al. (2009) also detected 16S rRNA gene sequences related
to the 16S rRNA genes of Methylotenera and Methylophilus.
Methylated amines are also a nitrogen source for a wide range
of nonmethylotrophic bacteria and the metabolic pathways
involved have been examined recently (Chen et al. 2010a,
2010c; Latypova et al. 2010). DNA stable isotope probing
experiments with
C-mono methylamine (CH
) revealed
Methylotenera mobilis as a dominant methylotroph utilizing
in Movile Cave mat samples, and also indicated the
presence of a novel facultative methylotroph which has now
been isolated and characterized (Wischer et al. unpublished).
As methylated amines can serve as both C and N source for mi-
crobial communities, a deeper understanding of the f unctional
diversity of methylated amines utilizers in this ecosystem will
add vital information on nutrient cycling in Movile Cave.
Nitrogen Cycling
Over the past two decades, studies targeting microbial nutri-
ent cycling within the Movile Cave ecosystem have focused
largely on sulfur and carbon, whereas microbial nitrogen cy-
cling has received little attention. Although the nitrifiers were
not the major focus, results from the DNA-SIP based study by
Chen et al. (2009) implied that ammonia- and nitrite-oxidizing
bacteria might be important primary producers in the Movile
Cave ecosystem alongside sulfur- and methane-oxidizing bac-
teria. Although ammonium concentrations in the cave waters
are relatively high (0.2–0.3 mM), nitrate has not been detected
(Sarbu 2000). This could be due to a rapid turnover of ni-
trate by either assimilatory nitrate reduction or denitrification.
Facultatively anaerobic sulfur oxidizers, such as Thiobacillus
denitrificans, are known to use NO
as an alternative electron
acceptor for respiration in oxygen-depleted conditions (Claus
and Kutzner 1985).
Therefore sulfur oxidation linked to denitrification could
be an important process in Movile Cave. This hypothesis was
also supported by Rohwerder et al. (2003), who detected high
numbers of SOB in enrichments with thiosulfate and nitrate
incubated under anoxic conditions. Chen et al. (2009) also re-
ported retrieval of 16S rRNA gene sequences related to den-
itrifiers from the phylum Denitratisoma from DNA isolated
from Movile Cave mat samples.
Microbial N
fixation may be another significant process of
the nitrogen cycle in the Movile Cave ecosystem. The ability
to fix N
is widespread among bacteria and archaea and many
bacteria present in Movile Cave are known N
fixers (such as
Beggiatoa and Methylocystis) (Murrell and Dalton 1983; Nel-
son et al. 1982). However, reduction of N
to NH
is highly
energy-consuming and generally carried out during nitrogen-
limited conditions (Postgate 1972). Although there are rela-
tively high standing concentrations of NH
in the cave water,
there may well be nitrogen-depleted niches within the micro-
bial mats where N
fixation could play a role. Process-based
-fixation measurements and assaying for nifH transcripts in
cave mat samples could determine whether N
fixation occurs
in this ecosystem.
Archaeal Microbial Communities
Archaeal microbial c ommunities are suggested to play an im-
portant role in nutrient recycling within cave ecosystems (Che-
lius and Moore 2004; Gonzalez et al. 2006; Northup et al.
2003). Over the years, the focus of research on microbial sys-
tems in Movile Cave has been on Bacteria whereas no in-depth
study has been performed on the contribution of Archaea to
nutrient cycling.
Recent results from Chen et al. (2009) revealed that the ar-
chaeal community in the Movile Cave (based on 16S rRNA
gene libraries) possessed some archaea that have been found in
deep-sea hydrothermal vents (originally shown by Takai and
Downloaded by [Deepak Kumaresan] at 19:52 31 January 2014
Microbiology of Movile Cave 191
Horikoshi 1999) although no sequences related to ammonia-
oxidizing archaea (major nitrifiers in deep-sea hydrother-
mal environments), methanogens, sulfur-oxidizing archaea
or anaerobic methane oxidizing-archaea were found in their
study. It is essential to target Archaeal communities in future
experiments to determine if they play a significant role in C,
N and S cycling.
Also, it should be noted that in the few studies investi-
gating the microbiology of Movile Cave using cultivation-
independent techniques (Chen et al. 2009; Hutchens et al.
2004; Porter et al. 2009) mat and water samples were used,
while microbial communities in Movile Cave sediment remain
largely unexplored. The likely lack of oxygen as an electron ac-
ceptor in sediments suggests that alternative electron acceptors
such as nitrate, nitrite, sulfate, CO
and metals such as Fe
and Mn
, together with organic matter deposited from float-
ing mats, could be important in sediment and anoxic zones
of the cave water. In fact we have evidence for the presence
of methanogens in Movile Cave sediments, based on amplifi-
cation of the mcrA gene from DNA extracted from sediment
Whole Genome Sequencing of Microbial Isolates
Whole genome sequencing of microorganisms has provided
important insights into their genetic capacity and the plethora
of available microbial genome sequences enables us to perform
comparative genomics between organisms that might occupy
different ecological niches but perform the same function.
Whereas molecular ecology studies using genes (16S rRNA
gene and metabolic genes) as biomarkers reveal the phylogeny
of microbes in a complex ecosystem, efforts should also be
focussed on isolating new microorganisms.
Rapidly reducing sequencing costs will enable us to se-
quence the genomes of novel and interesting microbial iso-
lates, which will not only provide access to their genetic and
metabolic potential, but also the ability to design focussed
biochemical and physiological experiments based on the in-
formation from the genome sequence. We have isolated a
Methylomonas strain from Movile Cave mat samples and the
genome is being sequenced. This will provide a comprehensive
overview of its genetic potential and also allow us to compare
it with the genomes of Methylomonas species from other envi-
Outlook: Microbial Community Composition Analysis
Using Next-Generation Sequencing Techniques
Next generation sequencing provides a new vista for molecular
microbial ecology research, allowing us to carry out a detailed
examination of microbial diversity in an ecosystem. Tremen-
dous progress has been made in understanding microbial sys-
tems by using either targeted gene sequencing (PCR-based
screen) or shotgun metagenomic sequencing that eliminates
any bias due to primer design and PCR (Gilbert and Dupont
A preliminary small-scale metagenomic sequencing of
DNA from Movile Cave mat samples yielded approximately
960,000 sequences, with a mean length of 360bp. Analysis of
the metagenomic data using MG-RAST (Meyer et al. 2008)
assigned the sequences to annotated proteins (36.8%), un-
known proteins (33.7%) and ribosomal sequences (1.9%). Of
the annotated sequences, 96.5% were of bacterial origin, 1.8%
eukaryotic, 1.3% archaeal and 0.2% were viral sequences.
Phylum-level phylogenetic classification revealed that 60%
of the total annotated sequences belong to Proteobacteria,
alongside bacteroidetes (12.1%) and firmicutes (7.6%). Inter-
estingly, nearly 3% of the total sequences retrieved were rep-
resentative of cyanobacterial sequences, which would not be
expected in a Movile Cave ecosystem devoid of light. In-depth
analysis of metagenomic data will yield better insights into
the functional diversity, genetic potential and role in nutrient
cycling of microbial communities in the ecosystem.
Using a combination of techniques such as SIP and metage-
nomic sequencing of
C-labelled-DNA (Chen et al. 2008;
Chen and Murrell 2010; Chen et al. 2010b) we can reduce
the complexity of sequence information obtained and partic-
ular functional groups can be targeted. Using this approach,
a composite genome of Methylotenera mobilis was extracted
from the metagenomic sequences from Lake Washington sed-
iment DNA (Kalyuzhnaya et al. 2008). Similar strategies can
be used in Movile Cave to obtain genome information for
microbes that are difficult to isolate and cultivate in the labo-
ratory. Although metagenomic analysis provides us with infor-
mation about the genetic potential of the microbial communi-
ties within an ecosystem, use of metatranscriptomics (Moran
et al. 2013) and metaproteomics (Seifert et al. 2012) will allow
access to the transcriptomes and proteomes, respectively, of
the active microbial communities.
Although we do have a basic understanding of individual
communities e.g., SOB, methanotrophs, further research is
required to understand the trophic interactions between dif-
ferent microbial functional guilds in Movile Cave. Sediment
and anoxic water microbial communities remain largely un-
explored and questions, such as whether there is any biogenic
methane production or anaerobic methane oxidation, remain
unanswered. Next-generation sequencing, combined with a
suite of molecular ecology techniques and a concerted effort
to isolate novel organisms, will improve our understanding
of the functional diversity of the microbial communities and
allow us to study the contributions of different functional
guilds in maintaining this self-sustaining chemoautotrophic
We thank Serban Sarbu and Rich Boden for valuable dis-
cussions, Vlad Voiculescu and Mihai Baciu for sampling and
Andrew Crombie for critical comments on the manuscript.
We acknowledge the funding from the Natural Environ-
ment Research Council (NERC) to JCM (NE/G017956),
Downloaded by [Deepak Kumaresan] at 19:52 31 January 2014
192 Kumaresan et al.
the NERC and Earth and Life Systems Alliance (ELSA) for
funding DK, a Warwick Postgraduate Research Fellowship
and University of East Anglia-ELSA funding for DW and a
NERC-CASE studentship for JS.
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... For the elaboration of the measurement protocol five samples from three different field sites were investigated; three samples (named RT1, RT2, RT6) from the river Río Tinto, Spain (Amils et al., 2007), one from the Movile Cave (named MC) (Chen et al., 2009;Kumaresan et al., 2014;Kumaresan et al., 2018) and one from the Sulphur Cave (named SC) (Sarbu et al., 2018), that are both located in Romania. These three field sites were selected for measurements because they reflect some environment conditions that occurred, or may still exist, on current Mars. ...
... The cave system is composed of an upper dry section, which connects to the deeper submerged passages that are flooded by mesothermal waters originating from an underlying sulfidic aquifer. Several airbells exist deeper into the cave which are separated from the upper section of the cave by the submerged passages (see, e.g., Fig. 1 in Kumaresan et al. (2014)). The water flowing through the cave contains high levels of H 2 S (0.2-0.3 mM), CH 4 (0.02 mM), NH 4 + (0.2-0.3 mM), and has a constant pH level of 7.4 at a constant temperature 20.9°C. ...
... A rich and diverse microbial community is present in the cave with high numbers of SOB and SRB, among many others. A more detailed description of this cave system can be found in, e.g., Chen et al. (2009), Kumaresan et al. (2014), Kumaresan et al. (2018). ...
Full-text available
The signatures of element isotope fractionation can be used for the indirect identification of extant or extinct life on planetary surfaces or their moons. Element isotope fractionation signatures are very robust against the harsh environmental conditions, such as temperature or irradiation, which typically prevail on solar system bodies. Sulphur is a key element for life as we know it and bacteria exist, such as sulphur reducing bacteria, that can metabolize sulphur resulting in isotope fractionations of up to −70‰ δ ³⁴ S. Geochemical processes are observed to fractionate up to values of −20‰ δ ³⁴ S hence, fractionation exceeding that value might be highly indicative for the presence of life. However, the detection of sulphur element isotope fractionation in situ , under the assumption that life has existed or still does exist, is extremely challenging. To date, no instrument developed for space application showed the necessary detection sensitivity or measurement methodology for such an identification. In this contribution, we report a simple measurement protocol for the accurate detection of sulphur fractionation δ ³⁴ S using our prototype laser ablation ionization mass spectrometer system designed for in situ space exploration missions. The protocol was elaborated based on measurements of five sulphur containing species that were sampled at different Mars analogue field sites, including two cave systems in Romania and the Río Tinto river environment in Spain. Optimising the laser pulse energy of our laser ablation ionization mass spectrometer (LIMS) allowed the identification of a peak-like trend of the ³⁴ S/ ³² S ratio, where the maximum, compared to internal standards, allowed to derive isotope fractionation with an estimated δ ³⁴ S accuracy of ∼2‰. This accuracy is sufficiently precise to differentiate between abiotic and biotic signatures, of which the latter, induced by, e.g., sulphate-reducing microorganism, may fractionate sulphur isotopes by more than −70‰ δ ³⁴ S. Our miniature LIMS system, including the discussed measurement protocol, is simple and can be applied for life detection on extra-terrestrial surfaces, e.g., Mars or the icy moons like Europa.
... This cave, located in south-eastern Romania (Dobrogea region), developed in oolitic and fossil-rich limestone of Sarmatian age (Late Miocene) and sealed off during the Quaternary by a thick and impermeable layer of clays and loess [17]. Movile Cave has a complex geological evolution with an ongoing speleogenesis driven by two main processes: the sulfuric acid corrosion in the partially submerged, lower cave level; and the condensation-corrosion processes active in the upper level of the cave [18]. The upper gallery (approx. ...
... Our current understanding of microbial life within the Movile Cave ecosystem was limited to the hydrothermal waters, as only microbial mats, water samples and Lake sediments were previously investigated [18,19]. Our study gives a first insight into the cave's chemoautotrophic and primary production potential beyond the hydrothermal waters, namely the cave's sediments. ...
Full-text available
Background Movile Cave (Dobrogea, SE Romania) hosts a subterranean chemoautotrophically-based ecosystem supported by a sulfidic thermal aquifer analogous to the deep-sea hydrothermal ecosystems. Our current understanding of Movile Cave microbiology has been confined to the thermal water proximity (no more than 2 m distant), with most studies focusing on the water-floating mat, which likely acts as the primary production powerhouse in this sulfidic ecosystem. To gain more insightful information on the functioning of the sulfidic Movile Cave ecosystem, we employed a metagenomics-resolved approach to reveal the microbiome diversity, metabolic potential, and interactions and infer its roles within the food webs in the sediments beyond the sulfidic thermal waters. Results A customized bioinformatics pipeline led to the recovery of 106 high-quality metagenome-assembled genomes from 7 cave sediment metagenomes. Assemblies’ taxonomy spanned 19 bacterial and three archaeal phyla with Acidobacteriota, Chloroflexota, Proteobacteria, Planctomycetota, Ca . Patescibacteria, Thermoproteota, Methylomirabilota , and Ca . Zixibacteria as prevalent phyla. Functional gene analyses allowed prediction of CO 2 fixation, methanotrophy, sulfur and ammonia oxidation as possibly occurring in the explored sediments. Species Metabolic Coupling Analysis of metagenome-scale metabolic models revealed the highest competition-cooperation interactions in the sediments collected at the farthest distance from the sulfidic water. As a result of simulated metabolic interactions, autotrophs and methanotrophs were hypothesized as major donors of exchanged metabolites in the sediment communities. Cross-feeding dependencies were assumed only towards ‘currency’ molecules and inorganic compounds (O 2 , PO 4 ³⁻ , H ⁺ , Fe ²⁺ , Cu ²⁺ ) in the sediment nearby sulfidic water, whereas hydrogen sulfide and methanol are predictably traded exclusively among communities dwelling in the distant gallery. Conclusions These findings suggest that the primary production potential of the Movile Cave expands way beyond its hydrothermal waters, enhancing our understanding of ecological interactions inside chemolithoautotrophically based subterranean ecosystems and their functioning.
... It should be emphasized that underground objects display harsh living conditions for microorganisms because they are heterotrophic ecosystems (with few exceptions, e.g., the Movile Cave "Peştera Movile" in Romania, a sulfur-based chemolitho-313 autotrophic ecosystem [81]) Despite this fact, in this study we reported the presence of 10 keratindependent genera, namely Aphanoascus, Arthroderma, Aspergillus, Chrysosporium, Cordyceps, Cosmospora, Keratinophyton, Metapochonia, Penicillium, and Pseudogymnoascus ( Table 2). All of these belong to the phylum Ascomycota, which is in agreement with Vanderwolf [12]. ...
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Despite speleomycological research going back to the 1960s, the biodiversity of many specific groups of micromycetes in underground sites still remains unknown, including keratinolytic and keratinophilic fungi. These fungi are a frequent cause of infections in humans and animals. Since subterranean ecosystems are inhabited by various animals and are a great tourist attraction, the goal of our research was to provide the first report of keratinophilic and keratinolytic fungal species isolated from three caves in Tatra Mts., Slovakia (Brestovská, Demanovská Lǎdová and Demanovská Slobody). Speleomycological investigation was carried out inside and outside the explored caves by combining culture-based techniques with genetic and phenotypic identifications. A total of 67 fungal isolates were isolated from 24 samples of soil and sediment using Van-breuseghem hair bait and identified as 18 different fungal species. The study sites located inside the studied caves displayed much more fungal species (17 species) than outside the underground (3 species), and the highest values of the Shannon diversity index of keratinophilic and keratinolytic fungi were noted for the study sites inside the Demänovská Slobody Cave. Overall, Arthroderma quadrifidum was the most common fungal species in all soil and/or sediment samples. To the best of our knowledge, our research has allowed for the first detection of fungal species such as Arthroderma eboreum, Arthroderma insingulare, Chrysosporium europae, Chrysosporium siglerae, Keratinophyton wagneri, and Penicillium charlesii in underground sites. We also showed that the temperature of soil and sediments was negatively correlated with the number of isolated keratinophilic and keratinolytic fungal species in the investigated caves.
... Hutchens et al. (2004) also identified aerobic methanotrophs using the methane present in the air bells (see Figure 1.12) as the other key primary producers. Both methylotrophs and methanotrophs have since been isolated and genome sequenced (Ganzert et al., 2014;Kumaresan et al., 2014). In terms of interations, Flot et al. (2014) investigated an association between Amphipods (Niphargus genus) and Thiothrix sulphur-oxidizing ectosymbiotic bacteria. ...
Microbial ecology is the science of micro-organisms and their biotic and abiotic interactions in a given ecosystem. As technology has advanced, molecular techniques have been widely used to overcome the limitations of classical approaches such as culturing and microscopy. Indeed, the development of Next Generation Sequencing (NGS) technologies in the past twenty years has largely helped to unravel the phylogenetic diversity and functional potential of microbial communities across ecosystems.Nonetheless, most of the environments studied through these techniques concentrated on relatively easily accessible, tractable and host-related ecosystems such as plankton (especially in marine ecosystems), soils and gut microbiomes. This has contributed to the rapid accumulation of a wealth of environmental diversity and metagenomic data along with advances in bioinformatics leading to the development of myriads of tools. Oxygen-depleted environments and especially their microbial eukaryote components are less studied and may lead to future phylogenetic and metabolic discoveries.In order to address this, we conducted analyses on two poorly studied suboxic ecosystems: Movile Cave (Romania) and lake Baikal sediments (Siberia, Russia). In this task, we aimed at unveiling the taxonomic and functional diversity of microorganims in these environments.To do so, I first evaluated the available bioinformatics tools and implemented a bioinformatics pipeline for 16S/18S rRNA gene-based metabarcoding analysis, making reasoned methodological choices. Then, as a case study, I carried out metabarcoding analyses of the water and floating microbial mats found in Movile Cave in order to investigate its protist diversity. Our study showed that Movile Cave, a sealed off chemosynthetic ecosystem, harbored a substantial protist diversity with species spanning most of the major eukaryotic super groups. The majority if these protists were related to species of freshwater and marine origins. Most of them were putatively anaerobic, in line with the cave environment, and suggesting that in addition to their predatory role, they might participate in prokaryote-protist symbioses.In a second study, I applied my metabarcoding pipeline to explore unique and relatively unexplored environment of Lake Baikal sediments. I first applied a metabarcoding approach using 16S and 18S rRNA genes to describe prokaryotic as well as protist diversity. Overall, the communities within these ecosystems were very diverse and enriched in ammonia-oxidizing Thaumarchaeota. We also identified several typical marine taxa which are likely planktonic but accumulate in sediments. Finally, our sampling plan allowed us to test whether differences across depth, basin or latitude affected microbial community structure. Our results showed that the composition of sediment microbial communities remained relatively stable across the samples regardless of depth or latitude.In a third study, we applied metagenomics to study the metabolic potential of communities associated to Baikal sediments and to reconstruct metagenome-assembled genomes (MAGs) of dominant organisms. This revealed the considerable ecological importance of Thaumarchaeota lineages in lake Baikal sediments, which were found to be the major autotrophic phyla and also very implicated in the nitrogen cycle. Chloroflexi and Proteobacteria-related species also appeared ecologically important.This PhD thesis reveals the taxonomic diversity of poorly studied suboxic ecosystems and therefore contributes to our knowledge of microbial diversity on Earth. Additionally, the analyses of surface sediment samples in lake Baikal adds new light on freshwater-marine transitions. The metagenomic analyses reported here allowed us to postulate a model of nutrient cycle carried out by microorganismsin these sediments. Overall, this work sheds light on the microbial ecology of oxygen-depleted environments, and most notably lake Baikal surface sediments.
... In cave soil, the load of the viral population is negligible, and only 0.2% of sequences were of the virus in 960,000 total sequences [88]. The study of virus diversity in cave environments remains a hot research topic. ...
Full-text available
The world is constantly facing threats, including the emergence of new pathogens and antibiotic resistance among extant pathogens, which is a matter of concern. Therefore, the need for natural and effective sources of drugs is inevitable. The ancient and pristine ecosystems of caves contain a unique microbial world and could provide a possible source of antimicrobial metabolites. The association between humans and caves is as old as human history itself. Historically, cave environments have been used to treat patients with respiratory tract infections, which is referred to as speleotherapy. Today, the pristine environment of caves that comprise a poorly explored microbial world is a potential source of antimicrobial and anticancer drugs. Oligotrophic conditions in caves enhance the competition among microbial communities, and unique antimicrobial agents may be used in this competition. This review suggests that the world needs a novel and effective source of drug discovery. Therefore, being the emerging spot of modern human civilization, caves could play a crucial role in the current medical crisis, and cave microorganisms may have the potential to produce novel antimicrobial and anticancer drugs.
... Due to the absence of photoautotrophs, the subterranean food webs are fundamentally dependent on allochthonous resources (Ferreira et al. 2007;Schneider et al. 2011). Less frequently, some communities are structured based on roots that reach the subterranean systems (Howarth 1983) and, more rarely, from organic matter originated from chemosynthesis (Kumaresan et al. 2014). In this context, caves were considered for a long time to be extreme environments unable to support a diverse fauna (Gilbert and Deharveng 2002;Simon et al. 2007). ...
Full-text available
Semiarid regions experience conspicuous seasonal variations, especially related to precipitation. Caves in these areas can be exceptions since they are less affected by dry seasons. In the north of the Brazilian semiarid, there are structurally heterogeneous karst areas with significant speleological potential and several anthropogenic impacts, with a neglected subterranean fauna. Therefore, we aimed to evaluate the influence of caves and external environmental features on the species richness and composition of cave invertebrates of this region. We expected that the caves would have high species richness and endemism, high dissimilarity among spatially discontinuous regions, and caves with water or guano would have greater overall richness and troglobitic species richness than those without water or guano. We collected invertebrates in 40 caves and recorded 416 species from 145 families and 45 orders (38.28 ± 13.88 spp./cave). We identified 57 species with troglomorphic traits, most having narrow distributions, including phylogenetic and/or geographic (oceanic) relicts, suggesting a lengthy and complex evolutionary history. In addition to the faunal dissimilarity among hydrographic basins and caves with or without water, our data indicate the variety of resources, the native vegetation surrounding the caves, the area, and the number of entrances as relevant variables of species composition and richness variation. Caves with water or guano had the highest richness of troglobites. The study area is unique in South America for having such a high concentration of troglobites associated with the presence of karstic aquifers and paleoclimatic changes (including oceanic transgressions and regressions), thus deserving emergency conservation actions.
... Microorganisms associated with dark and nutrient-poor cave areas are able to survive easily in this type of environment due to their chemoautotrophic mechanisms [44]. In fact, the majority of bacterial communities found in extreme environments are associated with nitrogen-, sulfur-, and methane-based chemoautotrophic pathways [45]. ...
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Carbon utilization of bacterial communities is a key factor of the biomineralization process in limestone-rich curst areas. An efficient carbon catabolism of the microbial community is associated with the availability of carbon sources in such an ecological niche. As cave environments promote oligotrophic (carbon source stress) situations, the present study investigated the variations of different carbon substrate utilization patterns of soil and rock microbial communities between outside and inside cave environments in limestone-rich crust topography by Biolog EcoPlate™ assay and categorized their taxonomical structure and predicted functional metabolic pathways based on 16S rRNA amplicon sequencing. Community level physiological profiling (CLPP) analysis by Biolog EcoPlate™ assay revealed that microbes from outside of the cave were metabolically active and had higher carbon source utilization rate than the microbial community inside the cave. 16S rRNA amplicon sequence analysis demonstrated, among eight predominant bacterial phylum Planctomycetes, Proteobacteria, Cyanobacteria, and Nitrospirae were predominantly associated with outside-cave samples, whereas Acidobacteria, Actinobacteria, Chloroflexi, and Gemmatimonadetes were associated with inside-cave samples. Functional prediction showed bacterial communities both inside and outside of the cave were functionally involved in the metabolism of carbohydrates, amino acids, lipids, xenobiotic compounds, energy metabolism, and environmental information processing. However, the amino acid and carbohydrate metabolic pathways were predominantly linked to the outside-cave samples, while xenobiotic compounds, lipids, other amino acids, and energy metabolism were associated with inside-cave samples. Overall, a positive correlation was observed between Biolog EcoPlate™ assay carbon utilization and the abundance of functional metabolic pathways in this study.
The area around the town of Mangalia in Southern Dobrogea (Romania) hosts a unique karst landscape represented mostly by large collapse dolines. Around one of these collapse dolines, named Obanul Mare, there is a peculiar morphology of hillocks that resemble the tropical labyrinth-cone karst but on a much-reduced scale. This peculiar relief was hypothesized to be residual, resulting from the ceiling collapse of a two-dimensional maze cave. This type of maze caves form when the water table remains stable for long periods of time, allowing groundwater to dissolve the carbonate host rock along all available discontinuities. The Obanul Mare structure contains a 200 m long cave (Movile cave), whereas a few kilometers away we can find Limanu maze cave, whose passages sum up to more than 3,500 m. Because the existence of a former maze cave is difficult to demonstrate, in this study we took an indirect approach to this issue. We hypothesize that if there was a maze cave and its ceiling collapsed, then the inner walls would be left standing as pillars. Subsequent subaerial evolution of these pillars would create slopes that would incline towards the former cave passages. By quantifying the slope aspect from a high-resolution digital surface model, we would be able to identify the direction of the original passages, hence the main hypothetical fracture lines along which those passages formed. By comparing these orientations to those of local fractures (deduced from the nearby valley and doline axes), as well as with the orientations of the Movile Cave passages, we found that they are well correlated. This strengthens the hypothesis that the hillocky terrain is tectonically conditioned and that it might be the result of cave ceiling collapse, bringing more information on the existence of maze caves in the region.
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Sulphidic cave ecosystems are remarkable evolutionary hotspots that have witnessed adaptive radiation of their fauna represented by extremophile species having particular traits. Ostracods, a very old group of crustaceans, exhibit specific morphological and ecophysiological features that enable them to thrive in groundwater sulphidic environments. Herein, we report a peculiar new ostracod species Pseudocandona movilaensis sp. nov. thriving in the chemoautotrophic sulphidic groundwater ecosystem of Movile Cave (Romania). The new species displays a set of homoplastic features specific for unrelated stygobitic species, for e.g., triangular carapace in lateral view with reduced postero–dorsal part and simplification of limb chaetotaxy (i.e., loss of some claws and reduction of secondary male sex characteristics), driven by a convergent or parallel evolution during or after colonization of the groundwater realm. P. movilaensis sp. nov. thrives exclusively in sulphidic meso-thermal waters (21°C) with high concentrations of sulphides, methane, and ammonium. Based on the geometric morphometrics-based study of the carapace shape and molecular phylogenetic analyses based on the COI marker (mtDNA), we discuss the phylogenetic relationship and evolutionary implication for the new species to thrive in groundwater sulphidic groundwater environments.
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A year-long study of the sulfidic aquifer in the Frasassi caves (central Italy) employed chemical analysis of the water and measurements of its level, as well as assessments of the concentration of H 2 S, CO 2 , and O 2 in the cave air. Bicarbonate water seepage derives from diffuse infiltration of meteoric water into the karst surface, and contributes to sulfidic ground-water dilution, with a percentage that varies between 30% and 60% during the year. Even less diluted sulfidic ground water was found in a localized area of the cave between Lago Verde and nearby springs. This water rises from a deeper phreatic zone, and its chemistry changes only slightly with the seasons with a contribution of seepage water that does not exceed 20%. In order to understand how the H 2 S oxidation, which is considered the main cave forming process, is influenced by the seasonal changes in the cave hydrology, the sulfide/total sulfur ratio was related to ground-water dilution and air composition. The data suggest that in the upper phreatic zone, limestone corrosion due to H 2 S oxidation is prominent in the wet season because of the high recharge of O 2 -rich seepage water, while in the dry season, the H 2 S content increases, but the extent of oxidation is lower. In the cave atmosphere, the low H 2 S content in ground water during the wet season inhibits the release of this gas, but the H 2 S concentration increases in the dry season, favoring its oxidation in the air and the replacement of limestone with gypsum on the cave walls.
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Cave development in the Madison aquifer of the Black Hills has taken place in several stages. Mississippian carbonates fi rst underwent eogenetic (early diagenetic) reactions with interbedded sulfates to form breccias and solution voids. Later sub-aerial exposure allowed oxygenated meteoric water to replace sulfates with calcite and to form karst and small caves. All were later buried by ~2 km of Pennsylvanian– Cretaceous strata. Groundwater fl ow and speleogenesis in the Madison aquifer were renewed by erosional exposure during Laramide uplift. Post-Laramide speleogenesis enlarged paleokarst voids. Most interpretations of this process in the Black Hills invoke rising thermal water, but they fail to account for the cave patterns. Few passages extend downdip below the present water table or updip to outcrops. None reaches the base of the Madison Limestone, and few reach the top. Major caves underlie a thin cover of basal Pennsylvanian–Permian Minnelusa Formation (interbedded quartzarenite and carbonates). Water infi ltrating through the Minnelusa Formation dissolves car-bonates in a nearly closed system, producing low pCO 2 , while recharge directly into Madison outcrops has a much higher pCO 2. Both are at or near calcite saturation when they enter caves, but their mixture is undersaturated. The caves reveal four phases of calcite deposition: eogenetic ferroan calcite (Mis-sissippian replacement of sulfates); white scalenohedra in paleovoids deposited during deep post-Mississippian burial; palisade crusts formed during blockage of springs by Oligocene–Miocene continental sediments; and laminated crusts from late Pleisto-cene water-table fl uctuations. The caves reveal more than 300 m.y. of geologic history and a close relationship to regional geologic events.
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Movile Cave, recently discovered in southern Romania, contains sulfide‐rich thermal waters in submerged passages, as well as isolated air pockets. The water surfaces within the air pockets are covered by substantial microbial biofilms, while the air bells contain an abundant and diverse community of terrestrial and aquatic animal species. Based on the results of dehydrogenase activity, fecal streptococci counts, and stable carbon isotope ratios, we propose that the cave community is biologically isolated and receives little, if any, organic carbon inputs from the surface environment. Several sulfide‐oxidizing chemoautotrophic bacteria were isolated from the cave waters. One putative Thiosphaera sp. strain, LV‐43, was further characterized. The presence and high level activity of RuBisCO was clearly demonstrated in this strain.
Stable isotope probing (SIP) is a technique that is used to identify the microorganisms in environmental samples that use a particular growth substrate. The method relies on the incorporation of a substrate that is highly enriched in a stable isotope, such as 13C, and the identification of active microorganisms by the selective recovery and analysis of isotope-enriched cellular components. DNA and rRNA are the most informative taxonomic biomarkers and 13C-labelled molecules can be purified from unlabelled nucleic acid by density-gradient centrifugation. The future holds great promise for SIP, particularly when combined with other emerging technologies such as microarrays and metagenomics
The thermomineral sulfurous waters at Mangalia in southeastern Dobrogea, Romania, have been known and used as spa facilities for well over 2,000 years (Feru and Capotà 1991). Hydrogeologieal studies performed during the last 60 years (Macovei 1912; Ciocîrdel and Protopopescu-Pache 1955; Moissiu 1968; Feru and Capotà 1991) identified a deep captive sulfurous aquifer located in Barremian-Jurassic limestones, extending 15 km to the North and 50 km to the South of Mangalia. In the Mangalia region, a system of geological faults allows the deep water to ascend toward the surface and mix with the Sarmatian oxygenated waters (Lascu et al. 1993). The biological investigation of the subsurface ecosystems associated with the sulfurous waters at Mangalia commenced in the late eighties, after the discovery of Movile Cave and its unique subterranean chemoautotrophically based ecosystem (Sarbu, 1990).
A number of representative species of obligate methane-oxidizing bacteria were surveyed for their ability to fix N2 by growth experiments and the acetylene reduction test. Although all strains exhibited growth on nitrogen-free plates, only type II organisms and the type X methanotroph Methylococcus capsulatus (Bath) grew well in nitrogen-free liquid medium and were capable of active acetylene reduction. N2-fixation in type II methanotrophs was less sensitive to O2 than in the type X methanotroph Methylococcus capsulatus (Bath) and batch cultures of type II organisms could be established at pO2 values of up to 0.2 bar. N2-fixation in Methylococcus capsulatus (Bath) was inhibited at pO2 values above 0.15 bar and the 'switch-off' of nitrogenase activity by ammonia was also observed in this organism.