4 Discussion
We first gave a thorough description of our workflow in the field,
explaining how we approach subterranean Martian analogue environments in
search for biosignatures. We then presented a case study of blue
microbial mats and their substrates from three lava tubes in Iceland
analyzed with a variety of analytical techniques, namely a portable XRF,
16S rRNA gene sequencing, Raman spectroscopy, and SEM/EDS. Here we
discuss the possible origins of the copper-rich secondary mineral
precipitates, the significance of the bacteria that inhabit them, and
the molecular markers they leave behind therein.
4.1 Cave geology and speleothems
We observed stark differences in elemental concentration (with
preliminary in situ XRF readings) between the substrates where
the microbial mats were sampled and the background areas, i.e.,
uncolonized areas nearby the sampling locations (Figure 5a-c). This
demonstrates an altered geological substrate that biofilms either
contribute to or benefit from in order to thrive in this environment.
Our mineralogical and elemental analysis of the lava rock substrate
(Figure 7) and blue secondary mineral precipitates (Figures 8-10) show
sequestering of elements more generally dispersed and scarce in basaltic
lava tubes, particularly copper. The boundaries between the unaltered
basalt and the precipitate rim, as seen in the SEM images of the thin
sections (Figures 8 & 9), are pristine, dictating that the elements
that make up the observed blue mineral precipitates must have filtered
in with the water through cracks in the cave walls or ceiling, instead
of being leached from the host rock.
4.1.1 Sources of copper in Iceland
The average copper content of recent Icelandic basaltic lava is low, in
the range of 10-200 ppm (μg/g) (Eason & Sinton, 2009; Gibson et al.,
1982), with occasional higher values found in older Miocene
volcaniclastic rocks (Schmincke et al., 1982). Similarly, the copper
content of Icelandic groundwater is also low, ranging from 0.00015 to
0.00209 ppm (Gunnarsdottir et al., 2015) where the Cu(II) ion is the
more common oxidation state (Schock et al., 1995). However, copper is a
semi-volatile element that can partition into a volatile-rich fluid that
can physically separate from magma (Candela & Holland, 1984). Copper
has been observed to be enriched in volcanic laze plumes from basaltic
intraplate volcanoes, which can transfer it directly into the marine
biosphere (Mason et al., 2021), and result in highly copper-enriched
groundwater in specific areas. In the case of Iceland, copper is
enriched in hyaloclastite deposits at the lava-ice interface (Furnes,
1978) and from there could be readily mobilized into groundwater that
can infiltrate the lava tubes through cracks. In addition, rain and
melting snow could be leaching copper from the volcanic ash that abounds
on the lava fields above, bringing it into the tubes through cracks and
cave openings. Kiernan et al. (2003) has noted the hydrogeological
significance of lava tubes in the Eldhraun lava field, with rainwater
and floods transporting glacio-aeolian deposits in these efficient
groundwater flow channels.
Volcanic ash, derived from explosive eruptions of evolved
intermediate-silicic volcanoes, has an evolved nature relative to
basaltic lava flows, characterized by an enrichment in many incompatible
elements, including copper. Hoffmann et al. (2012) showed that copper
was released from various volcanic ash samples in concentrations up to
0.00065 ppm after just 15 min of contact with seawater. Smith et al.
(1982) reported an average concentration of 0.39 ppm of Cu in leachates
from volcanic ash in experiments simulating its interaction with
rainfall and prolonged exposure with groundwater. Experiments with
volcanic ash from the 2000 eruption of the Mt. Hekla volcano in Iceland
showed a Cu flux of 0.069 ppm within 8 hours of mixing with deionized
water (Jones & Gislason, 2008). Though this eruption would not have
reached our areas of interest at the initial deposition stage, the
volcanic cloud spewed out 0.1 Tg of ash (Rose et al., 2003), allowing it
to be transported north-northeast by wind and depositing up to
5 kg/m2 on the headwaters of the Ytri–Rangá River
(Haraldsson, 2001).
There could thus exist many sources of copper in the Ódáðahraun and
Eldhraun lava fields, including the hyaloclastite formations in nearby
Mt. Laki and Mt. Herðubreið. The precise source of ash in the Ódáðahraun
lava field is difficult to determine, as wind plays a large role in
transportation of ash and sand, and a large proportion of it (some of
which may be copper-rich ash and shattered hyaloclastite material) has
been blown long distances over the lavas from sources closer to
Vatnajökull glacier or the Jökulsá á Fjöllum glacial river (Arnalds,
2015). Indeed both the Ódáðahraun and Eldhraun lava fields are subject
to severe wind erosion and very high aeolian deposition rates (Arnalds,
2010; Arnalds et al., 2001), constantly replenishing the areas with
volcanic ash.
4.1.2 Copper abundance on Mars
The surface of Mars is enriched in sulfur (Rudnick & Gao, 2003), and is
thus expected to show elevated concentrations of chalcophile elements
such as copper in the crust (Payré et al., 2019). Copper abundance
values up to 580 ppm were detected by the Curiosity rover in the Liga
sedimentary bedrock at Gale Crater (Berger et al., 2017). A chrysocolla
bearing unit, along with pseudomalachite and other copper mineral
phases, has been identified in the Shalbatana Valley palaeolacustrine
system on Mars, and is hypothesized to be a supergene alteration product
of copper sulfide minerals interacting with oxygen and water (Popa et
al., 2014). This indicates a redox system capable of oxidizing
Cu+ to Cu2+ in an oxidizing
environment in Mars’ past, which would likely have extended to lava
tubes. Copper enrichments seemingly precipitated from groundwater have
been found adsorbed to manganese deposits in the Kimberley Formation in
Gale Crater on Mars, and are thought to be deposited in oxidizing
conditions within fractures in the bedrock (Payré et al., 2019).
4.1.2 Blue speleothems in Iceland
In Figures 8-10 we described copper-rich secondary mineral precipitates
as elucidated by SEM/EDS. While Figure 8 and 10 show a single copper
phase, Figure 9 boasts a more complex precipitate, with discrete layers
of copper silicates, copper phosphates, manganese oxides, and
carbonate-bearing species. The copper phases present are expected to be
mostly in the Cu(II) oxidation state, as this is the ion more readily
available in the groundwater (Schock et al., 1995), and because of the
prominent blue color (Cu(I) minerals are generally red/brown).
Chrysocolla, an amorphous copper phyllosilicate, was found in samples C2
(Figure 8), B5 (Figure 9), and H7 (Figure 10). A similar finding was
previously reported in lava tubes in Kipuka Kanohina Cave Preserve in
Hawai’i by Northup et al. (2011), who also found a blue drop filled with
a precipitate with an Al:Cu:Si ratio of 0.15:1.8:1 as analyzed by EDS,
comparable to a typical chrysocolla ratio of 0.12:1.96:1 (Anthony,
1990). We also identified a layer of pseudomalachite in sample B5, which
has been hinted at often being associated with microbial mats (Little &
Wagner, 2018).
Manganese oxides can be deposited by microbes in waters with manganese
concentration as low as 10-20 ppb (Dickinson et al., 1996). Manganese
oxidation is associated with the metabolism of several genera of
bacteria. Leptothrix (a Proteobacteria associated with strictly
low concentrations of organic matter) has been shown to deposit
amorphous MnO2 (vernadite) and a black
MnO2 precipitate (birnessite) (Gounot, 1994), whileBacillus has been observed to recrystallize birnessite to
octahedral Mn3O4 (hausmannite) (Nealson
et al., 1988). Boston et al. (2001) found manganese oxides in limestone
caves to be linked to biogenic activity, identifying biogenically
precipitated manganese ’snow’ in limestone caves using a suite of
analyses, including culturing Mn-oxidizing bacteria isolated from the
‘snow’ and observing them produce amorphous manganese oxides in the
laboratory.
Ultimately, blue, copper-rich speleothems are attractive targets for
astrobiological research, as they house bacterial communities resistant
to elevated copper content. Should they occur in lava tubes on Mars,
they could also be thought of as a biotope within the caves, host to
extremophilic organisms.
4.2 Bacterial communities inhabiting blue mineral precipitates
The bacteria found in copper-enriched areas in the caves are present
there because they can tolerate such an environment, and they actively
sequester and adsorb copper ions. The significance of the bacteria
identified and their copper-coping mechanisms are discussed below.
Proteobacteria was the major phylum identified in the blue samples in
the southern cave (C2) and northern cave (H7). Families and genera were
generally similar across the samples, with some notable deviations.
Differences in environment (average cave temperature, age of lava tube,
humidity, etc.) may contribute to the differences in bacteria present
inside of the caves from north to south; however, there was a large
overlap in the major abundances found. A recent study (Selensky et al.,
2021) suggests that surface environment is not a major factor in organic
nutrient cycling in lava tubes, which further exemplifies them as
isolated environments that may harbor chemolithoautotrophic organisms
and biosignatures that are useful in the search for life in
extraterrestrial lava tubes. Furthermore, Northup et al. (2016) found
that bacterial diversity in Icelandic lava tubes differed substantially
from that in surface soil samples, with the most abundant cave phylum
being Actinobacteria, followed by Acidobacteria and Proteobacteria.
The majority of genera detected in the blue samples (C2 and H7)
represent copper-resistant oligotrophs. Ralstonia, Caulobacter,
Cupriavidus, and Corynebacterium accounted for 97% of the
relative genus abundance in sample C2 (Figure 6a). In sample H7Ralstonia, Cupriavidus, Caulobacter, andMethylobacterium-Methylorubrum accounted for a total of 99% of
the relative abundance (Figure 6b). Ralstonia, Caulobacter,
Cupriavidus , and Corynebacterium are reported to have high metal
resistance, which explains their presence in high copper concentration
regions of the caves (Janssen et al., 2010; Mergeay et al., 2015;
Morosov et al., 2018; Yang et al., 2019). Copper is essential for
bacteria in trace amounts as it is utilized as a micronutrient as well
as an enzymatic cofactor for redox activities (Giachino & Waldron,
2020). Copper resistance is found in different mechanisms within the
bacteria, the most ubiquitous being a) oxidation of Cu(I) to Cu(II), a
less toxic ion to bacteria; b) copper sequestration by metallothioneins
and c) transmembrane copper export (Ladomersky & Petris, 2015).
Additionally, the genome of Cupriavidus metallidurans containscop genes, which are highly induced by Cu(II) and encode for
copper exporting P1B-type ATPases (Monchy et al., 2020), and cueOgenes encoding for multicopper oxidase (Sanyal et al., 2020).
Much of the literature discusses the ability of both Cupriavidusand Ralstonia to adsorb metals to their cell membrane.
Morphological changes can be seen as they adjust to environments with
high concentrations of metals (Diels et al., 2009). This is also found
with extremely high concentrations of Mn(II), where a strain ofRalstonia picketti was able to survive and also remove the Mn(II)
from aqueous solution possibly by biosorption onto hydroxyl and carbonyl
groups on the bacterial surface, ultimately producing a precipitate
(Huang et al., 2018). These bacteria also induce efficient efflux of
copper transport using metallophores that can regulate uptake of metals
into the cell instead of relying on passive transport that can be deadly
(Kenney & Rosenzweig, 2018; von Rozycki & Nies, 2009).Cupriavidus is found colonizing metal abundant biotopes with
their well-adapted metal resistance (Janssen et al., 2010). The biotopic
relationship copper deposits have with these bacteria entwines their
fates as they grow concurrently alongside each other.
The capability of surviving in oligotrophic environments coupled with
high metal-resistance makes our reported genera particularly well-suited
for Martian and Martian analogue environments. Two novel species ofCupriavidus were discovered in mudflow deposits from Mt.
Pinatubo, where they exhibit chemolithoautotrophic growth using
hydrogen, oxygen, and carbon dioxide in an area deprived of organic
carbon (Sato et al., 2006). Cupriavidus, Ralstonia, andMethylobacterium were found on the Mars Odyssey Orbiter (prior to
flight) and other space industry settings as described by Mijnendonckx
et al. (2013). They described the metal resistance of Cupriavidus
metallidurans and Ralstonia picketti using select metals as
micronutrients. They found Cupriavidus metalliduransisolates to be able to withstand an excess of Cu2+typically toxic to many species of bacteria. These bacteria’s strong
resistance to the antimicrobial disinfection and sterilization
procedures of the space industry not only demonstrate their candidacy as
extremophiles, but further their candidacy as potential contaminants
that may be brought to Mars. The features of these bacteria that allow
them to persist in difficult environments on Earth and potentially on
Mars should also be considered when checking for contaminants in future
missions.
4.3 Molecular biosignatures
Although the presence of colorful pigments in an environment deprived of
light, such as lava tubes, could appear surprising, carotenoids and
other pigments can play different roles depending on the metabolisms of
the host cells. Carotenoid molecules have a wide distribution in very
diverse organisms (more than 750 chemical structures determined to
date), including extremophiles, where they serve several key functions
at the cellular level. In addition to serving as photoprotective
accessory pigments in phototrophic organisms, they have excellent
antioxidant properties acting as reactive oxygen species (ROS)
scavengers thus protecting cellular DNA and proteins (Stahl & Sies,
2003). They are also believed to help stabilize membranes at low
temperatures (Dieser et al., 2018), particularly relevant for Icelandic
microbial communities. It has even been proposed that carotenoids played
an important role in the early evolution of life on Earth (Alcaíno et
al., 2016; Klassen, 2010) by being involved in membrane stabilization,
prior to fatty acids (Ourisson & Nakatani, 1994). These functions and
properties, essential for the highly UV-irradiated early Earth, might
also be compatible with early Mars organisms (Cockell, 2002; Rothschild,
1990), adding to the relevance of carotenoids as a biosignature target
for our search for life on Mars. In addition, although we would not
normally expect phototrophic organisms in cave environments,
cyanobacteria have recently been reported in deep subsurface rock
samples on Earth, where they switch to a light-independent
hydrogen-based lithoautotrophic metabolism (Puente-Sánchez et al.,
2018).
Carotenoids may prove as a critical biosignature in our search for life
in extraterrestrial lava tubes as they are only found in living
organisms, are relatively easy to distinguish and detect, and have been
shown to be highly resistant to oxidative and radiative stress in
simulated Martian conditions (Baqué et al., 2016). Portable Raman
instruments could potentially detect carotenoid molecules in cavesin situ by humans or robotic explorers. As lava tubes are
shielded from radiation on Mars, carotenoids have the potential to be
preserved on even longer timescales. Potential pigmented species found
in our DNA analysis of sample C2 occur in the genera Cupriavidusand Methylobacterium. These are thought to be the source of the
carotenoids identified in the sample (Ramachandran et al., 2014; Osawa
et al., 2015; Heider et al., 2012). The identification of carotenoids in
our samples demonstrate a staying power in this copper-rich environment
that may lend itself to future identification in caves as evidence of
extant or extinct life.