Document [original]

Raman spectroscopic analysis of the calcium

oxalate producing extremotolerant

lichen Circinaria gyrosa

U. Böttger

, J. Meessen

, J. Martinez-Frias

, H.-W. Hübers

1,4

, F. Rull

, F. J. Sánchez

R. de la Torre

and J.-P. de Vera

German Aerospace Center DLR e.V,Institute of Planetary Research,Rutherfordstr. 2,12489 Berlin,Germany

e-mail: [email protected]e

Institut für Botanik,Heinrich-Heine-Universität,Universitätsstr.1,40225-Düsseldorf,Germany

Centro de Astrobiología (CSIC-INTA),Ctra. de Ajalvir km. 4,28850-Torrejón de Ardoz,Madrid,Spain

Technische Universität Berlin,Institut für Optik und Atomare Physik,Hardenbergstr. 36,10623 Berlin,Germany

Instituto Nacional de Técnica Aeroespacial (INTA),Ctra. de Ajalvir km. 4,28850-Torrejón de Ardoz,Madrid,Spain

Abstract: In the context of astrobiological exposure and simulation experiments in the BIOMEX project,

the lichen Circinaria gyrosa was investigated by Raman microspectroscopy. Owing to the symbiotic nature

of lichens and their remarkable extremotolerance, C. gyrosa represents a valid model organism in recent and

current astrobiological research. Biogenic compounds of C. gyrosa were studied that may serve as

biomarkers in Raman assisted remote sensing missions, e.g. ExoMars. The surface as well as different

internal layers of C. gyrosa have been characterized and data on the detectability and distribution of

β-carotene, chitin and calcium oxalate monohydrate (whewellite) are presented in this study. Raman

microspectroscopy was applied on natural samples and thin sections. Although calcium oxalates can also be

formed by rare geological processes it may serve as a suitable biomarker for astrobiological investigations. In

the model organism C. gyrosa, it forms extracellular crystalline deposits embedded in the intra-medullary

space and its function is assumed to balance water uptake and gas exchange during the rare, moist to wet

environmental periods that are physiologically favourable. This is a factor that was repeatedly demonstrated

to be essential for extremotolerant lichens and other organisms. Depending on the decomposition processes

of whewellite under extraterrestrial environmental conditions, it may not only serve as a biomarker of recent

life, but also of past and fossilized organisms.

Received 13 May 2013, accepted 30 July 2013, first published online 17 October 2013

Key words: biomarker, calcium oxalate, Circinaria gyrosa, Raman spectroscopy.

Introduction

The search for former, extant or recent life on other celestial

objects of our Solar system will be one of the priority goals of

future planetary missions (Steele & Beaty). The answer to the

question of the uniqueness of life on Earth will change man-

kind’s view in all ﬁelds of science, philosophy and society and

will be of the uttermost relevance for our perspective on bio-

science. Since its characteristics and constraints are roughly

unknown, the search for extraterrestrial life must be prepared

very carefully. It is essential to know what to look for before

ﬂying to other planets, satellites and moons. Methodological

diversity, objectiveness in interpretation and detailed back-

ground knowledge are crucial prerequisites for planning

scientiﬁc experiments on future planetary missions in the

Solar system. One astrobiological approach in reference to this

question is to investigate the chemical, physiological and

ecological characteristics of terrestrial extremotolerant and

extremophile organisms with respect to their adaptations to

predominant abiotic factors and harsh environmental con-

ditions. Such studies will broaden our knowledge on the limits

and limitations of terrestrial life and thus may help to focus our

efforts on the detection of life.

Several spaceﬂight and ground based experiments were

already performed with colonies of (cyano-)bacteria, fungi and

lichens that survived to a certain extent real space exposure or

simulated space conditions and were able to recover their

activity afterwards (de Vera et al.2003,2004a,b,2010;dela

Torre et al.2004; de la Torre Noetzel et al.2007,2010; Sancho

et al.2007; Sancho 2009; Raggio et al.2011; Onofri et al.2012;

Sánchez et al.2012). The Biology and Mars Experiment

(BIOMEX) is an international and interdisciplinary Low

Earth Orbit (LEO) exposure experiment which will be per-

formed on EXPOSE-R2 –the ESA exposure facility attached

to the Russian Module Zwesda on the International Space

Station (ISS). This facility is designed for the exposure of

samples to vacuum, temperature, solar and cosmic radiation as

well as the insolation regimen of the LEO (Rabbow et al.2009,

2012; Cottin 2011; de Vera et al.2012). The launch is scheduled

for April 2014. The exposure duration is scheduled to be

1.5 years. BIOMEX focuses on two main objectives: (1) The

ﬁrst objective of BIOMEX is to investigate the resistance and

International Journal of Astrobiology 13 (1): 19–27 (2014)

doi:10.1017/S1473550413000293 ©Cambridge University Press 2013

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possible decomposition of biomolecules like pigments and

cellular components under LEO-space and simulated Mars-

like conditions. (2) The second objective is to analyse to what

extent terrestrial extremophiles are able to survive LEO-space

and Mars-like conditions, and to determine which interactions

between biological samples and selected minerals (including

original terrestrial substrata as well as Moon- and Mars

analogues) could occur.

Biominerals –biogenically formed minerals –may serve as

biomarkers as they may be remnant for geological time-scales

even if the producing organisms itself vanished long before

(Thomas-Keprta et al.2000). As indicators of biological ac-

tivity, these minerals are of high interest for the search for

former life on other planetary objects, e.g. Mars. Several

extremotolerant micro-organisms were identiﬁed to excrete

oxalic acid into their surrounding where it disintegrates the

rock and binds leaching calcium ions to form calcium oxalate

deposits (Sohrabi 2012). This process allows that many organi-

sms grow into the rock to shelter from extreme environmental

conditions and facilitate bio-weathering, a potential biomarker

itself. In the context of future planetary missions, the charac-

teristics and detectability of biogenically formed oxalates need

to be understood in detail, especially if extremotolerant or-

ganisms prove to produce such compounds as an adaptation to

harsh environmental conditions. Among 13 other organisms,

Circinaria gyrosa is chosen as a biosample in BIOMEX.

C. gyrosa is a lichen, namely a symbiotic association composed

of a fungal and a photosynthetic partner. After the investi-

gation of this lichen species in space and in Martian-simulated

environment (Sancho et al.2007; de la Torre Noetzel et al.

2010; de Vera 2012), and the positive results of tolerance and

resistance obtained under these extreme conditions, it can be

conﬁrmed that it is a suitable model organism in Astrobiology.

We will take a step further trying to understand the process of

biomineralization under harsh environmental parameters.

In the present study, we will show that C. gyrosa is producing

calcium oxalate monohydrate in restricted areas of its thallus.

Thus, this lichen is suitable to investigate the products of bio-

mineralization under extreme environmental conditions.

We will present the Raman spectral characteristics of calcium

oxalate monohydrate, its precise distribution in the lichen

thallus, and suggest a hypothesis on its biological role. More-

over, we will demonstrate its role as a biomarker as well as the

Raman spectral characteristics of additional biomarker

compounds.

Raman spectroscopy is a non-destructive method to inves-

tigate the molecular and crystal structure of a sample. It is

based on inelastic scattering of monochromatic light, i.e. a

laser. The energetic shift of the reﬂected laser photons gives

the Raman spectrum of the sample. The Raman spectrum is

unique for the sample and thus allows its identiﬁcation. As the

Raman laser spectrometer (RLS) is scheduled to be part of the

future planetary exploration mission ExoMars (Esa robotic

exploration of mars), this technique is used in the present work

to determine biominerals as remanent biomarkers. This will

be demonstrated by investigating the characteristics and the

distribution of calcium oxalate within the lichen C. gyrosa by

Raman spectroscopy. The interpretation of Raman spectra

requires considerable a priori information and background

knowledge. We will contribute in the present study to the

background knowledge of Raman spectra of C. gyrosa. Besides

a valuable progress in understanding the internal structure and

Raman properties of a lichen model organism, the results can

be applied for future astrobiological experiments, especially in

the context of the BIOMEX project and the possible changes

that C. gyrosa might undergo during space exposure.

The following two steps have been applied to investigate and

interpret the Raman characteristics of C. gyrosa: (1) thallus

thin sections are used to determine the amount and distribution

of calcium oxalate and other lichen substances within the

lichen; (2) a fractured part of the thallus is examined to ﬁgure

out which of the previous ﬁndings can be identiﬁed as well on a

natural sample. To substantiate our ﬁndings, the distribution

of inner medullary crystals was veriﬁed by scanning electron

microscopy (according to Meeßen et al.,2013) and the identity

of calcium oxalate was checked by a complementary speciﬁc

reaction test (according to (Pizzolato 1964)).

The morphological and anatomical structures of C. gyrosa

are brieﬂy described in The lichen species C. gyrosa section.

The sample preparation and the experimental setup for the

Raman measurements are presented in the Methods section.

The measurement results will be presented and discussed in

the Results and discussion section with emphasis on the par-

ticular implications for C. gyrosa and the general role of

calcium oxalates in lichens followed by summary and an

outlook section.

The lichen species C. gyrosa

The subject of the present study is the lichen C. gyrosa

(nom. provis. (Sohrabi 2012), recently renamed from Aspicilia

fruticulosa). This lichen species naturally grows in steppe-like

and continental deserts of the Northern hemisphere, and

therefore, it is adapted to extreme environmental conditions

such as heat, long-term drought and high levels of insolation

(Sancho et al.2000). Our samples were collected at an open

forest area of the steppic highlands of Guadalajara (Spain),

near the locality of Zaorejas. They grow scattered in the clay-

like soil of a region characterized by drastic diurnal and

seasonal variations in terms of temperature and rainfall. It has

also survived simulated Mars- and space conditions (Sancho

et al.2007,2008; de la Torre Noetzel et al.2010; Sánchez et al.

2012). The coralloid to compact, vagrant thalli of C. gyrosa

are frequently relocated by wind and water and are often found

partially embedded in its clay-like substrate. Its fruticose thalli

are brownish to ochre and measure up to 2.5 cm in diameter.

At the branch tips, the pores (pseudocyphellae) could appear

as whitish openings and play a crucial role in the lichen’s

gas exchange (Sancho et al.2000; Meeßen et al.). Compared

with other lichen species used in astrobiological studies (as

Xanthoria elegans, Rhizocarpon geographicum and Buellia

frigida), C. gyrosa reveals intriguing morphological and ana-

tomical differences that may help to explain its high resistance

to harsh environmental but also to simulated Martian and to

20 U. Böttger et al.

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space conditions (Meeßen et al.): Thin sections of the lichen

branchings (Fig. 1) reveal distinct structures and a complex

internal stratiﬁcation. The outer cortex of the thalli is com-

posed of a brownish pigmented layer of dead cells followed by a

layer of vital isodiametric cells. Below, the lichen forms a

compact and extended subcortex consisting of tightly packed

fungal hyphae which are agglutinated by high amounts of

extracellular mucilage. That subcortex is supposed to contrib-

ute to the lichen’s mechanical stability which is necessary due

to its vagrant life-style. Nonetheless, naturally occurring thalli

are more or less frequently fractured. In addition, the subcortex

acts as a diffusion barrier for gas and water exchange, and as an

irradiation-protective layer. Below, algal cell clusters are distri-

buted evenly. Most inward, the central tube of each branching

as well as the layer in which the algal clusters are embedded are

ﬁlled with a medullary fungal tissue that is rich in inner aerial

spaces and connected to the surrounding air by the tip-located

pores.

Methods

Raman analysis of thin sections of C. gyrosa

Thin layer sections of 15–30 μmofC. gyrosa (Fig. 1) were

obtained from the distal branches of a representative specimen

by using a cryostatic microtome (Frigomobil 1206, Reichert-

Jung, −20 to −30 °C). Some specimens of the sections were

used to examine and document the morphological–anatomical

characteristics under the microscope (Axio Microscope and

Imager A1, Carl Zeiss AG) after staining with 5% lactoglycer-

ol/cotton blue. In addition, thin sections were ﬁxed on a glass

slide with 1% gelatine, air-dried for 8 hours and investigated by

Raman laser spectroscopy.

Raman measurements were performed with the confocal

WITec Alpha 300 system with a Nikon 10× (NA 0.25) objec-

tive (WITec 2008). The Raman laser excitation wavelength

of 532 nm was applied and a spectral resolution of about

4cm

−1

was achieved in a spectral range between 100 and

3800 cm

−1

. The ideal spot size on the sample is in focus at

about 1.5 μm. The laser power was set to 1 mW for the thin

section. The measurements were performed in air at room

temperature and under ambient air pressure. Single spectra,

line scans and image scans with up to 175 μm×175 μm and up

to 22.500 image points were measured. The image scans were

performed on ﬂat thin sections to identify the distribution of

the typical lichen substances within the different tissues of

C. gyrosa.

Raman analysis of the complete thallus of C. gyrosa

In a second step, Raman measurements were made on whole,

natural lichen thalli (Fig. 2). Taking into account that these

thalli are erratic and live freely in the ground, they are subjected

to all kinds of environmental inﬂuence and so could be found

fractured. Single spectra measurements and line scans were

obtained from the natural lichen with rough, uneven and

sometimes damaged surfaces. Here, it is of interest to ﬁnd out

which of the results from the thin section can be reproduced

with the natural sample.

Results and discussion

General characteristics of obtained Raman spectra of

C. gyrosa

Detailed investigation performed by image scanning revealed

differences in the distribution of Raman signatures for the

various anatomical structures and hyphal tissues of C. gyrosa.

Thus, physiologically different thalline layers were distinguish-

able by Raman imaging. Typical Raman spectra found in the

thin section and representing basic compounds of the lichen are

shown in Fig. 3. This investigation focuses on β-carotene as an

accessory pigment of the photosynthetic apparatus of the algal

symbiont and chitin as the main component of the cell wall of

the fungal symbiont, both with known Raman signatures. The

peak positions of the spectra representing the lichen com-

pounds are assigned correspondingly.

The spectrum of calcium oxalate monohydrate Ca

)×

O(Fig. 3), referred to as whewellite in mineralogical

Fig. 1. Thin section of a thalline branch tip of Circinaria gyrosa below

the distal pseudocyphellae. From the outside to the centre, the section

depicts the dark pigmented outer cortex, followed by the extended,

dense, and highly gelatinated subcortex and evenly distributed algal

clusters. The centre is made of loose medullary hyphal tissue. The thin

section measures 30 μm and is stained with 5% lactoglycerol-blue.

A digital light microscope (Axio imager A1, Zeiss) was used for the

analysis and recording of the micrographs.

Fig. 2. Fractured part of the thallus of Circinaria gyrosa; the circle

encloses the area investigated with Raman microspectroscopy,

including all internal stratiﬁcation (depicted in Fig. 1) for ulterior

reproduction of results.

Raman spectroscopic analysis of the calcium oxalate producing lichen C. gyrosa 21

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context, is characterized by the double peak at 1466 and

1493 cm

−1

, an additional peak at 1636 cm

−1

of the CO sym-

metric stretching vibration, the C–C stretching mode peak at

898 cm

−1

, and the CO

mode double peak at 505 and 524 cm

−1

(Shippey 1980; Frost & Yang 2003]. The double peak at 1466

and 1493 cm

−1

is well pronounced and suitable for interpret-

ation purposes. Nevertheless, polarization effects have to be

considered due to the orientation of the whewellite crystals.

The polarization effect inﬂuences the relative intensities of the

two peaks. On the other hand, this effect can be used as an

indication for the orientation of the whewellite crystals, e.g. in

image scans. The same is true for the less pronounced double

peak at 505 and 524 cm

−1

. The main, characterizing Raman

shifts of β-carotene (red spectrum in Fig. 3) are typically about

1008, 1158 and 1518 cm

−1

. The 1518 and 1158 cm

−1

bands are

strong and characteristic for the C=C and C–C stretching

vibrations (Edwards et al.2005). The smaller feature at

1008 cm

−1

corresponds to the rocking modes of the CH

groups (Vitek et al.2010).

The spectrum assigned to chitin (green spectrum in Fig. 3)

contains a large number of lines and bands. Only the main

Raman shifts are mentioned here: the bands between 1085 and

1125 cm

−1

represent C–O–C and C–O stretching, the band

at 1380 cm

−1

represents the rocking CH

vibrations, and the

2915 cm

−1

band is associated with the C–H

stretching mode

(Ehrlich et al.2007). The lines between 1260 and 1360 cm

−1

can be assigned to amide bands (Ehrlich et al.2007). It is also

important to take into account the Raman spectrum of the

background that is a glass slide on which the thin sections

are mounted. The spectrum of the glass slide is characterized

by a broad band around 1111 cm

−1

and two smaller but also

broad bands around 564 and 2405 cm

−1

. The main band of the

background overlaps with the main band of chitin. To decide if

there is a glassy background the band about 2405 cm

−1

is used.

In the following investigation, only the main lines and bands

are considered. Special care is taken for overlapping Raman

bands and lines among the compounds and the background.

The dotted squares around the speciﬁc Raman bands and lines

in Fig. 3 mark the spectral ranges used for the integral ﬁlters

for analysis and interpretation. These ﬁlters integrated over

Raman shift ranges that contained characteristic lines for

the lichen substances for calcium oxalate monohydrate

(whewellite), carotene and chitin. The integral ﬁlter ranges

were 1450–1482 cm

−1

for calcium oxalate monohydrate (whe-

wellite), 1130–1185 cm

−1

for carotene and 2835–3000 cm

−1

for chitin. As seen in Fig. 3, the chosen integral ﬁlter ranges do

not overlap and thus are appropriate to be used for the

representation of the substances in the colour-coded images

mentioned below. The range between 2340 and 2480 cm

−1

unaffected by the Raman spectra of the lichen substances. To

avoid false interpretation, this range is used to identify the

areas of the underlying glass slide in the images. Combining

these integral ﬁlter ranges, colour-coded images were derived

as represented in Figs 4-right, 5-right and 6-right. To determine

the type and distribution of lichen substances, several image

scans were performed at speciﬁc areas of the thallus: outer

cortex, subcortex, algal clusters and central medulla.

Raman spectra of the thin section of C. gyrosa

As mentioned in the Methods section the thin sections are

investigated ﬁrst. In the thin section of C. gyrosa, the layers of

the thallus can be identiﬁed clearly (Fig. 1). Starting from the

most external part the pigmented cortex is followed by the non-

pigmented subcortex. Algal clusters are embedded partially

Fig. 3. Typical Raman spectra found in Circinaria gyrosa; the dotted rectangles describe the spectral range ﬁltered for the discrimination of the

various compounds. All spectra were baseline corrected applying the correction procedure provided by the Raman spectrometer software

WITecProject.

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Fig. 4. Outer cortex of Circinaria gyrosa: Left –microscopic image. Right –Raman image of the scan, described by the red square in the

microscopic image (50 μm×50μm with 50×50 measurement points). The colours correspond to the different matter (tissue) of the lichen –green –

chitin, red –carotene, calcium monohydrate (whewellite) –yellow. Applied integral ﬁlters for substance identiﬁcation are taken as shown in Fig. 3.

Fig. 5. Algal clusters of Circinaria gyrosa: Left –microscopic image marked with a square for the colour-coded Raman image scan. Right –colour-

coded Raman image scan, described by the red square in the microscopic image (175 μm×175 μm with 150×150 measurement points). The

colours correspond to the different matter (tissue) of the lichen –green –chitin, red –carotene, calcium monohydrate (whewellite) –yellow. Applied

integral ﬁlters for substance identiﬁcation are taken as shown in Fig. 3.

Fig. 6. Central medulla of Circinaria gyrosa: Left –microscopic image marked with a square for the colour-coded Raman image scan. Right –

colour-coded Raman image scan, described by the red square in the microscopic image (175 μm×175 μm with 150×150 measurement points). The

colours correspond to green –chitin; red –carotene; yellow, white –calcium monohydrate (whewellite); blue –background (glass slide,

ﬂuorescence). Applied integral ﬁlters for substance identiﬁcation are taken as shown in Fig. 3.

Raman spectroscopic analysis of the calcium oxalate producing lichen C. gyrosa 23

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within or below the subcortex and are ensheathed by fungal

hyphae. This layer is followed by the inner medulla. The

hyphae of the central medulla are coated with crystals. Raman

image scans are performed for each of the layers.

The imaged area of the outer cortex (and subcortex) –the

surface of the thallus –can be seen in Fig. 4. On the left, the

microscopic image with the scanned area (red square) is shown.

On the right, the colour composite of the Raman scan of the

outer cortex could be seen. The different colours correspond to

the lichen compounds: green –chitin, red –β-carotene, yellow –

calcium monohydrate (whewellite). The spectra describing

these substances are shown in Fig. 3 with the same colour

code. Black areas indicate that no single substance can be

assigned and/or the spectra are highly ﬂuorescent. As expected

the outer cortex consists of chitin (green) and some β-carotene

(red) of uncertain origin is also present. The areal fraction

of chitin is increasing from the edge of the cortex in the

direction of the subcortex representing the extremely dense

packed hyphae in the subcortex. The presence of calcium

monohydrate (whewellite) is quite unexpected in the outer

cortex. Deeper analysis of the spectra showed that the yellow

areas in Fig. 4 are characterized by ﬂuorescence and thus the

assignment to whewellite is misleading. The spectra of the

black areas are, as already mentioned above, characterized

by high ﬂuorescence and/or a non-resolvable mixture of

different spectra. So the surface of the lichen C. gyrosa is

highly ﬂuorescent, which is important for measurements on the

natural sample.

Algal clusters embedded in hyphal tissue are shown in Fig. 5.

The square in the microscopic image (left) is marking the area

scanned with Raman microspectroscopy and corresponds to

the colour-coded image on the right. The spectra of whewellite

(yellow), chitin (green) and β-carotene (red) (see Fig. 3)

represent the main compounds of the algal cluster and attached

hyphae. Chitin gives avery clear Raman signal and is surround-

ing the algal cluster. In the colour-coded image (Fig. 5 right)

chitin of the subcortex is characterized by brighter green

compared with the chitin in the inner medulla. The spectra of

the latter area exhibit high ﬂuorescence connected to chloro-

phyll in the algae. However, the β-carotene signal (red) is still

visible on top of the ﬂuorescence and the spectrum reﬂects the

areal distribution of the photobionts and the shape of the algal

cluster. Spectra of calcium monohydrate (whewellite) can be

found apart from the subcortex and the algal cluster in the

direction of the inner medulla. The yellow patches in the white-

rimmed area in the Raman colour-coded image in Fig. 5 (right)

are whewellite. The spectra of whewellite here show two strong

lines about 1466 and 1493 cm

−1

in contrast to the spectra of the

outer cortex where only one strong line exists at 1466 cm

−1

The following layer, located in the centre of the thallus

represents the inner medulla. In Fig. 6 (left), the microscopic

image of the inner medulla with the red square marking the

Raman scanned area is shown. The colour-coded image (right)

derived from the Raman scan shows the distribution of chitin

(green), the glass slide (blue) and whewellite (yellow), which

is always attached to the chitin. β-carotene (red) is present as

well, but only in a very small portion. The morphological–

anatomical structure of the inner medulla (Fig. 6) is not as

compact as the outer cortex and subcortex (Fig. 4). That’s why

the (blue) background is visible in the inner medulla. The

hyphal chitin signal (green) is clearly observed forming a loose

medullary tissue. Whewellite (yellow, white) is remarkably

present in the inner medulla. Interestingly, calcium oxalate is

always attached to the hyphae, which means that C. gyrosa

forms extracellular whewellite in the inner medullary space.

This is new and important to take into account for the inves-

tigation of the natural sample. The corollary of this peculiarity

will be discussed in Calcium oxalate and its role in C. gyrosa

section. The different colours for whewellite (yellow and white)

correspond to the different ratios of the intensity of the main

lines around 1466 and 1493 cm

−1

. For the yellow areas the line

about 1466 cm

−1

is much stronger than the 1493 cm

−1

line.

For the white areas an opposite ratio is observed. The change

in the ratio cannot be explained by polarization and thus,

orientation effects. Further investigation is needed to study this

change, which is outside the scope of this paper and will be

the aim of a new publication.

Raman spectra of natural thalli of C. gyrosa

In a second analysis, the question was answered which of

the ﬁndings of Raman microspectroscopy can be conﬁrmed

for the fractured part of a complete thallus of the lichen

C. gyrosa. Several single spectra and line scans were measured

on various parts of the lichen thallus. Image scans were not

possible to be performed because of the too rough structure of

the surface.

Spectra measured on the brownish areas of C. gyrosa

showed mainly ﬂuorescence. In very few cases, some indication

for Raman signatures of β-carotene and whewellite could

be detected. This coincides with the results presented for the

outer cortex of the thin section, which showed strong

ﬂuorescence.

Fig. 7. Microscopic image of the subcortex with algal clusters and

some parts of the subcortex and the inner medulla. The red arrow

represents the line scan.

24 U. Böttger et al.

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Whitish areas of the thallus of C. gyrosa that appear in Fig. 2

were identiﬁed either as pseudocyphellae or as branches layed

bare by fracturing. In Fig. 7, the microscopic image of the sub-

cortex with algal clusters and some parts of the inner medulla

can be seen. This area is the surface of a fractured part and no

additional manipulation was done with the lichen in order to

perform Raman measurements. The red (A E) line rep-

resents the line scan sweeping over the subcortex and the inner

medulla and algal clusters. The corresponding raw Raman

spectra are shown in Fig. 8. Raman spectra of whitish areas are

characterized by ﬂuorescence. In addition, Raman features of

β-carotene, chitin and whewellite can be observed rather well.

The interpretation of the Raman spectra with respect to the

corresponding line scan positions shows that chitin is present

in almost all positions, except at the ﬁrst points of the scan.

This can be derived from the Raman band of the C–H stretch-

ing mode around 2911 cm

−1

. Raman bands of 1008, 1158 and

1523 cm

−1

can be found on positions with algal clusters

indicating β-carotene, which is in agreement with the results of

the thin section. Whewellite signatures are present in Raman

spectra of the positions of the ﬁrst part of the line scan near

algal cluster positions and starts where chitin (of the inner

medulla) arises in the spectrum as well (Fig. 8). The Raman

spectra of whewellite contain also signatures of chitin and

carotene (Fig. 9). One reason can be that the Raman signal

comes from the surface as well as from layers below that can

contain all compounds of the inner medulla. Another explana-

tion can be that the border area between the medulla and the

algal clusters is observed during the scan and all three signals

exist in parallel.

Thus, the conclusions drawn from Raman measurements on

the thin section can be applied to the natural sample: the outer

cortex (brownish in the natural sample) is highly ﬂuorescent

and difﬁcult to investigate with Raman spectroscopy. On parts

with damaged surface, the underlying layers can be investi-

gated and give decisive Raman signals. The algal clusters can

be seen clearly by the β-carotene Raman signature. The sub-

cortex and the inner medulla (whitish in the natural sample)

are characterized by the spectrum of chitin, especially the

C–H stretching mode around 2911 cm

−1

, and whewellite.

In summary, whewellite can be measured by Raman spectro-

scopy mainly together with chitin and carotene and is located

in the inner medulla and near the algal clusters. This co-

occurrence of three biogenic substances within one lichen

thallus and their peculiar spatial distribution could be seen in

the Raman spectra for the thin section as well as for the pristine

thallus sample. Such results give valuable information for

astrobiological modelling and for the search and deﬁnition of

bio-signatures.

Calcium oxalate and its role in C. gyrosa

Whewellite is discussed as a product of biological as well as of

mineralogical processes. Its appearance in hydrothermal

deposits indicates abiogenic origin, while the biogenic origin

is supported by its appearance in coal and sedimentary nodules

(Hoefs 1969; Zák & Skála 1993; Vassilev & Vassilev 1996;

Ward 2002). In plants, calcium oxalate crystals are formed as

Fig. 8. Raman spectra of the line scan are shown in Fig. 7. The Raman spectrum of the position marked with an asterisk is in shown in more detail

in Fig. 9.

Fig. 9. Raman spectrum of the position marked in Fig. 8 with an

asterisk. The assignments represent whewellite, β-carotene and chitin.

Raman spectroscopic analysis of the calcium oxalate producing lichen C. gyrosa 25

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virtually insoluble vacuolic deposits to compensate the surplus

of calcium supply and act as herbivore defence (Sitte et al.

2002). Some extremotolerant lichens excrete oxalic acid and

degrade their rock habitats by bioweathering (Edwards et al.

1997), allowing the symbionts to colonize more sheltered

endolithic habitats. As a result, calcium oxalates are formed by

the reaction of oxalic acid with the rock. Thus, its abundance

and multiple biogenic formation make calcium oxalate a valu-

able biomarker for astrobiological research.

For C. gyrosa an alternative function of calcium oxalate

monohydrate is proposed. Its medullary location, its hyphal

attachment as demonstrated in the above Raman investi-

gation, together with its virtual insolubility and the gas ex-

change promoting function of the medulla (Sancho et al.2000;

Sánchez et al.2012) indicate a role in water balance. As all

lichens, C. gyrosa is a poikilohydric organism, i.e. their water

content will tend to equilibrium with the water status of the

environment, under wet conditions they become hydrated

and active and under dry conditions they dry out and become

dormant. It tolerates dehydration without physiological

damage and is metabolically active under moist environmental

conditions. Under wet conditions lichens take up water, lead-

ing to the soaking of interhyphal spaces. Thus, liquid water

strongly decreases the diffusion rate of CO

towards the algae

and inhibits photosynthesis at the very moment when water is

not a limiting factor (Lange et al.1997). Besides other stra-

tegies found in lichens, extracellulary deposited crystals on

loosely connected medullary hyphae are discussed to prevent

complete soaking of the medullary gaseous space (Lange et al.

1997; Honegger 2009). In this context calcium oxalate is sug-

gested to enhance the air/hyphae surface in C. gyrosa,to

prevent increased diffusion resistance of CO

(Sancho et al.

2000) and thus facilitates efﬁcient gas exchange when wet.

Summary and outlook

The lichen C. gyrosa, chosen as a model organism for

BIOMEX, has been investigated by Raman spectroscopy.

Different parts of the lichen were characterized on a thin

section and on a natural sample. Calcium oxalate mono-

hydrate (whewellite) has been in focus of this investigation.

The present study revealed that C. gyrosa forms extracellular

whewellite deposits upon the medullary hyphae inside the

thallus. This is a peculiar ﬁnding for lichens and suggests a

speciﬁc role of this internally localized biomineral. The func-

tion of whewellite in C. gyrosa is assumed to reduce the

wettability of the loose medullary hyphae, and thus to hinder

the complete soaking of the thallus under wet environmental

conditions. Keeping gas-ﬁlled spaces in the medulla is

suggested to facilitate sufﬁcient gas exchange and to ensure

-provision for photosynthezising algae in the wet, meta-

bolically active thalli. Depending on the decomposition pro-

cesses of whewellite, it may not only serve as a biomarker of

recent life, but also of past and fossilized organisms. Although

whewellite can also be formed by rare geological processes,

it serves as a suitable biomarker for astrobiological investi-

gations.

Acknowledgements

The authors would like to express their sincere gratitude to

the German Federal Ministry of Economics and Technology

(BMWi) and the German Aerospace Center (DLR) for fund-

ing the work of Joachim Meeßen (50BW1153), to the Spanish

Instituto Nacional de Técnica Aeroespacial (INTA) for grant-

ing a PhD scholarship to Francisco Javier Sánchez Iñigo, to the

Spanish Ministry of Science and Innovation (ESP AYA2010-

11422-E, 2010–2012, from 2010 to 2012) and INTA for the

economic support of BIOMEX, and to the German Aerospace

Center (DLR) for supporting the ESA-space experiment

BIOMEX (ILSRA ESA-ILSRA 2009-0834, P-I Dr J.-P. de

Vera). We would also like to thank the reviewers for their

comments and suggestions.

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