Microbial Pigments and Their Degradation Products as Biosignatures

Carotenoids are a class of vibrant
biological pigments that have a characteristic chemical
structure centered around a polyene core (Lu et al.
2018). Carotenoids and their derivatives are candidate
biosignatures because they can persist in the terrestrial
geologic record for up to 1.73 billion years
(Vinnichenko et al. 2020), have specific structures that
are likely the result of complex pathways, mediate the
survival of many microorganisms in Mars and Ocean
Worlds analog environments, and are detectable with
multiple techniques, including Raman spectroscopy. In
this project, we aim to investigate the detectability of
carotenoid pigments with different spectroscopic
methods to inform future instrument selection. We
compare the spectra of five unaltered carotenoids, two
model compounds, and carotenoid-forming archaeon
with visible and deep UV Raman spectroscopy and UVVis
absorption spectrophotometry. We then use one
model pigment, beta-carotene, to evaluate the likelihood
that unique spectral properties of carotenoids, or their
refractory byproducts, would be preserved and
detectable on a remote planetary surface by exposing it
to simulated conditions for Mars.
Sample Acquisition. Pigments betacarotene,
lutein, zeaxanthin, astaxanthin, and lycopene
were purchased from Sigma Aldrich. Halobacterium
salinarum NRC-1 was acquired from Carlina Biological
and grown in Halobacterium media. Mineral salts
including sodium sulfate, sodium carbonate, and halite
were used to form matrices in which the beta-carotene
was embedded before exposure. Pigment-mineral mixes
were at a 1:10 ratio in water.
Analytical Techniques. Deep UV Raman data were
collected on a custom laboratory mapping spectrometer
called MOBIUS (Mineral and Organic Based
Investigations using Ultraviolet Spectroscopy), which is
an analog to the SHERLOC instrument on the Mars
2020 Perseverance rover (Bhartia et al. 2021). It
features a 248.56 nm NeCu pulsed laser, liquid
nitrogen-cooled detector, and tunable optical setup.
Visible Raman data were collected using a Horiba Jobin
Yvon LabRam HR spectrometer with a frequencydoubled
Nd:YAG laser (532 nm) and a HeNe laser (633
nm). A VWR 6300 PC UV/Visible Spectrophotometer
was used to collect absorption data for carotenoid
solutions, model compounds, and solvents in UVpermissible
capped cuvettes. Data were collected from
190-1100 nm at 1 nm increments. All spectral data were
analyzed using Igor Pro 9 (Wavemetrics).
Irradiation. We used a vacuum chamber equipped
with a cryostat and a flood electron gun to simulate
Martian surface temperatures, low pressures, and
ionizing radiation (10keV, 10μA for 6h at 200K for our
initial tests). The samples were prepared by drying the
pigment-mineral mix onto polished metal tabs, then
mounted on the cryostat for processing. Samples were
then analyzed directly on the tabs after exposure.
Results: In comparing the visible and deep UV
Raman spectra of unaltered pigments, we found that they
differed drastically. Carotenoids are often studied with
visible Raman and typically have peaks at 1525 cm-1 and
1157 cm-1, due to the stretching of the C=C and C-C
bonds in the polyene structure. However, in deep UV, the
strongest feature is at ~1630 cm-1 and is broad, possibly
indicating that multiple peaks are forming this feature.
This stark difference is likely due to different preresonant
enhancement effects. The UV-Vis results show
that there is an absorption band in the deep UV <300 nm,
which supports the hypothesis that the 248.6 nm
excitation is interrogating another aspect of carotenoids
than visible Raman. Our preliminary exposure tests
indicated that pigments – even without minerals present -
were largely unaltered in the applied conditions, with
only a slight broadening in the primary polyene peaks
apparent in the visible Raman data.
Figure 1. A) Visible vs. deep UV Raman spectra of
unaltered beta carotene. B) Schematic of exposure.
Conclusions: Our results to date indicate that deep
UV and visible Raman spectroscopy, both techniques
with planetary mission heritage from Mars 2020 (Wiens
et al. 2021, Bhartia et al. 2021), may be used in a
complementary manner to observe carotenoids. In
addition, we find that beta-carotene is largely resistant to
our current exposure conditions, though there may be
some amount of amorphization of the material which
could cause the broadening of the peaks at 1525 and 1157
cm-1. As a next step, we aim to increase the dosage and
duration of exposure to observe degradation of the parent
pigment, possibly add UV as a factor via an Ar mini-arc
UV lamp and use GC-MS to characterize possible
degradation products.