Raman spectroscopy for future planetary exploration

Research article Received: 30 September 2014

Revised: 19 November 2014

Accepted: 9 December 2014

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jrs.4647

Raman spectroscopy for future planetary exploration: photodegradation, self-absorption and quantification of carotenoids in microorganisms and mineral matrices Jan-Hein Hooijschuur,a,b Mattheus F.C. Verkaaik,a Gareth R. Daviesb and Freek Ariesea* Carotenoids are among the key biomarkers in the search for life on other planets, and non-destructive Raman spectroscopy on future rover missions is a potential sensitive detection method, especially under resonant conditions. In this research, reflectance spectra of minerals and microorganisms were measured using ultraviolet/visible diffuse reflectance spectroscopy in order to evaluate potential resonance Raman conditions and the possible degree of sample damage during laser irradiation. We report a photodegradation and semi-quantitative Raman study of β-carotene and the carotenoid-containing extremophile Deinococcus radiodurans mixed with calcite at excitation wavelengths of 440 nm, 532 nm and 785 nm. A different type of carotenoid was detected in a culture of Chroococcidiopsis. Carotenoids embedded in bacterial membranes were found to be less sensitive to photodegradation than in a mineral matrix. Corrections for self-absorption effects were performed using the 1085 cm1 peak of calcite as an internal standard. Carotenoid-type signals from 1 mg g1 D. radiodurans in calcite could be detected, corresponding to about 5 μg g1 β-carotene in calcite (≈0.5% cell weight). This research emphasizes the potential suitability of Raman spectroscopy in the detection of organic biomarkers in future planetary exploration. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: (max 5) Resonance Raman scattering; internal standard; diffuse reflectance spectroscopy; Deinococcus radiodurans; Chroococcidiopsis

Introduction Raman spectroscopy (RS) is a non-invasive technique that can provide highly specific chemical information and can do so in combination with low instrumental weight and low power consumption. This potentially makes RS an important component for planetary exploration. The ExoMars rover of the European Space Agency (ESA) will be equipped with a 532-nm RS instrument as part of the Pasteur instrument suite and is scheduled for launch in 2018.[1] The National Aeronautics and Space Administration (NASA) payload for the Mars 2020 mission will include a 248.6-nm deep ultraviolet (UV) Raman spectrometer as part of the Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals (SHERLOC) instrumentation.[2] Both Raman spectrometers will focus on finding biosignatures: molecules that provide unambiguous proof of past or present life. The current environmental conditions for life on Mars and elsewhere in our Solar System are more extreme than on Earth. Compared to Earth, Mars is characterized by lower temperatures, atmospheric pressure and humidity and higher levels of UV radiation. Initially, however, Mars had wetter conditions more favourable for life.[3] The exact duration of these warmer–wetter conditions is a matter of some debate, but it appears that conditions on early Mars were comparable to those on Earth when life started to develop.[4] This is the rationale behind the major research efforts to assess if life also developed on early Mars and to search for biomarkers in current and future planetary missions. Life on Earth can exist even under the most extreme conditions: at high or low temperatures,

J. Raman Spectrosc. (2015)

extreme pH values and dry environments.[5] Even polyextremophiles exist, able to survive different combinations of such extreme conditions, for example a combination of a hot, alkaline and saline environment.[6] These environments are direct analogues of past and current Martian environments as well as conditions thought to prevail on the moons of the giant outer planets.[7] This is one of the reasons that ESA has scheduled a major mission to the Jupiter system (JUpiter ICy moons Explorer; JUICE). In order to develop and test new instrumentation for Martian biomarkers, Earth extremophiles are commonly used as proxies. Mars lacks a magnetic field, has no ozone layer and only has a thin atmosphere (on average 600 Pa of pressure). Under these circumstances organic molecules can easily become (photo)degraded, due to high fluxes of UV light and charged solar particles. A high flux of ionizing radiation influences the viability of microorganisms, like Deinococcus radiodurans and Natronorubrum strain HG-1.[8,9] In addition, the photoanalytical properties of molecules such as fluorescence are influenced by high UV dosages.[10]

* Correspondence to: F. Ariese, LaserLaB, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. E-mail: [email protected] a LaserLaB, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV, Amsterdam, The Netherlands b Deep Earth and Planetary Science, Faculty of Earth and Life Sciences, VU University Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands

Copyright © 2015 John Wiley & Sons, Ltd.

J. Hooijschuur et al. Furthermore, differences in degradation rates of the Raman signals of D. radiodurans and Synechocystis were observed following exposure to 15 kGy of gamma radiation, with the signal of the carotenoids in D. radiodurans diminishing more rapidly than in Synechocystis.[11] These processes make the analysis of many biomarkers by RS problematic, and specific methodologies will be needed to optimize detection and for quantification. Life forms have developed different strategies to protect themselves against the effects of harmful radiation. Microorganisms build protective shields via biotransformation of a mineral.[12,13] Organisms like endoliths and chasmoliths live in cracks or pore spaces inside minerals, effectively using the rock substrate as protection.[14] Other bacteria produce photoactive compounds like scytonemin and carotenoids that absorb harmful UV radiation.[15,16] Due to the near-ubiquitous occurrence of carotenoids in extremophiles and their tendency to use minerals for protection, this work will focus on methods to detect carotenoids within mineral matrices as a potential proxy for the detection of past or present life in the context of planetary exploration and the search for the earliest life forms on Earth. Carotenoids represent a family of over 600 different compounds[17] that are only produced by living organisms and are therefore ideal biomarkers for the detection of life. In general, carotenoids like lycopene, β-carotene and astaxanthin absorb in the blue–green part of the electromagnetic spectrum and are red to the human eye.[17] The blue complex of astaxanthin and protein found in the carapace of lobsters illustrates the large influence of the environment on the UV/visible (VIS) electronic absorption properties of carotenoids. Astaxanthin as a molecule in solution has its absorption maximum at 488 nm, and therefore appears as a red colour. When astaxanthin forms a complex with a protein, for example in lobster, the absorptions shifts to 632 nm and appears as blue colour.[18] If an electronic transition of the analyte and an available laser wavelength (partially) overlap, resonance enhancement can be expected, resulting in higher sensitivity for the target analyte and at the same time improved selectivity over the matrix. To assess the applicability of resonance Raman spectroscopy (RRS) to a specific analyte, its absorption spectrum must be known. Such UV/VIS spectra, however, should be measured in the natural environment of the analyte as transition maxima may shift with matrix polarity, molecular stacking effects, etc. Since conventional UV/VIS spectroscopy is only suitable for transparent solutions, UV/VIS diffuse reflective spectroscopy (DRS) was used in this work to obtain reflectance spectra of bacteria embedded in highly scattering minerals. In the literature, mixtures of quartz and D. radiodurans or quartz and Chroococcidiopsis have been analysed with UV/VIS DRS at concentrations of 108 organisms per cm3, as proxies for fertile soils or aqueous environments.[19] In that study of mineral–organic mixtures, UV/VIS DRS was combined with near-infrared (NIR). Data from the UV/VIS/NIR DRS were processed using chemometrics to determine the composition of mineral–organic mixtures.[20] Previous published work on carotenoids in biological samples has shown that the vibrations of an individual carotenoid compound may show variable spectral shifts (up to 16 cm1) dependent upon the host organism. For example, the C¼C stretch vibration of β-carotene is at 1515 cm1 in purple onion and at 1531 cm1 in orange peel.[21] It therefore appears that the environment and the aggregation state (microcrystals or in solution) of a specific molecule are of great influence on the Raman signal. This complicates the identification of carotenoids on the basis of subtle wavenumber shifts, even if pure standards are available.

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In RRS, part of the excitation light may also be absorbed, resulting in unwanted effects such as heating. Photodegradation has been reported for histidine when performing UV-RRS.[22] This was also observed for single living cells when using 514 nm but not at 660 nm.[23] Obviously, photodegradation strongly depends on excitation wavelength and laser power. Photodegradation can also be matrix dependent: Abramczyk et al. found that carotenoids in a carrot matrix are more stable than in acetonitrile solution, and Withnall et al. reported that carotenoids in shells are more stable than in saffron.[24,25] In addition to identification, RS or RRS can be used to quantify carotenoid levels in a sample. The potential occurrence of carotenoids in extremophiles on other planets is most likely to be heterogeneously distributed among minerals. Therefore, experimental methodologies need to be developed to optimize the sensitivity and selectivity of the methodology to detect low abundances. Previous RS research showed that β-carotene in mineral matrices could be detected down to 0.1 ppm under resonant conditions.[26] In quantitative RRS measurements, self-absorption can be a confounding factor. Self-absorption of radiation by a sample occurs when the wavelength of the incident light or that of the resulting Raman emission overlaps with its absorption spectrum. This is especially the case when a compound has a high molecular absorption coefficient, occurs at relatively high concentrations and/or in the case of long optical path lengths. The latter may not be known in the case of turbid liquids or scattering solids. Wu et al. evaluated different methods for correction of self-absorption in quantitative Raman spectroscopic analysis of a catalysis.[27] In a solid sample the catalytic support (δ-Al2O3) served as an internal standard during the quantitative analysis of VOx. In the experiments reported here, a series of carotenoids and extremophiles are examined after mixing with a calcite matrix, where the calcite peak intensity will be used as an internal standard. This study elaborates on our previous work regarding Raman detection of minerals and molecules behind different mineral layers.[28,29] Here, UV/VIS diffuse reflectance spectra were recorded for β-carotene–calcite mixtures to determine optimal RRS excitation wavelengths and to measure spectral changes of the β-carotene following laser irradiation. Photodegradation was observed under resonance conditions, and this phenomenon must be considered when applying RRS for quantitative studies. Finally, RRS spectra of a mixture of D. radiodurans with calcite will be compared with spectra of standard samples with known concentrations of β-carotene in a calcite matrix. The suitability of RRS for the semi-quantitative analysis of carotenoids in microorganisms will be discussed.

Experimental Samples The mineral matrix used in the experiments consisted of calcite (calcium carbonate, CaCO3, 98% pure, light powder) purchased from Fisher Scientific, Geel, Belgium. β-Carotene, purum was purchased from Sigma-Aldrich, St. Louis (MO), USA, and cyclohexane 99 + % for spectroscopy was purchased from Acros Organics, Fisher Scientific—Geel, Belgium. β-Carotene–calcite mixtures ranging from 104 μg to 101 μg of β-carotene per gram calcite were prepared by mixing weighed amounts of β-carotene with the calcite powder. The mixture was homogenized using a ceramic mortar and pestle. D. radiodurans strain was donated by dr. Wilfred Röling, VU University Amsterdam. After incubation on a nutrient agar (NA) plate,



Raman spectroscopy for future planetary exploration the D. radiodurans was grown for two days at 30 °C and stored in the refrigerator at 4 °C for up to one month before incubation of a fresh plate. The microbiologically tested NA was purchased from Sigma-Aldrich, St. Louis (MO), USA. The D. radiodurans suspension of 10 mg ml1 was prepared by carefully scraping off 10 mg of colonies from the NA-plate, adding 1 ml of a normal saline solution and mixing using a vortex. This suspension was used to prepare a D. radiodurans–calcite mixture of 1 mg cells per gram calcite by transferring 100 μl of the suspension to 100 mg of calcite and homogenizing it using a ceramic mortar and pestle. Small amounts of this mixture were transferred to a microscope slide and measured immediately after drying. The pure 10 mg ml1 suspension was used for the experiments using a rotating capillary. The approximate number of organisms in the suspension was determined by the Raman mapping of dried spots of 0.2-μl droplets from a tenfold dilution series of the 10 mg ml1 D. radiodurans suspension. For high biomass measurements, the D. radiodurans was cultivated in aqueous 13 g l1 Nutrient Broth No 3 (Sigma-Aldrich, St. Louis (MO), USA), placed under continuous swirling in a dark room at 25 °C for 7 days. Bacteria were concentrated by a first centrifugation step (3 min, 2520 ×g) followed by washing with normal saline to remove medium and a second centrifugation step (3 min, 2520 ×g). This yielded a deeply coloured orange–pink bacterial paste. The Chroococcidiopsis was obtained from the Culture Collection Yerseke (CCY) by dr. Gerard Muyzer of the University of Amsterdam. A bacterial stock was cultivated in aqueous BG-11 medium + trace metal mix A5, under continuous sterile bubble aeration in low intensity direct fluorescent light for several months. Bacteria were concentrated by centrifugation as described above for D. radiodurans. This yielded a dark green bacterial paste. A high biomass D. radiodurans–calcite mixture was prepared by adding 2.48 g of wet bacterial paste to 6.00 g of calcite and homogenization using a razor blade. A sample and a control plate were prepared by pressing 3.50 g of the mixture into two spectrophotometer slides. The slides were dried overnight in a 25 °C room. A high biomass Chroococcidiopsis–calcite mixture was prepared in the same way, starting with 2.50 g of wet bacterial paste. For the matrix-free photodegradation experiments, the wet bacterial paste of D. radiodurans and Chroococcidiopsis was pressed into glass capillaries of ~0.6-mm internal diameter. Instrumentation UV/VIS DRS measurements were performed using a Shimadzu UV2501PC UV/VIS spectrophotometer equipped with an Ulbricht integrating sphere, model ISR-240A. Pure CaCO3 powder was used as a reference, the spectral bandwidth was 5 nm and the scan speed was set to 600 nm min1. In order to measure the UV/VIS spectral changes due to photodegradation, carotenoid mixtures with calcite were irradiated at four different wavelengths, and reflectance measurements were recorded before and after irradiation. For exposure at 532 nm, a frequency-doubled Nd:YVO4 laser (Coherent Verdi-V8, Santa Clara, CA, USA) operated at 0.2 W was used. The optical parametric oscillator (OPO, see below) setup was used to produce 15 mW of fundamental 785-nm light in combination with a 785 band-pass filter to block the 532 nm of the pump laser. For short-wavelength irradiation, the frequency-doubled OPO was used, producing 5 mW of 385 nm or 124 mW of 439-nm light. In all cases the beam of laser light was expanded with a 16× microscope objective to a diameter of 17 mm to match the size of the sample holder of the UV/VIS


diffuse reflectance spectrometer. In addition, photodegradation was also studied by monitoring the changes in peak intensity during consecutive Raman experiments. Raman spectra at excitation wavelengths of 532 and 785 nm were recorded using a Renishaw InVia Reflex confocal Raman microscope (Wotton-under-Edge, United Kingdom) with a Peltiercooled CCD detector (203 K). The output power of the 532- and 785-nm lasers was 80 and 300 mW, respectively. The instrument included a Leica light microscope with 5×, 20× and 50× air objectives. The 521 cm1 Raman shift of an internal silicon standard was used to verify the spectral calibration of the system. Lorentzian fits were used to determine peak centres and intensities. The OPO setup for resonance excitation in the blue consisted of a 532-nm, frequency doubled Nd:YVO4 laser (Coherent Paladin Advanced 532-20000, Santa Clara, CA, USA) operated at 20 W and pumping an OPO laser (APE Levante Emerald, APE Angewandte Physik & Elektronik GmbH, Berlin, Germany) with tuneable wavelength range for the Signal from 690 to 990 nm and an Idler range from 1150 to 2300 nm. Both Signal and Idler can be frequency doubled/tripled (APE HarmoniXX). To record the RRS spectra the OPO signal was set to 880 nm and frequency doubled to 440 nm. The signal was collected in backscatter mode through a 20×, 0.65NA microscope objective (Partec GmbH, Münster, Germany) and a dielectric stack long-pass filter, Semrock 450 AELP (Semrock Inc., Lake Forest, IL, USA), focused at a spectrograph (Andor Shamrock, SR-303i-A, Belfast, UK) and detected with an Andor Newton 920 CCD detector (Andor DU920P-BR-DD) cooled to 60 °C.

Results and discussion To be able to monitor the photodegradation of the carotenoidcalcite samples and check if it would also lead to changes in the spectral shape, UV/VIS DRS was used to determine the reflectivity of all samples. Blanks were determined before and after irradiation with laser light. The samples of 10 mg g1 β-carotene in a matrix of calcite were irradiated for 30 min at different laser wavelengths, as shown in Fig. 1. The laser wavelengths and powers used were 5 mW at 385 nm; 124 mW at 439 nm; 200 mW at 532 nm and 15 mW at 785 nm. In all cases the spot size diameter was 17 mm. A control sample with the same concentration of 10 mg g1 βcarotene in a calcite matrix was measured 30 min after preparation but in the absence of laser irradiation (black dotted curve in Fig. 1). As expected, the same spectrum (i.e. no change) was observed for the sample irradiated at 785 nm, since the mixture does not absorb at this wavelength. Bleaching was observed after laser irradiation at shorter wavelengths: the overall reflectance of the sample increased over the entire spectral window between 300 and 600 nm. The shorter the wavelength of the laser the more photodegradation was observed, despite the marked differences in laser power. This indicates photodegradation of the β-carotene in the mineral matrix, and the most marked increase in reflectance was observed around 358 nm and 450 nm. Interestingly, the long-wavelength shoulder at ~535 nm was much less affected. To monitor possible photodegradation during the actual Raman experiments, 100 consecutive measurements of 10 accumulations of 1-s exposure time (1000 s in total) were recorded from a sample containing 0.1 mg g1 β-carotene in calcite. Spectra were obtained with a Raman confocal microscope at an excitation wavelength of 532 nm with a 20× objective and 0.6-mW laser power. The intensity



J. Hooijschuur et al. of the C¼C stretch vibration of carotene at 1515 cm1 (determined by Lorentzian peak fitting) was used to monitor the photodegradation over time, as shown in Fig. 2. The decreasing intensities show an exponential decay that requires more than one exponent to be fitted. A bi-exponential decay curve is in better agreement with the experimental data, and monitoring the signal with ten times longer exposure times (100 × 100 s) shows a third, even slower component with a decay time of ~3000 s (not shown). This means that there are several populations of β-carotene molecules with different decay rates, which could be related to differences in chemical environment. Since we are, however, dealing with multiple scattering samples and poorly defined focal volumes, it is more likely due to local differences in laser intensity. Molecules close to the point of excitation receive a higher photon flux and decay faster than molecules that are located further away, but still contribute to the signal. In agreement with the latter explanation, no photodegradation was observed when β-carotene was

1

Figure 1. UV/VIS diffuse reflectance spectra of 10 mg g β-carotene in a calcite matrix, following 30-min irradiation at 385 nm (purple), 439 nm (blue), 532 nm (green) or 785 nm (red), along with the non-irradiated control (black, dotted). All spectra were recorded against a blank (= calcite) reference. Note that generally the shorter the laser wavelength the more photodegradation is observed.

1

Figure 2. Photodegradation of β-carotene in calcite (0.1 mg g ) during 1 RRS measurements, resulting in a decrease in intensity of the 1515 cm C¼C stretch peak over time. One hundred consecutive spectra of 10 accumulations of 1-s exposure time were recorded at an excitation wavelength of 532 nm and 0.6-mW laser power. The plotted peak intensities were fitted with a bi-exponential decay function with lifetimes of 47 and 502 s.


irradiated with the same setup at 532 nm through 2.5 mm of translucent calcite. The photodegradation of the carotenoid compounds inside D. radiodurans was studied by mixing the bacteria 1 mg g1 with calcite and irradiating with the confocal Raman setup at 532 nm. The resulting decay is shown in Fig. 3. It demonstrates that the bacterial carotenoids are more photoresistant than those in the pure standard. In spite of a ten times higher laser power, the signal intensity decreases more slowly than that of β-carotene powder mixed with calcite. The data points of the sample containing D. radiodurans are more scattered due to the overall lower intensities of the C¼C stretch vibration. An even slower decay (ca. 15% decrease in 1000 s; not shown) was observed for a 10 mg ml1 D. radiodurans suspension inside a rotating capillary excited at 532 nm. We assume that both the constant movement of the sample as well as the heat dissipation by water help to reduce the degradation rate, but such measures would be difficult to implement in the field. In marked contrast, when using an excitation wavelength of 785 nm no photodegradation was observed for β-carotene (data not shown), in agreement with the results of the UV/VIS diffuse reflectance spectroscopy (Fig. 1). At that wavelength there is no absorption but of course also no resonance enhancement effect. As a result, the detection sensitivity is lower. The bacterial samples also gave no indications of photodegradation effects at 785 nm. The Raman spectra of D. radiodurans and Chroococcidiopsis are shown in Fig. 4, together with standard spectra of β-carotene as pure powder and in cyclohexane. The bacteria spectra yield clear signs of carotenoid-like compounds, with prominent peaks in the 1520 cm1 range for the C¼C stretch vibration and near 1160 cm1 for the C―C stretch vibration of the polyene chain. The Chroococcidiopsis spectrum also has a strong fluorescence background. Raman band frequencies have been used in the literature to identify different carotenoids and especially the length of the conjugated chain.[24] The two β-carotene spectra in Fig. 4 were taken in different environments. Interestingly, under resonance conditions (440 nm) their spectra do not show any major differences due to matrix polarity or aggregation state: The C¼C stretching band of β-carotene is found at 1528 cm1 for the pure

Figure 3. Photodegradation during RRS measurements of the carotenoids 1 in D. radiodurans (top; 1 mg g in calcite, measured at 6-mW laser power) in 1 comparison with β-carotene (bottom, 0.1 mg g in calcite measured at 0.6mW laser power). Peak intensity plots show the C¼C stretch band at 1 1 1511 cm and 1515 cm , respectively. Ten consecutive spectra of 10-s exposure time were recorded at an excitation wavelength of 532 nm. The plotted peak intensities were fitted with a bi-exponential decay function with lifetimes of 3.3 and 19 s for β-carotene in calcite, but no good fit was obtained for D. radiodurans in calcite.



Raman spectroscopy for future planetary exploration

Figure 4. Resonance Raman spectra of four carotenoid containing 1 samples: β-carotene 1 mg ml solution in cyclohexane (black), β-carotene 1 pure powder (red), D. radiodurans 10 mg ml suspension (green) and Chroococcidiopsis suspension (magenta). Measured at an excitation wavelength of 440 nm; 18-mW laser power; exposure 10 × 1 s. Spectra are vertically offset for legibility.

powder and at 1526 cm1 in cyclohexane. Using 532 or 785 nm excitation, however, this band shifts to 1515 cm1 for the powder sample but not for the cyclohexane solution (spectra not shown). This unusual phenomenon appears to be due to the C¼C peak consisting actually of two adjacent bands that show resonance enhancement at different wavelength ranges.[30] Such data suggest that carotenoid identification on the basis of only Raman band positions should be performed with caution, and the measurement conditions of samples and standards should always be clearly reported. Interestingly, the Raman spectrum of pure β-carotene (Fig. 4) was of lower intensity than that of the 1 mg ml1 solution in cyclohexane, in spite of the higher β-carotene concentration. This is mostly due to self-absorption: the dark crystalline material absorbs very strongly at the 440-nm laser wavelength, and only molecules close to the surface will contribute to the Raman signal. At 785-nm excitation, this effect plays no role, and the pure powder gives much stronger Raman signals (spectra not shown) In an attempt to quantify the level of carotenoids in D. radiodurans in the calcite matrix, a set of calibration samples was prepared consisting of β-carotene mixed with calcite. Concentrations ranged from 10 mg g1 down to 0.01 mg g1. For these mixtures, the intensity of the 1085 cm1 peak of calcite was used as an internal standard to correct for self-absorption as well as possible differences in laser power or alignment. All these factors should affect the Raman intensities of the analyte and those of the internal standard to the same extent. The calcite content was essentially the same for all samples since the matrix makes up more than 99% of the D. radiodurans-calcite and β-carotene–calcite mixtures. Therefore, the calcite peak was used to normalize the spectra. When using an excitation wavelength of 440 nm the Raman emission wavelength of the calcite peak at 1085 cm1 is 462 nm. At these wavelengths the absorption of light by β-carotene is at its maximum. Self-absorption by the sample becomes less when longer excitation wavelengths are used. In Fig. 5, Raman spectra of 10, 1, 0.1 and 0.01 mg g1 β-carotene in calcite are shown. Spectra of D. radiodurans samples mixed with calcite measured under identical circumstances are shown in Fig. 6. The relevant peak intensities of the calibration samples and bacteria samples are listed in Table 1. The 1085 cm1 calcite peak intensity is also given, and all calcite peaks are normalized to the calcite


Figure 5. Raman spectra of four calibration samples containing different concentrations of β-carotene in calcite: 10 (black), 1 (red), 0.1 (blue) and 1 1 0.01 (magenta) mg g . The 1085 cm peak of calcite illustrates the selfabsorption (inner filter effect) at higher β-carotene levels. Measured at an excitation wavelength of 440 nm; 33-mW laser power; exposure 10 × 1 s. Spectra are vertically offset for legibility.

Figure 6. Raman spectra of two samples containing different 1 concentrations of D. radiodurans in calcite: 10 (black) and 1 (red) mg g . 1 The 1085 cm peak of calcite is used as internal standard. Corrected 1 intensities of the 1511 cm C¼C stretch peak are 2994 and 289 for 10 1 and 1 mg g , respectively. Measured at an excitation wavelength of 440 nm; 33-mW laser power; exposure 10 × 1 s.

peak of the lowest concentration of the β-carotene–calcite calibration series. Hooijschuur et al. reported that peak maxima of the UV/VIS diffuse reflectance spectrum of D. radiodurans on calcite overlap with the peak maxima of the β-carotene–mineral mixtures.[29] Furthermore, the measured peak intensities and normalized peak intensities of the C¼C stretch vibration of the carotenoid in D. radiodurans (at 1513 cm1) and of the β-carotene– calcite mixtures (at 1515 cm1) are given. A suspension of 10 and 1 mg ml1 of D. radiodurans (10 mg ml1 corresponds with approximately 108 organisms per ml) was carefully mixed with calcite. The concentration of 10 mg g1 D. radiodurans in calcite is relatively high from a planetary exploration point of view but realistic for fertile soils or aqueous environments. Raman spectra of carotenoid-type signals of a ten times diluted sample (1 mg g1) of D. radiodurans in calcite could still be detected (see Fig. 6). The signal intensity corresponded to about 5 μg g1 βcarotene of the external calibration series. For quantification of the signal several factors should be taken in account, which will also play a role when dealing with real samples.



J. Hooijschuur et al. 1

1

Table 1. Raman peak intensities (counts) of calcite (1085 cm ) and carotene (1511–1525 cm ) in calibration samples and bacteria at an excitation wavelength of 440 nm 1

Sample in calcite (mg g ) β-Carotene 10 1 0.1 0.01 D. radiodurans 10 1

Intensity calcite 1 1085 cm

Calcite normalized

Intensity C¼C stretch 1 1511–1525 cm

Intensity C¼C stretch IS corrected

1933 4113 14 388 19 090

0.101 0.215 0.754 1.000

5717 4189 3653 778

56 604 19 484 4845 778

15 612 8074

0.812 0.425

2431 123

2994 289

First, many carotenoid derivatives exist, and often the chemically identical standard may not be available. In this case, deinoxanthin is the most abundant carotenoid in the cell membrane of D. radiodurans and has 12 conjugated C¼C double bonds[31]; βcarotene is our model carotenoid with 11 such bonds. A small red shift should be expected in the deinoxanthin spectra due to an increased length of the double bond, and therefore the RRS cross sections at 440-nm excitation will not be the same for these carotenoids. In addition, the environment of the carotenoids in the two samples differs; β-carotene in the calcite standards is present as solid crystallites, whereas deinoxanthin is uniformly distributed throughout the cell membrane of the D. radiodurans. The self-absorption losses at 440 nm will be identical, but will be slightly different for the calcite peak at 462 nm and the carotenoid C¼C stretch peak at 471 nm. Finally, although the exposure times were kept very short (10 × 1 s), some degree of photodegradation may have occurred and may be different for membraneembedded and crystalline carotenoids (see Fig. 3). For these reasons, the finding that the Raman spectra of carotenoid-type signals of concentrations of 1 mg g1 D. radiodurans in calcite corresponds with about 5 μg g1 β-carotene in calcite should be considered as a semi-quantitative estimate at best. Inside the bacteria, this carotenoid content would constitute about 0.5% of the cell weight.

Conclusions In this work, samples containing β-carotene in calcite were irradiated for 30 min with wavelengths of 385, 439, 532 and 785 nm. As expected for the photoactive compound β-carotene, the shorter the wavelength the more photodegradation was observed. In accordance with the reflectance spectra, no photodegradation was observed at 785 nm for the standard nor for the carotenoids in D. radiodurans, but sensitivity and selectivity over matrix components are of course worse at that wavelength. Different degradation rates were observed for β-carotenecalcite and D. radiodurans–calcite mixtures at 532 nm. The carotenoids inside the bacteria showed greater photostability, probably because of differences in carotenoid structure and because of a more favourable environment for the carotenoids in D. radiodurans to dissipate energy. In the case of β-carotene–calcite mixtures, a range of photodegradation rates was observed, presumably due to different photon intensities in the scattering sample. No photodegradation was observed when measuring through 2.5 mm of translucent calcite. We conclude that it is important to take the risk of photodegradation into account when


RS or RRS is used for detection of life on other planets, especially if one attempts to quantify these compounds on the basis of Raman intensities. We showed differences in the C¼C stretch vibrations for different carotenoids (β-carotene and carotenoids in D. radiodurans and Chroococcidiopsis), different aggregation states of the same molecule (β-carotene in solution and as solid) and for the same compound at different excitation wavelengths (β-carotene at 440, 532 and 785 nm). All of these factors can cause shifts in the peak position of up to 10 cm1, and therefore care must be taken when using Raman frequencies for carotenoid identification. We also conclude that irradiation conditions and the form of the samples need to be rigorously reported in all cases. One of the potential drawbacks of RRS is the possibility of selfabsorption by the sample, which can be corrected for by using an internal standard. In this study a major Raman peak of the calcite matrix surrounding the target compound was used. In real samples the matrix components will also have to be identified. In order to quantify carotenoids in D. radiodurans its Raman spectrum was compared with standards of β-carotene in calcite mixtures. Concentrations down to 1 mg g1 D. radiodurans in calcite could be detected. This corresponds to approximately 107 individual organisms per cm3, one order lower than the number of organisms per cm3 in fertile soil or in aqueous environment. The amount of carotenoid in 1 mg g1 D. radiodurans in calcite corresponds to about 5 μg g1 β-carotene in calcite (assuming similar RRS cross sections). This would indicate that about 0.5% of the bacterium’s cell weight consists of carotenoids. Acknowledgements We would like to thank Wilfred Röling of the Department of Molecular Cell Physiology of the VU University Amsterdam for providing us with a strain of wild type Deinococcus radiodurans, and to Gerard Muyzer of Microbial Systems Ecology at the University of Amsterdam for providing the Chroococcidiopsis from the Culture Collection Yerseke. We also thank NWO—Netherlands Space Office for funding this project ALW-GO-PL/14 and for the OPO system funded by NWO-Middelgroot Chemical Sciences grant # 700.59.103.

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