Detection of carotenoids of halophilic prokaryotes in solid inclusions inside laboratory-grown chloride and sulfate crystals using a portable Raman spectrometer: applications for Mars exploration

ABSTRACT

Inclusions in evaporitic minerals sometimes contain remnants of microorganisms or biomarkers, which can be considered as traces of life. Raman spectroscopy with resonance enhancement is one of the best analytical methods to search for such biomarkers in places of interest for astrobiology, including the surface and near subsurface of planet Mars. Portable Raman spectrometers are used as training tools for detection of biomarkers. Investigations of the limits and challenges of detecting biomolecules in crystals using Raman spectroscopy is important because natural occurrences often involve mineral assemblages as well as their fluid and solid inclusions. A portable Raman spectrometer with 532 nm excitation was used for detection of carotenoid biomarkers: salinixanthin of Salinibacter ruber (Bacteroidetes) and α-bacterioruberin of Halorubrum sodomense (Halobacteria) in laboratory-grown artificial inclusions in compound crystals of several chlorides and sulfates, simulating entrapment of microorganisms in evaporitic minerals. Crystals of halite (NaCl), sylvite (KCl), arcanite (K₂SO₄) and tschermigite ((NH₄)Al(SO₄)₂·12H₂O) were grown from synthetic solutions that contained microorganisms. A second crystalline layer of NaCl or K₂SO₄ was grown subsequently so that primary crystals containing microorganisms are considered as solid inclusions. A portable Raman spectrometer with resonance enabling excitation detected signals of both carotenoid pigments. Correct positions of diagnostic Raman bands corresponding to the specific carotenoids were recorded.

halophilic prokaryotes, carotenoids, inclusions, salts, miniature Raman spectrometer

INTRODUCTION

Evaporites are rock assemblages formed during the drying of lakes, seas or other water bodies typically in arid climates. Brines precipitate crystals. The mineralogy depends upon the composition of the parent brines (Hardie and Eugster 1970). Evaporitic series of gypsum and halite of Tertiary age are known from the Mediterranean (Central Sicily, Upper Miocene, 5 Mya (Decima and Wezel 1973) and Spain, Upper Eocene, 37 Mya (Ayora et al. 1994)). Much older deposits of bedded halite are known from Kansas, USA (Hutchinson Salt, Permian, 280 Mya (Andeskie and Benison 2019)) and Australia (Browne Formation of central Australia, Neoproterozoic, 800 Mya (Blamey et al. 2016)). Some evaporitic systems, dry lakes or desert zones are located in dry areas (California, Western Australia, the Atacama desert in Chile, the Namib Desert in Namibia (Stoertz and Ericksen 1974; Watson 1988; Crowley and Hook 1996; Benison et al. 2007). The minerals halite (NaCl), sylvite (KCl), gypsum (CaSO₄·2H₂O), epsomite (MgSO₄·7H₂O), arkanite (K₂SO₄) and tchermigite ((NH₄)Al(SO₄)₂·12H₂O) are examples of evaporitic phases. Bedded evaporites formed as salt crystals as large as several centimeters precipitate from surface brines in natural saline lakes and lagoons, as well as man-made salterns, of varying depths. Learning about the composition of mineral crusts and their possible content of biomarkers is a crucial task for geomicrobiology and future planetary search for life (Broly, Parnell and Bowden 2018).

Diverse microbial communities have been found thriving in saline lakes, saline lagoons, and salterns of various compositions (Oren 2002a; Zaikova et al. 2018). In general, halophilic microorganisms inhabit a wide range of different aqueous environments with high salinities (Oren 2002b), and they are often characterized by specific biomolecules such as pigments. Examples are the carotenoids bacterioruberin and its derivatives synthesized by haloarchaea and salinixanthin, which is typical for the halophilic bacterium Salinibacter ruber (Oren 2002a). Presence of such pigments can be investigated and identified in extracts by HPLC but Raman spectroscopy is an ideal tool for direct pigment analysis in situ (Winters, Lowenstein and Timofeeff 2013; Jehlička et al. 2014b).

Raman spectroscopy is a versatile technique for the identification of minerals and biomolecules. Its advantage consists in non-destructive and fast and possibly also in situanalysis of samples without the need of laborious sample preparation. Raman spectra reflect the molecular transitions and contain Raman bands whose wavenumber shifts are indicative of particular chemical bonds as well as their molecular environments, molecular interactions and overall molecular structures. A particular advantage of Raman spectroscopy is that the bands obtained from host inorganic mineral matrices, and even from water based fluids such as in fluid inclusions (Benison et al. 1998), can also be accessed simultaneously with the organic parts. In this case, the molecular interactions between the biological material and the host lattices can be determined. The use of an optical microscope in laboratory benchtop spectrometers additionally allows to investigate micrometer-sized solid, liquid or gas inclusions in minerals (Wang and Lowenstein 2017). Similarly, organic inclusions (Orange et al. 1996) or ancient organic matter in the form of disordered graphite (Pasteris and Wopenka 2003; Benison et al2008) can be detected and identified in minerals. Studying biological and microbiological pigments of extremophiles with Resonance Raman spectroscopy was intensively pursued in recent years (Jehlička, Edwards and Oren 2013; Jehlička et al. 2014a,b; Jehlička, Edwards and Oren 2014; Osterrothová et al. 2019).

An alternative approach is to employ miniaturized Raman spectrometers. In portable Raman spectrometers the illumination and collection of scattered light occurs through the optical head of the device generally without the use of a microscope. Portable Raman spectrometers are used in mineralogy, geobiology or cultural heritage studies where their main advantage of in situ analysis is exploited. Portable spectrometers may be applied for detection of pigments of microorganisms under field conditions (Miralles et al. 2012; Jehlička and Oren 2013; Jehlička, Culka and Nedbalová 2016).

Detection of carotenoids utilizing their signal enhancement through Resonance Raman spectroscopy using a laser excitation wavelength around 500 nm results in an increase of band intensity of several orders of magnitude over the normal non-resonance situation. Wavelength selection for excitation is extremely important for accessing quality Raman spectral data from microbiological samples. Red lasers can be suitable for detecting scytonemin, chlorophyll and phycobiliproteins. Green excitation allows detecting and discriminating carotenoids (Vítek et al. 2012).

Carotenoids have two strong Raman bands due to in-phase ν₁(C = C) and ν₂(C–C) stretching vibrations of the polyene chain (Gill, Kilponen and Rimai 1970; Merlin 1985). The Raman spectrum of β-carotene (11 conjugated double bonds) contains the ν₁ band located at 1515 cm⁻¹ and the ν₂ band at 1157 cm⁻¹. A feature of medium intensity occurs at 1008 cm⁻¹ (ν₃), corresponding to the in-plane rocking modes of the CH₃ groups attached to the polyene chain. The wavenumber positions of both ν₁ and ν₂ bands depend on the length of the polyene chain of carotenoids (the number of conjugated double bonds) (37–40) (Koyama 1995; Withnall et al. 2003). The shift in band position is much more pronounced in the case of the ν₁ band; a longer conjugated polyene chain causes a shift in the ν₁ band to lower wavenumber positions and vice versa.

A few previous studies reported details on pigment distribution in artificial and natural inclusions in salt crystals. Fendrihan, Musso and Stan-Lotter (2009) showed how to detect bacterioruberin and additional biomolecular components in halophilic archaea (Halococcus dombrowskii, Hcc. morrhuae, Halobacterium salinarum, Hbt. noricense, Halorubrum sp. and Haloarcula japonica) trapped in laboratory-grown halite and then analyzed by laser Raman spectroscopy (FT-1064 nm and dispersive 514.5 nm). A portable Raman system (532 nm excitation) allows detecting carotenoids in laboratory grown inclusions of Hrr. sodomense, Hbt. salinarum and S. ruber in chlorides and sulfates (Jehlička et al. 2018).

Schubert, Lowenstein and Timofeeff (2009) described carotenoid-producing halophilic microorganisms similar to modern Dunaliella preserved in fluid inclusions trapped inside halite from Death Valley and Saline Valley, California, USA. Raman spectroscopy was used to identify pigmented matter in these fluid inclusions as well: carotenoids were found with their diagnostic bands at 1000–1020 cm⁻¹ (v₃), 1150–1170 cm⁻¹ (v₂) and 1500–1550 cm⁻¹ (v₁) (Winters, Lowenstein and Timofeeff 2013). Raman microspectrometry also allowed detecting β-carotene aggregates from Dunaliella in halite inclusions from shallow deposits in the extremely acidic and saline Lake Magic, Western Australia (Conner and Benison 2013).

Collecting and reporting Raman spectra of microbiological biomolecules accompanied by suggested assignments of diagnostic bands is an important activity for better future use of this analytical technique. Raman spectrometers will be deployed in the frame of robotic exobiology-focused missions to Mars. Set for launch in 2020, NASA and ESA (Mars 2020 and Exomars 2020) will send robotic rovers to better investigate Martian rocky outcrops or subsurface rocks through a combination of imaging and spectroscopic techniques. Miniaturized Raman spectrometric tools will be integrated on-board of rovers of both space agencies (SHERLOC is the NASA's Raman and LIBS instrument, RLS is the ESA's spectrometer). Detecting and identifying minerals and their solid and fluid inclusions as well as potential trace biomarkers (e.g. carotenoids and other pigments) will be among the main tasks during the in situ investigations on the Martian surface. Some important characteristics of these spectrometers on-board of the Martian rovers are larger laser spot size and lower spectral resolution, compared to the bench-top instruments. Therefore, using portable or handheld Raman spectrometers for the studies involving biomarker detection in mineral or rock matrices could provide more relevant results and experience.

Here, we show that portable Raman spectrometers can be used with advantage to detect and discriminate carotenoids of S. ruber and Hrr. sodomense within solid inclusions in both simple and complex salts grown under laboratory conditions. In this study, core seed crystals (NaCl, KCl, K₂SO₄, and (NH₄)Al(SO₄)₂·12H₂O) were used as nuclei for growth of larger crystals of salt. Resulting Raman spectra provided information both about the microbial pigments trapped in the inclusions and about the mineralogical composition of the crystalline masses. The results and experience obtained using the portable Raman spectrometer, instead of the bench-top spectrometer, can provide a better approximation to the expected outcome and setbacks of pigment detection by Raman spectrometers aboard the Martian rovers.

MATERIALS AND METHODS

Bacterial strains and growth conditions

S. ruber M31 (DSM 13855^T) was grown in medium containing (g L⁻¹): NaCl, 195; MgSO₄·7H₂O, 25; MgCl₂·6H₂O, 16.3; CaCl₂.2H₂O, 1.25; KCl, 5.0; NaHCO₃, 0.25; NaBr, 0.625, and yeast extract, 1.0, pH 7.0. The medium for Hrr. sodomense (ATCC 33755^T) contained: NaCl, 125; MgCl₂.6H₂O, 160, K₂SO₄, 5.0; CaCl₂.2H₂O, 0.1; Yeast extract, 1.0, casamino acids, 1.0, and soluble starch, 2.0; pH 7.0. All media were sterilized by autoclaving. Cultures (1 liter) were grown with shaking in 2-liter Erlenmeyer flasks (35 °C). Late-exponential-growth phase cultures were harvested by centrifugation (8000 × g, 4 °C, 20 min). Cell pellets were washed in 20 ml 25% NaCl, centrifuged, and resuspended in 20 ml 25% NaCl and centrifuged again.

Seed crystals preparation

Cell pellets derived from 200 ml culture volume were suspended in 40 ml of saturated solutions of NaCl, KCl, K₂SO₄ and (NH₄)Al(SO₄)₂·12H₂O (Sigma) in 100 ml Erlenmeyer flasks. The flasks were incubated open in the dark without shaking at 35 °C until dry (for about 1 week). Then the crystals were washed in saturated solutions of the same salts and air-dried.

Preparation of secondary crystals

To cover the seed crystals with a layer of NaCl, a stock solution of NaCl was prepared by adding an appropriate amount of NaCl to hot deionized water until all salt was dissolved. The supersaturated solution was let to deposit excess solid at ambient temperature (ca. one day).

For the growth of a crystal cover on NaCl seed crystals, the stock solution was used directly without additional treatment. The seed crystals were placed into a glass beaker filled with 50 ml of the stock solution and let to evaporate at ambient temperature. After ca. seven days, newly grown salt crystals incorporating the original seed crystals were handpicked and air dried. For seed crystals composed of KCl, K₂SO₄, and NH₄Al(SO₄)₂·12H₂O, an additional step was required to prevent dissolution of the seed crystals. An excess amount of the respective salt was added to the stock solution and stirred for ca. 12 hours at ambient temperature to prepare solutions saturated with KCl, K₂SO₄, or NH₄Al(SO₄)₂·12H₂O, respectively. The subsequent stages of the procedure were the same as for NaCl crystals. Fig. 1 shows examples of the types of crystals used for Raman analysis.

Figure 1.

Macrophotographs of grown crystals with visible entrapped microorganisms: Salinibacter ruber trapped in NaCl (A), Salinibacter ruber trapped in K₂SO₄ and covered by NaCl (B), Halorubrum sodomense trapped in K₂SO₄(C), Halorubrum sodomense trapped in K₂SO₄ and covered by NaCl (D), Macro focus-stacked images.

Raman spectrometric analyzes

An EnSpectr RaPort (San José, CA, USA) portable Raman spectrometer was used. The instrument weighs 2.5 kg and uses a doubled Nd:YAG laser at 532 nm, a suitable excitation wavelength for carotenoid studies. The typical laser spot size is around 100–500 μm, which is much larger compared to the usual micrometric laser spot sizes of bench-top spectrometers. The laser power as controlled by the software supplied was set to around 40 mW as measured at the sample. This relatively high energy was needed for the through-the-crystal analyzes. The instrument was operated using a laptop computer, linked by means of a USB 2.0 interface, for the acquisition of spectra. Instrument was powered from the electrical grid, and the analyzes were conducted under laboratory conditions. The measurement range was set to 200 to 3200 cm⁻¹, with a maximum spectral resolution of 7 cm⁻¹. Primary crystals containing the microorganisms were analyzed with several settings, including the ‘auto’ settings where the instrument itself determines the accumulation time and number of individual accumulations (frames). Optimalized manual settings were established as 1 s accumulation time and 30 of these accumulations were acquired for each spectrum. For the complex crystals the manual settings were optimized as 5 s accumulation time and 30 of these accumulations acquired for each spectrum. Six replications of the analysis were taken on each sample, with a miniature shift of the analyzed crystal, to counter the sample inhomogeneity. The resulting six Raman spectra for each specimen were averaged. This instrument has a very good stability of the Raman lines between the different measurements (usually precise up to 1 cm⁻¹). Therefore, repeated analyzes on the same spot were not necessary. Moreover, this actually makes a process of averaging feasible, because the Raman lines are found at identical wavenumber positions and the averaging is mainly to address the different relative intensities of bands of carotenoids and crystals. No calibration was required before the measurements, but the correct wavenumber positions were checked using benzonitrile and polystyrene standards. The spectra were saved in a plaintext *.esp format and after conversion to *.spc files, they were viewed and manipulated using the GRAMS/AI 9.1 spectroscopy software. The spectra are presented without any manipulations, unless specified otherwise.

Primary crystals including microorganisms were analyzed directly as they were positioned on top of the aperture of the flat instrument probe-head. For small crystals (diameter less than ca 1.5 mm) samples were analyzed through a thin glass cover-slip, to avoid accidental drop of the samples through the aperture. This was realized as an instrumental improvement for analyzes and had no noticeable effect on the resulting spectra. The complex samples of secondary crystals grown over the primary seed crystals were analyzed directly as positioned over the aperture. The size of the crystals was in the range of 2–5 mm, and the measurement spots were selected as a combination of favorable geometry (crystal orientation and shape and the flat probe-head) and a minimum of 1–2 mm of crystalline matter positioned between the aperture and the inner seed crystal with the trapped microorganisms. The actual laser spot size and precise position of laser beam reaching the seed crystal in the interior is hard to determine, the control was provided by naked eye only, and therefore the analyzes must be considered blind. This fact was mitigated by repeated analyzes at slightly different positions, of which average spectra were computed. The question of the precise focus depth is probably less important for the miniaturized instruments than the bench-top spectrometers. The combination of much wider laser spot size, and possibly higher laser power used, low confocality, and almost transparent samples, results in a much bigger volume from which the Raman signal is collected. In our experience, the focus depth for the miniaturize Raman spectrometers usually ranges from 0 to 1 mm.

RESULTS AND DISCUSSION

Raman spectra obtained from biomass of microorganisms entrapped in crystals are presented in Figs 2 and 3. The main Raman bands detected are listed in Table 1. The most characteristic Raman bands of carotenoids are denoted in bold, while bands due to the host crystals are denoted in italics in both the Table and Figures. Based on the optical inspection of the studied samples, the resulting Raman spectra are considered to contain a majority of Raman signal from solid mineral matrix, and solid organic matter. The detected Raman bands of carotenoid pigments, enhanced by the resonance, are the dominant signals of the organic biomolecules. However, the contribution of the signal originating from fluid inclusions cannot be entirely ruled out due to a relatively large volume of analyzed sample using the portable Raman spectrometer. The characteristics of the portable spectrometers such as laser spot size and lower resolution are closer to the parameters of spectrometers on board of the Martian rovers than the parameters of the bench-top instruments. Additionally, the sample handling and subsequent precision of the focus is going to be less well controlled. A larger laser spot size could offer some advantages in specific scenarios.

Figure 2.

Raman spectra of salinixanthin of Salinibacter ruber entrapped in crystal samples.

Figure 3.

Raman spectra of carotenoids of Halorubrum sodomense entrapped in crystal samples.

Table 1.

Detected Raman bands of carotenoid pigments and host crystals.

Salinibacter ruber
Primary crystal	Sec. crystal	Raman bands
NaCl	–						872	964		1001					1153	1186		1283		1511	2150	2302		2656
NaCl	NaCl						872	963		1001					1153	1187	1208	1284	1447	1511	2157	2302	2508	2656	3022
KCl	–						871	963		1001					1154	1187	1209	1283	1446	1511	2151	2300	2512	2654	3018
KCl	NaCl							963		1002					1153			1285	1447	1510	2157	2300	2507	2655
K2SO4	–		447sh	455	618	624sh			982	1001	1092	1107			1154	1189	1208	1285		1511	2148	2301		2655
K2SO4	NaCl			452	619	627sh			992					1082	1153		1202	1285		1511	2156	2301	2509	2656
K2SO4	K2SO4			454	619	627sh			983	999sh	1092	1107	1145		1153sh			1281		1511	2154	2300		2657
NH4 sulf	–		446	457	615	624	874		989	1001sh					1153	1188	1209	1282	1445	1510	2150	2300	2509	2656	3016
NH4 sulf	NaCl	435		459	615				989	1001sh			1133sh		1153	1192	1213	1284	1446	1511	2157	2303	2512	2655
Halorubrum sodomense
Primary crystal	Sec. crystal	Raman bands
NaCl	–							946			1000			1151	1188	1209	1284	1446	1507	2002	2102	2147	2295	2503	2649
NaCl	NaCl							949			999			1151	1190		1285	1445	1507	2002		2149	2298	2504	2647
NaCl	K2SO4								982sh	992				1152					1508				2298	2502	2651
KCl	–						877	951			1000			1152	1191	1210	1284	1446	1507	2004	2104	2148	2295	2505	2648
KCl	NaCl							945			1000			1152					1508			2151	2299	2507	2646
K2SO4	–			454	619	625sh		946	982		1000	1108		1152	1187	1209	1285	1446	1507	2003	2101	2149	2296	2504	2651
K2SO4	NaCl			452	619			945		991			1081	1152		1202	1284	1446	1509			2149	2299	2506	2650
NH4 sulf	–		441	460	615				989				1132sh	1150			1282		1507			2147	2294	2501	2647
NH4 sulf	NaCl			459	614	623			989				1133sh	1150			1283		1507			2147	2294	2503	2646

Strong intensity Raman bands are denoted in bold, bands due to the host crystals are denoted in italics

Crystals containing S. ruber

Since pure NaCl and KCl have no Raman active bands in the studied range, the only detected Raman signals are derived from the carotenoid pigments. This signal is enhanced by the selected laser excitation of 532 nm, which enables in-resonance measurements for carotenoids. The resulting amplification of the Raman signal (typically a 10³ enhancement) enables detection of relatively low contents of these pigments in the studied samples.

Strong carotenoid signals including the three characteristic Raman bands located at 1511, 1153–4 and 1001 cm⁻¹ were observed. Furthermore, the second order bands were detected. These bands are located in the spectral region above the 2000 cm⁻¹, and can be interpreted as a linear combination of the three characteristic Raman bands of carotenoids. For instance, the 2656 cm⁻¹ band is a combination (1511 + 1153 cm⁻¹) with a small downshift. These bands are generally much weaker and their detection confirms that a strong signal of carotenoid pigments is present. Obtained values of Raman bands correspond well to the carotenoid salinixanthin as investigated previously by Jehlička, Edwards and Oren (2013). The right part of Fig. 2 presents Raman spectra of the samples in which primary crystals of NaCl and KCl containing S. ruber were overgrown by newly formed crystals of NaCl. The effect on the resulting Raman spectra was generally an increased level of noise, as the carotenoid Raman signal decreased due to attenuation because the excitation laser beam needed to pass through several millimeters of crystal. The scattered photons also need to pass back through the crystal to reach the detector. The carotenoid signature was clearly detected, with wavenumber values corresponding to those obtained from simple crystals. A high signal-to-noise ratio for the carotenoid Raman bands was obtained in the NaCl–NaCl system (primary—secondary crystal), comparable to the quality of the spectra of primary crystals with inclusions. For the KCl–NaCl system the bands are still located at the correct positions, although the spectra contain a significant level of noise (even after averaging) and a moderate fluorescence background (Fig. 2).

For samples where primary host crystals are sulfates, superposition of Raman spectra of the organic and inorganic parts of the sample occurs. Raman spectra of sulfates typically have one strong intensity ν₁(SO₄) band at around 1000 cm⁻¹ that can overlap with the ν₃ band of carotenoids at a similar position. This is especially true when portable instruments with a lower spectral resolution are used, such as in this study. Similarly, the ν₃(SO₄) band due to the anti-symmetric vibration can partially overlap with the ν₂(C = C) band of carotenoids, which are typically located at around 1150 cm⁻¹. The bands located between 450 and 650 cm⁻¹ are due to the bending vibration of the sulfate tetrahedra fall in the spectral region without carotenoids features. As Fig. 2 illustrates, a strong signal due to the unique carotenoid of S. ruber was detected in the simple crystals samples. The wavenumbers obtained correspond to salinixanthin values reported (Jehlička et al. 2014a; Jehlička, Edwards and Oren 2014).

For the samples that were covered with a NaCl crystal layer, increased level of noise was noted again. Otherwise, the Raman signal of salinixanthin is still rather intense, and the two strongest carotenoid Raman bands are located at correct positions (1511 and 1153 cm⁻¹) with second order bands clearly distinguishable. Interesting Raman spectra were recorded for the system of K₂SO₄–NaCl complex crystals. While the carotenoid signal was detected with a high intensity, a new band located at 992 cm⁻¹ suggests that a new potassium/sodium sulfate also crystallized from the solution in addition to the original potassium sulfate. This newly formed sulfate can be identified as (K,Na)₃Na(SO₄)₂ (Marszałek 2016), which naturally occurs as mineral aphthitalite (glaserite). The Raman bands due to this mineral were detected at 452, 619, 627, 992, 1082 and 1202 cm⁻¹, which is in a good agreement with reference values of 452, 620, 629, 991, 1087 and 1202 cm⁻¹ reported by Prieto-Taboada et al. 2019. This sulfate commonly appears as solid phase along with NaCl in the Na⁺-K⁺-Cl⁻/SO₄²⁻-H₂O system at temperatures higher than 4°C (Braitsch 1971). This suggests that the sulfate of aphthitalite composition replaced the K₂SO₄ phase of the original primary crystal to a high degree. In the case of the K₂SO₄–K₂SO₄ system, the signal of the crystal matrix dominated the resulting spectra. The intensity of ν₁ and ν₂ carotenoid bands at 1511 and 1153 cm⁻¹ was weak and of the same intensity scale as the surrounding ν₃ bands of the sulfate. The 1153 cm⁻¹ band is manifested only as a shoulder on the closely located 1145 cm⁻¹ mineral band. However, due to a low level of noise, additional second order bands can be clearly distinguished. For the NH₄Al(SO₄)₂·12H₂O–NaCl system the carotenoid signal was also clearly detected as medium intensity bands of 1511 and 1153 cm⁻¹ and several weak intensity combination bands in the second order spectral region. No changes were observed in the Raman spectrum of the primary crystal.

Crystals containing Hrr. sodomense

Raman spectra obtained when analyzing the primary chloride crystals containing trapped Hrr. sodomense manifest only strong signals due to the carotenoid α-bacterioruberin, which is the characteristic and generally dominant carotenoid of halophilic Archaea (Oren 2002a). The wavenumber values of the detected Raman bands were typically several reciprocal centimeters lower compared to the values of the S. ruber carotenoid: at 1507, 1151–2 and 1000 cm⁻¹ (compare Figs 2 and 3). This is consistent with previous spectroscopic studies using the two microorganisms (Jehlička et al. 2018; 2019). A strong Raman signal of α-bacterioruberin was detected for the potassium sulfate primary crystal sample, and also the weaker Raman bands were clearly distinguished at their correct positions. The carotenoid signals were significantly lower (relative to the signal of crystal phase) in the ammonium sulfate sample, but again at the correct positions.

Raman spectra of samples where the primary crystals were covered by a newly formed layer of NaCl are presented on the right side of Fig. 3. A relatively strong carotenoid signal was detected in the NaCl–NaCl system, although significantly lower than for the similar sample containing the S. ruber culture. The lowest carotenoid signal was detected in the KCl–NaCl system, where the resulting spectra contained moderately high backgrounds in addition. For the K₂SO₄–NaCl system, very similar results were obtained when compared to the S. ruber samples: a low level of noise, a strong signal of the newly formed phase of aphthitalite composition, and moderately strong carotenoid signals. In this particular case, however, the value for the ν₁ band position was 1509 cm⁻¹, which is 2 cm⁻¹ more than the usual value, as was detected for all other samples. We currently do not have an explanation for this difference, but the shift was consistent at several spots on the sample we analyzed. For the NH₄Al(SO₄)₂·12H₂O–NaCl system the carotenoid signal was also clearly detected with the positions of the two strongest pigment bands located at 1507 and 1150 cm⁻¹. Samples in which the primary crystals were sulfates show partial overlap of the Raman bands due to carotenoids and sulfates (bands around 1000 and 1152 cm⁻¹). In the NaCl–K₂SO₄ system, with the outer layer being the sulfate mineral, the level of noise was significant; however, the characteristic carotenoid signal was still detected at positions of 1508 and 1152 cm⁻¹ (see Table 1).

General analytical observations

When this type of complex crystal–biomarker system samples were analyzed, several challenges were encountered. Firstly, the inhomogeneity of the distribution of the organic biomass even at the small scale (the typical size of the primary crystals at the center of grown outer crystals was frequently less than 0.5 mm). This was addressed by the multiple analyzes at slightly different positions and subsequent averaging of the spectra. The crystallographic orientation of the macroscopic outer crystals also played a role during the analyzes. The laser power used was quite high, and in some cases of crystal orientation towards the instrument aperture from which the laser is directed at the sample, the detector was saturated by the excessive signal of the mineral matrix. This is probably due to different optical properties (dispersion, diffraction, absorption of both primary excitation and Raman signal) of the crystals in different directions. This was especially encountered for the sulfate crystals samples and was remedied by avoiding these problematic crystal–laser orientations. The fact that this instrument has a completely flat and wide probe-head was actually advantageous as it allowed easy positioning of the samples at orientations favorable for analysis. However, this is useful only for small samples (even more so because the region to be analyzed is close to their center) in direct contact with the aperture of the instrument. Frequently safety goggles were necessary as the positioning of the crystals was performed during ongoing laser interaction with the sample. More often, this flat probe-head is a complication, for example, for in situ field analyzes of microbial communities on outcrops, as there precise positioning of the aperture and simultaneous good focus in contact with the rock can be very difficult to achieve (Jehlička et al. manuscript in preparation).

CONCLUSIONS

Characteristic Raman spectra of the carotenoids salinixanthin and α-bacterioruberin were unambiguously detected in complex two-layered laboratory-grown artificial crystals of sulfates and chlorides containing trapped microorganisms S. ruber and Hrr. sodomense, respectively. The results demonstrate the ability of the miniature portable Raman spectrometer (equipped with the excitation enabling the resonance effect) to identify traces of life buried within a multi-component evaporitic matrix. Such complex samples can be good approximations for the often-complicated natural systems of evaporitic sedimentary rocks, which are considered as good candidates for preservation of traces of life, and may thus be suitable targets for in situ planetary astrobiology research in the near future.

ACKNOWLEDGEMENTS

This study was funded by the Czech Science Foundation Project 17–04270S and was also supported by the Center for Geosphere Dynamics (UNCE/SCI/006). AO was supported by Grant no. 2221/15 from the Israel Science Foundation. This study was further supported by the Erasmus + inter-institutional agreement between the Charles University, Prague and the Hebrew University of Jerusalem.

Conflicts of interest. None declared.