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DOI: 10.1039/C5AN00438A
Characterization of carotenoids in soil bacteria and investigation of their photodegradation by UVA radiation via resonance Raman spectroscopy Vinay Kumar B.N., ǂ,Փ Bernd Kampe, ǂ,Փ Petra Röschǂ,Փ and Jürgen Popp*,ǂ,±,Փ 5
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Institute of Physical Chemistry and Abbe Center of Photonics, University of Jena, Helmholtzweg 4, D-07743 Jena, Germany ± Leibniz Institute of Photonic Technology, Albert-Einstein-Straße 9, D-07745 Jena, Germany Փ InfectoGnostics, Forschungscampus Jena, Philosophenweg 7, D-07743 Jena, Germany 10
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ABSTRACT: A soil habitat consists of an enormous number of pigmented bacteria with the pigments mainly composed of diverse carotenoids. Most of the pigmented bacteria in the top layer of the soil are photoprotected from exposure to huge amounts of UVA radiation on a daily basis by these carotenoids. Photostability of these carotenoids depends heavily on the presence of specific features like a carbonyl group or an ionone ring system on its overall structure. Resonance Raman spectroscopy is one of the most sensitive and powerful techniques to detect and characterize these carotenoids and also monitor processes associated with it in its native system at a single cell resolution. However, most of the resonance Raman profiles of carotenoids have very minute differences thereby making it extremely difficult to confirm if these differences are attributed to the presence of different carotenoids or if it’s a consequence of its interaction with other cellular components. In this study, we device a method to overcome this problem by monitoring also the photodegradation of the carotenoids in question by UVA radiation wherein a differential photodegradation response will confirm the presence of different carotenoids irrespective of the proximities in their resonance Raman profiles. Using this method, the detection and characterization of carotenoids in pure cultures of five species of pigmented coccoid soil bacteria is achieved. We also shine light on the influence of the structure of the carotenoid on its photodegradation which can be exploited for use in characterization of carotenoids via resonance Raman spectroscopy.
INTRODUCTION 30
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A soil habitat consists of a huge diversity of microorganisms with diverse features and performing different functional roles.1 Many of these microorganisms are pigmented with most of their pigments being constituted of carotenoids. Different types of carotenoids are produced by microorganisms ranging from fungi to bacteria. 2 Carotenoids are isoprenoid compounds that possess a characteristic polyene chain that is mainly responsible for imparting color via its visible wavelength absorption capabilities. Carotenoids apart from imparting color play many important roles in bacteria like scavenging free radicals as efficient antioxidants, photoprotection from harmful ionizing radiation, light harvesting in photosynthetic bacteria and also function to strengthen membrane integrity.3 The diverse nature of carotenoids makes it an ideal marker of taxonomic importance in some of the bacteria at times even within a single genus. Some of the novel photostable carotenoids might also be of industrial application for use in cosmetics and food industry as an important constituent in sun screening creams and colorants in food.4 Carotenoids role as antioxidants finds many applications in the health and cosmetic industries throughout the world.5
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Carotenoids are also considered as one of the most sensitive biomarkers to look for as an indication for the presence of organics in inter planetary surfaces like that of mars.6 Carotenoids with this high range of applications are intensely studied worldwide via a range of techniques. Here, resonance Raman spectroscopy is an extremely sensitive technique for the analysis of pigments and has been used extensively for the detection and discrimination of carotenoids at a single cell level.7,8,9 Analysis of carotenoids both on a qualitative and quantitative basis has been accomplished quite well in the past few decades using various techniques like HPLC, GC-MS, LC-MS, NMR and UV-VIS absorption spectroscopy.10,11 These techniques are very sensitive and offer very good resolution with respect to separation of individual carotenoids constituted in a sample. However, these techniques have drawbacks in terms of the need for complex sample extraction and preparation procedures which in turn increases the time consumed for the analysis and also drastically reduce the chances of carrying out onsite real time analysis. Raman spectroscopy in contrast does not require the need for any sample preparation or extraction of pigments allowing for onsite real time analysis of native samples and also at a single cell resolution in the
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laboratory’s if needed.12,13 However, the technique does have a drawback of at times generating data that lead to contradictory interpretations in terms of a definitive characterization of carotenoids. In some cases, the conformational changes resulting from the carotenoid interaction with the native matrix components like proteins and carbohydrates leads to a major change in its Raman profile resulting in false interpretations about its structure and eventual assignments.14 Thereby, raising doubts if the carotenoid resonance Raman profile of close proximities observed in similar pigmented bacteria is indicative of the presence of a different carotenoid or not. To overcome this problem we probe the photodegradation of the carotenoids by UVA radiation via resonance Raman spectroscopy which can be considered as an approach to confirm the presence of differing major carotenoids constituting the bacteria irrespective of the proximities in the resonance Raman profile. In this study, we demonstrate the identification and discrimination of carotenoids in pure cultures of five species of pigmented coccoid soil bacteria using Raman spectroscopic single cell analysis and Raman mapping of a cluster of cells. Probing of the photodegradation of these carotenoids by UVA radiation proves to be influential in terms of confirming the presence of different carotenoids. It may also be possible to predict additional structural details of a novel carotenoid by comparison to the photodegradation responses of carotenoids whose structures are well studied.
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MATERIALS AND METHODS
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Bacterial species: The following species of pigmented bacteria; Micrococcus luteus (DSM 348), Kocuria rosea (DSM 20447), Kocuria turfanensis (DSM 22143), Kocuria salsicia (DSM 24776) and Deinococcus radiodurans (DSM 46620) from three Genera Micrococcus, Kocuria and Deinococcus were used in this study. For comparison, the non pigmented bacteria Micrococcus lylae (DSM 20315) was used as the control. All these bacterial cultures were obtained from the German collection of Microorganisms and cell cultures (Leibniz Institute DSMZ, Germany). Sample preparation: All the bacterial cultures were inoculated into a suitable growth media. Trypticase soy yeast agar medium was used for K. salsicia, K. turfanensis and D. radiodurans; M. luteus was cultured on nutrient agar media; K. rosea and M. lylae was cultured on corynebacterium agar media. All the bacterial cultures were incubated for 72 hr at an incubation temperature of 30 ºC barring M. lylae which was incubated at 37 ºC. A single colony of cells growing on the petri dishes is picked using a sterilized loop and transferred into distilled water contained in a small eppendorf vial. The cells are washed twice using distilled water by centrifuging the samples at 12500 rpm for three minutes to remove any residual media which would hinder the Raman spectra signatures. The pellet thus obtained is again suspended into distilled water and vortexed vigorously at 2500 rpm for two minutes in order to achieve separation of the cluster of cells so as to be able to analyze single cells. The vortexing step is skipped for preparation of bulk samples and the optical density is set to around 0.1 ensuring that the distribution of cells in all the samples are approximately even. 5 µl of these samples thus obtained are placed on a fused silica slide and allowed to air dry prior to the Raman analysis.
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Exposure to UVA radiation: All the pigmented bacteria and the non pigmented control bacteria were exposed to UVA radiation using a 366 nm UVA (6W) lamp. For this a single colony of cells is transferred into distilled water collected in a small petri dish. This petri dish with its lid removed is then placed on a shaking incubator with the set up to agitate cells at 100 rpm ensuring all cells on the petri dish are exposed to the radiation. The UVA lamp is placed at a distance of approximately 1.5 cm above the upper tip of the petri dish. The total exposure time for each sample was 240 minutes wherein 5 µl of the sample was withdrawn at every 30 minutes and Raman analyses was performed as mentioned above. Raman instrumentation: Raman spectroscopic measurements were carried out for single cells and bulk samples using a commercial micro-Raman device (HR LabRam inverse system, JobinYvon Horiba). A frequency doubled Nd:YAG laser with a wavelength of 532 nm was used as an excitation source. The laser power used was approximately 150 µW at the sample. The laser beam was focused on the sample by means of a 50X Leica PL Fluor objective down to a spot diameter of ~ 0.7-1 µm. The entrance slit of the dispersive spectrometer was set to 400 µm and the Raman scattered light was detected by a peltier cooled back illuminated CCD (1024 x 512 pixel) camera. The focal length of the spectrometer is 800 mm and is equipped with a grating of 1800 lines mm-1 set up to give a resolution of about ~1.3 cm-1. The Raman scattered light was detected by a Peltier cooled CCD detector. The total integration time per spectrum was ~1-3 s. For the Raman mapping experiments of the bulk samples were moved by an x/y motorized stage (Marzhauser, Wetzlar, Germany) with a minimal step size of 0.1 µm. The step size of the Raman map was configured to 1 µm in x and y directions and the integration times were chosen to be 1s for each point on the map during the Raman mapping so as to avoid photobleaching of the carotenoids. Data analysis: All Raman spectra were processed with the LabSpec software (Horiba Jobin-Yvon, Benzheim, Germany) and GNU R version (2.15.3)15 using scripts developed by our group. The Raman spectra acquired from single cells were calibrated using acetamidophenol as a reference which was measured regularly prior to the sample analysis. All the Raman spectra represent an averaged spectrum of 100 individual spectra acquired during the course of three batches of analysis. For intensity plots of the Raman maps, the spectra were baseline corrected and the images were smoothened by the LabSpec software. Integrated area scatter plots involving the two marker bands corresponding to carotenoids in all the five species of bacteria were estimated using Origin (OriginLab, Northampton, MA) data analysis software.
RESULTS AND DISCUSSION
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A large variety of microbes and plants in nature synthesize different types of carotenoids mainly serving purpose as light harvesting complexes in plants and highly efficient antioxidants in microorganisms like bacteria and fungi.16 These pigments contribute to the vibrant coloration of many plants and microbes in nature. Their unique structure consisting of a characteristic polyene chain comprising alternating single and double bonds contribute heavily to its color imparting nature.3 The tendency of carotenoids to be present in most of the microbes that live in extreme conditions
Analyst Accepted Manuscript
DOI: 10.1039/C5AN00438A
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has even drawn a lot of interest towards them being used as vital organic markers in extraterrestrial environments.17 Carotenoids have been proved to aid the microbes exposed to extreme stress like UV radiation, high salt concentrations etc in resisting free radical damage resulting from such extreme conditions.18-21 Resonance Raman spectroscopy has in the last two decades emerged as an important technique to study carotenoids because of the specificity with which carotenoids can be probed in any complex sample at a single cell resolution.22,23 Resonance Raman bands of carotenoids are observed at 1004 cm-1 corresponding to the in plane rocking vibrations of the CH3 groups attached to the polyene chain, 1157 and 1500-1550 cm-1 which correspond to the ν(C-C) and ν(C=C) vibrations respectively constituting the polyene chain of carotenoids.24,25,26 Overtones of these bands are also mostly visible at the higher wavenumber range. The identification and characterization of the carotenoids are based on the resonance Raman signals at 1157 and 1500-1550 cm-1 and are the most sensitive markers wherein the position of these signals is highly influenced by their overall structure. The position of the Raman signal at 1500-1550 cm-1 is influenced specifically by the conjugated chain length in the carotenoid structure.27 These properties have enabled the successful characterization of carotenoids from different bacteria via resonance Raman spectroscopy. Using resonance Raman spectroscopy we have analysed five species of pure cultures of pigmented soil bacteria for the characterization of the carotenoids constituted in them. Fig. 1 shows the resonance Raman spectra of D. radiodurans, K. rosea and M. luteus acquired from single cells with each Raman spectrum representing an average of 100 individual measurements taken during the course of three batches. And Fig. 1(f) represents the integrated Raman intensity standard deviation of the Raman signals of the carotenoid bands at 1153-57 and 1500-30 cm-1 for all the five species of bacteria respectively. The red pigmented soil bacterium K. rosea has characteristic resonance Raman signals at 1153 and 1506 cm-1 which corresponds to the carotenoids unique structural features mentioned previously. K. rosea is known to be constituted of different configurations of bacterioruberin as the major carotenoid in its cells which has a structure composed of 13 conjugated double bonds.28 D. radiodurans which is also a red pigmented bacterium has resonance Raman signals at 1153 and 1510 with a red shift of 4 cm-1 when compared to the carotenoid profile of K. rosea. D. radiodurans is constituted of deinoxanthin as the major carotenoid in its cells which has a structure composed of 11 conjugated double bonds.29 M. luteus which is a yellow pigmented bacterium has resonance Raman signals at 1157 and 1530 cm-1 with a red shift of 24 and 20 cm-1 respectively when compared to K. rosea and D. radiodurans. Sarcinaxanthin is the major carotenoid in M. luteus and has a structure composed of 9 conjugated double bonds.30 The resonance Raman profile representing carotenoids present in these three pigmented bacteria is in good correlation with their structures based on previous observations that the Raman signal at the 1500 cm-1 position is red shifted to lower wavenumber positions with the increase in the conjugated chain length in the carotenoids structure. The Raman spectra precisely correlates with the conjugated chain length features of the carotenoids in these three species of bacteria and can
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be clearly distinguished from one another even though the differences are minute in the case of D. radiodurans and K. rosea. Fig. 1 also shows the resonance Raman spectra of K. salsicia and K. turfanensis, the major carotenoids present in these two species of pigmented bacteria has not been studied or reported previously to the best of our knowledge. The resonance Raman profile of the major carotenoid present in the yellow pigmented bacteria K. salsicia shows Raman signals at 1157 and 1523 cm-1. The position of the Raman signal at 1523 cm-1 suggests that the major carotenoid present may have about 7 or 8 conjugated double bonds in its structure as deduced by comparing it with the Raman spectra of standard canthaxanthin carotenoid which has 9 conjugated double bonds and has a Raman signal at 1520 cm-1 as shown in Fig. 1(g). Similarly, the red pigmented K turfanesis shows resonance Raman signals at 1153 and 1514 cm-1, the Raman signal at this position suggests that the major carotenoid present may have about 11 conjugated double bonds as deduced by comparing it with the Raman spectra of lycopene which also has 11 conjugated double bonds and the Raman signal at 1514 cm-1 as shown in Fig. 1(g). However, a recent study by Vanessa de Oliveira et al14 suggested that such assignments should be made with caution as they proved using different plant sources that the position of the 1500-1550 cm-1 Raman signal is also influenced by the interaction of the carotenoids with the surrounding biomolecular components like proteins and relevant cell matrix components. And also the small changes in the position of the ν(C=C) Raman signal observed must be considered with caution irrespective of the resolution of the spectrometer used for the study. Such an influence could easily lead to misinterpretation of carotenoids under investigation. To overcome this problem and to confirm that the minute resonance Raman spectral changes we observe in similarly pigmented bacteria is indeed resulting from a different carotenoid and not by other influences, we have probed the response of the carotenoids against UVA radiation. As mentioned earlier, carotenoids are efficient UV radiation protectants due to its antioxidant and light harvesting properties. Most of the bacteria that grow in regions of extreme sunlight exposure are equipped with carotenoids to survive against large doses of UVA radiation exposure.31 We considered that a carotenoid with a different overall structure must exhibit different kinetics of photodegradation by UVA radiation irrespective of its interaction with other cellular components.36, 41 Fig. 2(a) shows the resonance Raman kinetics of the major carotenoid present in D. radiodurans which has a Raman signal at 1510 cm-1 acquired from single cells at time intervals of 30 minutes each collected over the course of 240 minutes. Fig. 2(c) shows the response of the Raman bands at 1153 and 1510 cm-1 corresponding to the major carotenoid to UVA radiation exposure at different time points. It is clearly seen that the resonance Raman signal resulting from the major carotenoid Deinoxanthin whose structure has 11 conjugated double bonds and a keto group in the ionone ring terminal is extremely photo stable throughout the exposure time. It is well known that D. radiodurans is one amongst the toughest bacterium on the planet and can resist extreme conditions due to its unique features of which its major carotenoid has a vital contribution. Fig. 2(b) shows the consequent Raman maps acquired from a bulk of cells depicting the relative intensity ratio distribution of the
Analyst Accepted Manuscript
DOI: 10.1039/C5AN00438A
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carotenoids present at time points 30 and 240 minutes for D. radiodurans. The Raman maps were acquired from the same sample but from different areas; as we draw out 5 µl of the sample at different time points and dry the sample prior Raman analysis, there was no possibility of analysing the same area at a different time point. The maps are color coded from blue to yellow in the increasing order wherein blue regions represent minute or no presence of carotenoids and yellow represents a substantial presence. These time points for mapping were chosen based on the observations made in single cell measurements. The intention of acquiring a Raman map was solely to show that the photodegradation pattern is a collective response occurring in approximately all the cells and to detect if the carotenoid is stable or degraded at those respective time points in the bulk of cells. In general, the relative intensity ratio distribution of Raman signals 1153 and 1510 cm-1 corresponding to carotenoids observed in all the Raman maps quite clearly depicts the presence or absence of carotenoids at the respective time points, with the Raman map corresponding to D. radiodurans showing the presence of carotenoids even after 240 minutes of exposure to UVA radiation indicating its stability and resistance to photodegradation within the time points considered in this study. There wasn’t any definite pattern to indicate substantial degradation of carotenoids with time in this case as seen from both the single cell measurements and consequent Raman maps in Fig. 2. Fig. 2(c) shows the response of the Raman bands at 1153 and 1506 cm-1 corresponding to the major carotenoid to UVA radiation exposure at different time points. We also detected intense carotenoid signals in some of the cells at 240 minutes which matched the intensities of those observed at 30 minutes as shown in Fig. 2(c) clearly confirming that the carotenoid in D. radiodurans was overall unaffected during the time span of 240 minutes. A completely different photodegradation profile was observed in K. rosea. The major carotenoid bacterioruberin present in K. rosea has a Raman signal corresponding to ν(C=C) at 1506 cm-1 and an overall open chain structure consisting of 13 conjugated double bonds was completely photodegraded within a span of 150 minutes as shown in Fig. 3(a). However the samples were monitored for the complete duration of 240 minutes to confirm that there is no upregulation in carotenoid synthesis. The subsequent Raman maps shown in Fig. 3(b) acquired at 30 and 150 minutes illustrating the photodegradation of carotenoid in a large number of cells. Fig. 3(c) shows the response of the Raman bands at 1153 and 1506 cm-1 corresponding to the major carotenoid to UVA radiation exposure at different time points. This differential photodegradation of the carotenoids present in these two species of red pigmented bacteria towards UVA radiation clearly confirms the presence of different carotenoids irrespective of the proximities in their resonance Raman spectra. In general, it has been observed in previous studies via transient absorption spectroscopic techniques that a carotenoid with a keto group has a lower photodegradation quantum yield and is less reactive and more photostable.32 The rate constants of photobleaching have also been observed to be lower in a carotenoid that possesses a keto functional group in their structure.33 Our results as shown in Fig. 2(c) and Fig. 3(c) are in good correlation with these studies in that the carotenoid in D. radiodurans apparently is composed of such features and happens to be clearly
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more photostable displaying an extensively lower photodegradation level when compared to K. rosea. Fig. 4(a) illustrates the carotenoid photodegration profile of another red pigmented bacteria K. turfanensis analyzed in this study wherein the carotenoids are quite stable but photodegrade drastically post 180 minutes and are almost completely undetectable after 210 minutes of UVA exposure. The corresponding Raman maps in Fig. 4(b) at time points 30 and 210 minutes showing precise photodegradation of the carotenoids in the bulk of cells. Fig. 4(c) shows the response of the Raman bands at 1153 and 1514 cm-1 corresponding to the major carotenoid to UVA radiation exposure at different time points which clearly indicates a delayed degradation when compared to the carotenoid in K. rosea and highly less photostability when compared to the carotenoid in D. radiodurans. We can deduce from this pattern that the carotenoid present in this species could possibly be an open chain structure similar to that of the carotenoid present in K. rosea which is bacterioruberin. Such patterns have been observed previously wherein the carotenoids without ionone ring terminals and no carbonyl groups are degraded at higher rates when compared to their counterparts possessing these features.34,35 Such a pattern is also seen in Fig. 4(c) in comparison to the carotenoid in D. radiodurans as shown in Fig. 2(c). Although, it probably does have fewer numbers of double bonds in its structure when compared to bacterioruberin by taking into consideration also the position of the ν(C=C) Raman signal at 1514 cm-1 as mentioned previously. This could be one of the reasons associated with its delayed degradation when compared to the carotenoid in K. rosea. Nevertheless, the differential carotenoid photodegradation profile when compared to the other two red pigmented bacteria confirms the presence of a unique carotenoid in K. turfanensis. Similarly, the carotenoid photodegradation profile of two of the yellow pigmented bacteria M. luteus and K. salsicia were also analysed. The carotenoid degradation profiles of both these bacteria were very similar and the carotenoids were not photodegraded completely during the course of 240 minutes as seen from the single cell measurements and the consequent Raman maps shown in Fig. 5. And also the time dependent response of the marker band of carotenoids to UVA radiation confirms that they share an overall similar photodegradation pattern as shown in Fig. 6. Fig. 6 depicts that carotenoids in both these bacteria show a sharp decrease during the first 30 minutes of exposure and later is relatively stable until the 240 minutes of UVA radiation exposure. This pattern suggests that the carotenoids in these two bacteria have similar structures, however the position of the Raman signal corresponding to the conjugated chain length which appears at 1530 cm-1 for M. luteus and at 1523 cm-1 for K. salsicia respectively may be suggesting the presence of a difference in the conjugated double bond system of their structure which might be misleading. However, their similar photodegradation profile happens to signal that these two species of bacteria have similar carotenoids; the major carotenoid present in K. salsicia could possibly have a very similar structure as sarcinaxanthin which is the major carotenoid present in M. luteus as described previously. And the prominent red shift observed in the Raman signal corresponding to the conjugated chain length observed in their resonance Raman profile could be indeed resulting from an influence of the carotenoid interaction with
Analyst Accepted Manuscript
DOI: 10.1039/C5AN00438A
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other cell components as observed by Vanessa de Oliveira et al previously.14 This clearly advocates the need for reservations when using resonance Raman spectroscopy alone for decisive characterization of carotenoids which have not been studied previously. It must also be mentioned here that the substantial degradation of carotenoids observed in both these species when compared to the carotenoid in D. radiodurans suggests overall that D. radiodurans has the most photostable carotenoid amongst the five species analyzed in this study. Interestingly, the photodegradation pattern of the carotenoids we observed in the pigmented bacteria used in this study correlates well with a study based on UV B photodegradation of carotenoids present in human skin involving a closed chain β-carotene and open chain lycopene carotenoids.36 We could also deduce based on the photodegradation profile that carotenoids with ionone ring terminals and carbonyl groups, hydroxyl groups associated with ionone ring terminals and a lower conjugation chain length are possibly less susceptible to photodegradation than their counterparts as observed based on the profiles of D. radiodurans and M. luteus which both have carotenoids with such features. However, it should be noted that there is no link between the photodegradation profile and the antioxidant property of a carotenoid. A vast variety of bacteria and other microorganisms in general have evolved to upregulate the synthesis of carotenoids in the presence of ample nutrients when growing under stressful conditions like large doses of UV radiation which possibly nullifies the effect of possessing less photostable carotenoids.37,38 Additionally, to check the effect of UVA photodegradation of carotenoids on the other biomolecular components of the bacterial cells, we took the UVA exposed fraction of cells from all the samples after 240 minutes and spread plated this fraction in their respective media. Those species of bacteria wherein the carotenoids remained stable throughout the UVA exposure period i.e. in D. radiodurans, M. luteus and K. salsicia, were all viable and new colonies were formed. Whereas, in K. rosea and K. turfanensis wherein the carotenoids were degraded, there was no growth observed. And also, when the non pigmented bacterium M. lylae was exposed to UVA radiation, the UVA exposed fraction collected after just 30 minutes of exposure showed no growth, this is because of the obvious damages to DNA, protein etc induced by the UVA radiation which makes the bacteria non viable.39 This clearly demonstrated the protective role of the carotenoids against UVA radiation, its presence along with enzymatic antioxidant systems prove to be critical in the survival of bacteria against stressful conditions.40 In conclusion, our results precisely demonstrate the potential of resonance Raman spectroscopy in the detection and characterization of carotenoids from pure cultures of soil bacteria at a single cell resolution. Probing the photodegradation profile of the carotenoids against UVA radiation proves valuable as an important analysis to confirm the presence of different carotenoids amongst bacteria with similar colored pigments irrespective of the proximities in their resonance Raman profiles. A basic provisionary understanding of the structure of novel carotenoids can be possibly made by comparing their photodegration profile with carotenoids whose structures are well known. Anyhow, it must be mentioned that the separation of structurally very closely related carotenoids within a
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single and different systems is still not achievable using resonance Raman spectroscopy because of the overlap of the main Raman signals corresponding to carotenoids as may be seen in the case of M. luteus and K. salsicia and thereby must be supplemented with techniques like HPLC and LC-MS.
SUMMARY 65
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In the present study, we report the characterization of carotenoids in five species of pure of cultures of pigmented soil bacteria. We demonstrate for the first time the influence of probing the photodegradation of carotenoids by UVA radiation in order to confirm the presence of different carotenoids irrespective of the proximities in their resonance Raman profiles. This method can be used to investigate if there is any influence of other cell components on the position of resonance Raman signals which is vital for the characterization of carotenoids in order to avoid missassignments. And it also has the potential to be applicable for gaining basic information about the overall structure of a novel carotenoid as shown in this study. However it is necessary to also analyze more pigmented bacteria preferably from those isolated from different environmental sources to validate further the utility of probing photodegradation patterns to assist in the characterization of carotenoids which will be addressed in our future studies. Nevertheless, an accurate and comprehensive prediction of a novel carotenoid would have to be supplemented with the analysis via gold standard techniques like HPLC and LC-MS.
AUTHOR INFORMATION 85
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Corresponding author * E-mail: [email protected], Phone: +49-3641-948320, +49-3641-206300, Fax: +49-3641-948302.
ACKNOWLEDGEMENTS This research is financially supported by the German Research Foundation (DFG) under the grant GRK 1257/2: “Alteration and element mobility at the microbe-mineral interface”.
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Notes The authors declare no competing financial interest. REFERENCES:
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1) W. B. Whitman, D. C. Coleman and W. J. Wiebe, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 6578-6583. 2) S. Liaaen-Jensen and A. G. Andrewes, Annu. Rev. Microbiol., 1972, 26, 225-248. 3) G. Britton, FASEB J., 1995, 9, 1551-1558. 4) W. Stahl and H. Sies, Am J ClinNutr., 2012, 96, 1179s-1184s. 5) W. Stahl, U. Heinrich, H. Jungmann, H. Sies and H. Tronnier, Am J Clin Nutr., 2000, 71, 795-798. 6) H. G. Edwards, I. B. Hutchinson, R. Ingley, J. Parnell, P. Vitek and J. Jehlicka, Astrobiology., 2013, 13, 543-549.
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7) J. Jehlicka, H. G. Edwards and A. Oren, Appl. Environ. Microbiol., 2014, 80, 3286-3295. 8) I.A. Weissflog, K. Grosser, M. Bräutigam, B. Dietzek, G. Pohnert and J. Popp. ChemBioChem., 2013, 14, 727-732. 9) S. Kumar, N. Matange, S. Umapathy and Sandhya S. Visweswariah, FEMS Microbiol. Lett., 2015, 362, 1-6. 10) S. M. Rivera, P. Christou and R. Canela-Garayoa, Mass Spectrom Rev., 2014, 33, 353-372. 11) H. C. Furr, Journal of Nutrition., 2004, 134, 281s-285s. 12) J. Jehlicka, H. G. Edwards and A. Oren, Spectrochimicaacta. Part A, Molecular and biomolecular spectroscopy, 2013, 106, 99-103. 13) A. Silge, E. Abdou, K. Schneider, S. Meisel , T. Bocklitz, H. W. LuWalther, R. Heintzmann, P. Rosch and J. Popp. Cell. Microbiol., 2014. 41 14) E. Vanessa de Oliveira, H.V. Castro, H.G. Edwards and L.F. de Oliveira. J. Raman Spectrosc., 2010. 642-650. 15) R Development core team R: a language and environment for statistical computing. R foundation for statistical computing, 2011, Vienna. 16) G. A. Armstrong and J. E. Hearst, Faseb Journal., 1996, 10, 228-237. 17) H. G. M. Edwards, I. B. Hutchinson and R. Ingley, Int J Astrobiol., 2012, 11, 269-278. 18) C. S. Cockell, P. Rettberg, G. Horneck, K. Scherer and M. D. Stokes, Polar Biol., 2003, 26, 62-69. 19) D. Libkind, P. Perez, R. Sommaruga, M.D.C. Dieguez, M. Ferraro, S. Brizzio, H. Zagarese and M Broock Van. Photochem. Photobiol. Sci., 2004, 3, 281-286. 20) M. Moline, M. R. Flores, D. Libkind, C. DieguezMdel, M. E. Farias and M. van Broock, Photochemical &photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology., 2010, 9, 1145-1151. 21) M. De Lourdes Moreno, C. Sanchez-Porro, M.T. Garcia and E. Mellado. Methods Mol. Biol., 2012, 892, 207-217. 22) P. Rösch, M. Harz, M. Schmitt, K.D. Peschke, O. Ronneberger, H. Burkhardt, H.W. Motzkus, M. Lankers, S. Hofer, H. Thiele and J. Popp, Appl.Environ.Biol., 2005, 71, 1626-1637. 23) C. Matthäus, S. Schubert, M. Schmitt, C. Krafft, B. Dietzek, Ulrich S. Schubert and J. Popp, ChemPhysChem, 2013, 14, 155-161. 24) R. Withnall, B. Z. Chowdhry, J. Silver, H. G. Edwards and L. F. de Oliveira, Spectrochim. Acta, Part A., 2003, 59, 2207-2212. 25) S. Schlücker, A. Szeghalmi, M. Schmitt, J. Popp and W. Kiefer, J. Raman Spectrosc., 2003, 34, 413-419. 26) M. Schmitt, B. Leimeister, L. Baia, B. Weh, I. Zimmermann, W. Kiefer and J. Popp, ChemPhysChem, 2003, 3, 296-299. 27) K. Grosser, L. Zedler, M. Schmitt, B. Dietzek, J. Popp and G. Pohnert, Journal of Bioadhesion and Biofilm Research, 2012, 28, 687-696. 28) A. Strand, S. Shivaji and S. Liaaen Jensen, Biochem. Syst. Ecol., 1997, 25, 547-552. 29) J. Hong-Fang, Int. J. Mol. Sci., 2010, 11(11), 4506-4510. 30) R. Netzer, M. H. Stafsnes, T. Andreassen, A. Goksoyr, P. Bruheim and T. Brautaset, J. Bacteriol., 2010, 192, 5688-5699. 31) Y. Zhu, J. E. Graham, M. Ludwig, W. Xiong, R. M. Alvey, G. Shen and D. A. Bryant, Arch BiochemBiophys., 2010, 504, 86-99. 32) J.T Landrum, Ed. Carotenoids: Physical, Chemical and Biological Functions and Properties; CRC Press, Florida, US, 2009. 33) A. Mortensen and L. H. Skibsted, Free Radic Res., 1997, 26, 549-563. 34) B. R. Nielsen, A. Mortensen, K. Jorgensen and L. H. Skibsted, J Agr Food Chem., 1996, 44, 2106-2113. 35) D. Cvetkovic and D. Markovic, J Serb ChemSoc., 2008, 73, 15-27. 36) M. E. Darvin, I. Gersonde, H. Albrecht, W. Sterry and J. Lademann, Laser Phys., 2006, 16, 833-837. 37) M. Ehling-Schulz, W. Bilger and S. Scherer, J. Bacteriol., 1997, 179, 1940-1945. 38) B. Mogedas, C. Casal, E. Forjan and C. Vilchez. J BiosciBioeng., 2009, 108, 47-51. 39) P.M. Girard, S. Francesconi, M. Pozzebon, D. Graindorge, P. Rochette, R. Drouin and E. Sage. E. Journal of Physics: Conference Series., 2011, 261, 1-10. 40) X. Lin, X. Xu, C. Yang, Y. Zhao, Z. Feng and Y. Dong, Ecotoxicol. Environ. Saf., 2009, 72, 1899-1904.
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41) T.R. Hata, T.A. Scholz, I.V. Ermakov, R.W. McClane, F. Khachik, W. Gellermann and L.K. Persching. J. Invest. Dermatol. 2000, 115, 441448.
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Figures Fig.1 5
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Fig 1. Resonance Raman spectra of carotenoids in (a) red pigmented K. rosea (b) red pigmented D. radiodurans (c) red pigmented K. turfanensis (d) yellow pigmented M. luteus (e) yellow pigmented K. salsicia (f) Integrated Raman intensity standard deviation of the two marker bands of carotenoids in all species (g) lycopene and canthaxanthin standards.
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Fig. 2
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Fig 2. (a) Single cell resonance Raman photodegradation profile of the carotenoid in D. radiodurans exposed to UVA radiation up to 240 minutes (b) Relative intensity ratio distribution of the Raman bands at 1510 and 1153 cm-1 representing carotenoids at time point 30 minutes (1) and 240 minutes (2); (c) Integrated peak area of the marker bands corresponding to the carotenoids as a function of UVA radiation exposure.
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Fig 3. (a) Single cell resonance Raman photodegradation profile of the carotenoid in K. rosea exposed to UVA radiation up to 240 minutes (b) Relative intensity ratio distribution of the Raman bands at 1506 and 1153 cm-1 representing carotenoids at time point 30 minutes (1) and 240 minutes (2); (c) Integrated peak area of the marker bands corresponding to the carotenoids as a function of UVA radiation exposure.
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Fig.4
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Fig 4. (a) Single cell resonance Raman photodegradation profile of the carotenoid in K. turfanensis exposed to UVA radiation up to 240 minutes (b) Relative intensity ratio distribution of the Raman bands at 1514 and 1153 cm-1 representing carotenoids at time point 30 minutes (1) and 240 minutes (2); (c) Integrated peak area of the marker bands corresponding to the carotenoids as a function of UVA radiation exposure.
Fig.5
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Fig 5. (a) Single cell resonance Raman photodegradation profile of the carotenoids in M. luteus exposed to UVA radiation up to 240 minutes. (b) Relative intensity ratio distribution of the Raman bands at 1530 and 1157 cm-1 representing carotenoids at time point 30 minutes (1) and 240 minutes (2). (c) Single cell resonance Raman photodegradation profile of the carotenoid in K. salsicia exposed to UVA radiation up to 240 minutes. (d) Relative intensity distribution of the Raman bands at 1523 and 1157 cm-1 representing carotenoids at time point 30 minutes (1) and 240 minutes (2).
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Fig.6
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Fig 6. (a) Integrated peak area of the marker bands corresponding to the carotenoids in M. luteus as a function of UVA radiation exposure. (b) Integrated peak area of the marker bands corresponding to the carotenoids in K. salsicia as a function of UVA radiation exposure.
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Characterization of carotenoids in soil bacteria and investigation of their photodegradation by UVA radiation via resonance Raman spectroscopy Vinay Kumar B.N., ǂ,Փ Bernd Kampe, ǂ,Փ Petra Röschǂ,Փ and Jürgen Popp*,ǂ,±,Փ 5
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Institute of Physical Chemistry and Abbe Center of Photonics, University of Jena, Helmholtzweg 4, D-07743 Jena, Germany ± Leibniz Institute of Photonic Technology, Albert-Einstein-Straße 9, D-07745 Jena, Germany Փ InfectoGnostics, Forschungscampus Jena, Philosophenweg 7, D-07743 Jena, Germany 10
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ABSTRACT: A soil habitat consists of an enormous number of pigmented bacteria with the pigments mainly composed of diverse carotenoids. Most of the pigmented bacteria in the top layer of the soil are photoprotected from exposure to huge amounts of UVA radiation on a daily basis by these carotenoids. Photostability of these carotenoids depends heavily on the presence of specific features like a carbonyl group or an ionone ring system on its overall structure. Resonance Raman spectroscopy is one of the most sensitive and powerful techniques to detect and characterize these carotenoids and also monitor processes associated with it in its native system at a single cell resolution. However, most of the resonance Raman profiles of carotenoids have very minute differences thereby making it extremely difficult to confirm if these differences are attributed to the presence of different carotenoids or if it’s a consequence of its interaction with other cellular components. In this study, we device a method to overcome this problem by monitoring also the photodegradation of the carotenoids in question by UVA radiation wherein a differential photodegradation response will confirm the presence of different carotenoids irrespective of the proximities in their resonance Raman profiles. Using this method, the detection and characterization of carotenoids in pure cultures of five species of pigmented coccoid soil bacteria is achieved. We also shine light on the influence of the structure of the carotenoid on its photodegradation which can be exploited for use in characterization of carotenoids via resonance Raman spectroscopy.
INTRODUCTION 30
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A soil habitat consists of a huge diversity of microorganisms with diverse features and performing different functional roles.1 Many of these microorganisms are pigmented with most of their pigments being constituted of carotenoids. Different types of carotenoids are produced by microorganisms ranging from fungi to bacteria. 2 Carotenoids are isoprenoid compounds that possess a characteristic polyene chain that is mainly responsible for imparting color via its visible wavelength absorption capabilities. Carotenoids apart from imparting color play many important roles in bacteria like scavenging free radicals as efficient antioxidants, photoprotection from harmful ionizing radiation, light harvesting in photosynthetic bacteria and also function to strengthen membrane integrity.3 The diverse nature of carotenoids makes it an ideal marker of taxonomic importance in some of the bacteria at times even within a single genus. Some of the novel photostable carotenoids might also be of industrial application for use in cosmetics and food industry as an important constituent in sun screening creams and colorants in food.4 Carotenoids role as antioxidants finds many applications in the health and cosmetic industries throughout the world.5
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Carotenoids are also considered as one of the most sensitive biomarkers to look for as an indication for the presence of organics in inter planetary surfaces like that of mars.6 Carotenoids with this high range of applications are intensely studied worldwide via a range of techniques. Here, resonance Raman spectroscopy is an extremely sensitive technique for the analysis of pigments and has been used extensively for the detection and discrimination of carotenoids at a single cell level.7,8,9 Analysis of carotenoids both on a qualitative and quantitative basis has been accomplished quite well in the past few decades using various techniques like HPLC, GC-MS, LC-MS, NMR and UV-VIS absorption spectroscopy.10,11 These techniques are very sensitive and offer very good resolution with respect to separation of individual carotenoids constituted in a sample. However, these techniques have drawbacks in terms of the need for complex sample extraction and preparation procedures which in turn increases the time consumed for the analysis and also drastically reduce the chances of carrying out onsite real time analysis. Raman spectroscopy in contrast does not require the need for any sample preparation or extraction of pigments allowing for onsite real time analysis of native samples and also at a single cell resolution in the
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laboratory’s if needed.12,13 However, the technique does have a drawback of at times generating data that lead to contradictory interpretations in terms of a definitive characterization of carotenoids. In some cases, the conformational changes resulting from the carotenoid interaction with the native matrix components like proteins and carbohydrates leads to a major change in its Raman profile resulting in false interpretations about its structure and eventual assignments.14 Thereby, raising doubts if the carotenoid resonance Raman profile of close proximities observed in similar pigmented bacteria is indicative of the presence of a different carotenoid or not. To overcome this problem we probe the photodegradation of the carotenoids by UVA radiation via resonance Raman spectroscopy which can be considered as an approach to confirm the presence of differing major carotenoids constituting the bacteria irrespective of the proximities in the resonance Raman profile. In this study, we demonstrate the identification and discrimination of carotenoids in pure cultures of five species of pigmented coccoid soil bacteria using Raman spectroscopic single cell analysis and Raman mapping of a cluster of cells. Probing of the photodegradation of these carotenoids by UVA radiation proves to be influential in terms of confirming the presence of different carotenoids. It may also be possible to predict additional structural details of a novel carotenoid by comparison to the photodegradation responses of carotenoids whose structures are well studied.
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MATERIALS AND METHODS
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Bacterial species: The following species of pigmented bacteria; Micrococcus luteus (DSM 348), Kocuria rosea (DSM 20447), Kocuria turfanensis (DSM 22143), Kocuria salsicia (DSM 24776) and Deinococcus radiodurans (DSM 46620) from three Genera Micrococcus, Kocuria and Deinococcus were used in this study. For comparison, the non pigmented bacteria Micrococcus lylae (DSM 20315) was used as the control. All these bacterial cultures were obtained from the German collection of Microorganisms and cell cultures (Leibniz Institute DSMZ, Germany). Sample preparation: All the bacterial cultures were inoculated into a suitable growth media. Trypticase soy yeast agar medium was used for K. salsicia, K. turfanensis and D. radiodurans; M. luteus was cultured on nutrient agar media; K. rosea and M. lylae was cultured on corynebacterium agar media. All the bacterial cultures were incubated for 72 hr at an incubation temperature of 30 ºC barring M. lylae which was incubated at 37 ºC. A single colony of cells growing on the petri dishes is picked using a sterilized loop and transferred into distilled water contained in a small eppendorf vial. The cells are washed twice using distilled water by centrifuging the samples at 12500 rpm for three minutes to remove any residual media which would hinder the Raman spectra signatures. The pellet thus obtained is again suspended into distilled water and vortexed vigorously at 2500 rpm for two minutes in order to achieve separation of the cluster of cells so as to be able to analyze single cells. The vortexing step is skipped for preparation of bulk samples and the optical density is set to around 0.1 ensuring that the distribution of cells in all the samples are approximately even. 5 µl of these samples thus obtained are placed on a fused silica slide and allowed to air dry prior to the Raman analysis.
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Exposure to UVA radiation: All the pigmented bacteria and the non pigmented control bacteria were exposed to UVA radiation using a 366 nm UVA (6W) lamp. For this a single colony of cells is transferred into distilled water collected in a small petri dish. This petri dish with its lid removed is then placed on a shaking incubator with the set up to agitate cells at 100 rpm ensuring all cells on the petri dish are exposed to the radiation. The UVA lamp is placed at a distance of approximately 1.5 cm above the upper tip of the petri dish. The total exposure time for each sample was 240 minutes wherein 5 µl of the sample was withdrawn at every 30 minutes and Raman analyses was performed as mentioned above. Raman instrumentation: Raman spectroscopic measurements were carried out for single cells and bulk samples using a commercial micro-Raman device (HR LabRam inverse system, JobinYvon Horiba). A frequency doubled Nd:YAG laser with a wavelength of 532 nm was used as an excitation source. The laser power used was approximately 150 µW at the sample. The laser beam was focused on the sample by means of a 50X Leica PL Fluor objective down to a spot diameter of ~ 0.7-1 µm. The entrance slit of the dispersive spectrometer was set to 400 µm and the Raman scattered light was detected by a peltier cooled back illuminated CCD (1024 x 512 pixel) camera. The focal length of the spectrometer is 800 mm and is equipped with a grating of 1800 lines mm-1 set up to give a resolution of about ~1.3 cm-1. The Raman scattered light was detected by a Peltier cooled CCD detector. The total integration time per spectrum was ~1-3 s. For the Raman mapping experiments of the bulk samples were moved by an x/y motorized stage (Marzhauser, Wetzlar, Germany) with a minimal step size of 0.1 µm. The step size of the Raman map was configured to 1 µm in x and y directions and the integration times were chosen to be 1s for each point on the map during the Raman mapping so as to avoid photobleaching of the carotenoids. Data analysis: All Raman spectra were processed with the LabSpec software (Horiba Jobin-Yvon, Benzheim, Germany) and GNU R version (2.15.3)15 using scripts developed by our group. The Raman spectra acquired from single cells were calibrated using acetamidophenol as a reference which was measured regularly prior to the sample analysis. All the Raman spectra represent an averaged spectrum of 100 individual spectra acquired during the course of three batches of analysis. For intensity plots of the Raman maps, the spectra were baseline corrected and the images were smoothened by the LabSpec software. Integrated area scatter plots involving the two marker bands corresponding to carotenoids in all the five species of bacteria were estimated using Origin (OriginLab, Northampton, MA) data analysis software.
RESULTS AND DISCUSSION
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A large variety of microbes and plants in nature synthesize different types of carotenoids mainly serving purpose as light harvesting complexes in plants and highly efficient antioxidants in microorganisms like bacteria and fungi.16 These pigments contribute to the vibrant coloration of many plants and microbes in nature. Their unique structure consisting of a characteristic polyene chain comprising alternating single and double bonds contribute heavily to its color imparting nature.3 The tendency of carotenoids to be present in most of the microbes that live in extreme conditions
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has even drawn a lot of interest towards them being used as vital organic markers in extraterrestrial environments.17 Carotenoids have been proved to aid the microbes exposed to extreme stress like UV radiation, high salt concentrations etc in resisting free radical damage resulting from such extreme conditions.18-21 Resonance Raman spectroscopy has in the last two decades emerged as an important technique to study carotenoids because of the specificity with which carotenoids can be probed in any complex sample at a single cell resolution.22,23 Resonance Raman bands of carotenoids are observed at 1004 cm-1 corresponding to the in plane rocking vibrations of the CH3 groups attached to the polyene chain, 1157 and 1500-1550 cm-1 which correspond to the ν(C-C) and ν(C=C) vibrations respectively constituting the polyene chain of carotenoids.24,25,26 Overtones of these bands are also mostly visible at the higher wavenumber range. The identification and characterization of the carotenoids are based on the resonance Raman signals at 1157 and 1500-1550 cm-1 and are the most sensitive markers wherein the position of these signals is highly influenced by their overall structure. The position of the Raman signal at 1500-1550 cm-1 is influenced specifically by the conjugated chain length in the carotenoid structure.27 These properties have enabled the successful characterization of carotenoids from different bacteria via resonance Raman spectroscopy. Using resonance Raman spectroscopy we have analysed five species of pure cultures of pigmented soil bacteria for the characterization of the carotenoids constituted in them. Fig. 1 shows the resonance Raman spectra of D. radiodurans, K. rosea and M. luteus acquired from single cells with each Raman spectrum representing an average of 100 individual measurements taken during the course of three batches. And Fig. 1(f) represents the integrated Raman intensity standard deviation of the Raman signals of the carotenoid bands at 1153-57 and 1500-30 cm-1 for all the five species of bacteria respectively. The red pigmented soil bacterium K. rosea has characteristic resonance Raman signals at 1153 and 1506 cm-1 which corresponds to the carotenoids unique structural features mentioned previously. K. rosea is known to be constituted of different configurations of bacterioruberin as the major carotenoid in its cells which has a structure composed of 13 conjugated double bonds.28 D. radiodurans which is also a red pigmented bacterium has resonance Raman signals at 1153 and 1510 with a red shift of 4 cm-1 when compared to the carotenoid profile of K. rosea. D. radiodurans is constituted of deinoxanthin as the major carotenoid in its cells which has a structure composed of 11 conjugated double bonds.29 M. luteus which is a yellow pigmented bacterium has resonance Raman signals at 1157 and 1530 cm-1 with a red shift of 24 and 20 cm-1 respectively when compared to K. rosea and D. radiodurans. Sarcinaxanthin is the major carotenoid in M. luteus and has a structure composed of 9 conjugated double bonds.30 The resonance Raman profile representing carotenoids present in these three pigmented bacteria is in good correlation with their structures based on previous observations that the Raman signal at the 1500 cm-1 position is red shifted to lower wavenumber positions with the increase in the conjugated chain length in the carotenoids structure. The Raman spectra precisely correlates with the conjugated chain length features of the carotenoids in these three species of bacteria and can
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be clearly distinguished from one another even though the differences are minute in the case of D. radiodurans and K. rosea. Fig. 1 also shows the resonance Raman spectra of K. salsicia and K. turfanensis, the major carotenoids present in these two species of pigmented bacteria has not been studied or reported previously to the best of our knowledge. The resonance Raman profile of the major carotenoid present in the yellow pigmented bacteria K. salsicia shows Raman signals at 1157 and 1523 cm-1. The position of the Raman signal at 1523 cm-1 suggests that the major carotenoid present may have about 7 or 8 conjugated double bonds in its structure as deduced by comparing it with the Raman spectra of standard canthaxanthin carotenoid which has 9 conjugated double bonds and has a Raman signal at 1520 cm-1 as shown in Fig. 1(g). Similarly, the red pigmented K turfanesis shows resonance Raman signals at 1153 and 1514 cm-1, the Raman signal at this position suggests that the major carotenoid present may have about 11 conjugated double bonds as deduced by comparing it with the Raman spectra of lycopene which also has 11 conjugated double bonds and the Raman signal at 1514 cm-1 as shown in Fig. 1(g). However, a recent study by Vanessa de Oliveira et al14 suggested that such assignments should be made with caution as they proved using different plant sources that the position of the 1500-1550 cm-1 Raman signal is also influenced by the interaction of the carotenoids with the surrounding biomolecular components like proteins and relevant cell matrix components. And also the small changes in the position of the ν(C=C) Raman signal observed must be considered with caution irrespective of the resolution of the spectrometer used for the study. Such an influence could easily lead to misinterpretation of carotenoids under investigation. To overcome this problem and to confirm that the minute resonance Raman spectral changes we observe in similarly pigmented bacteria is indeed resulting from a different carotenoid and not by other influences, we have probed the response of the carotenoids against UVA radiation. As mentioned earlier, carotenoids are efficient UV radiation protectants due to its antioxidant and light harvesting properties. Most of the bacteria that grow in regions of extreme sunlight exposure are equipped with carotenoids to survive against large doses of UVA radiation exposure.31 We considered that a carotenoid with a different overall structure must exhibit different kinetics of photodegradation by UVA radiation irrespective of its interaction with other cellular components.36, 41 Fig. 2(a) shows the resonance Raman kinetics of the major carotenoid present in D. radiodurans which has a Raman signal at 1510 cm-1 acquired from single cells at time intervals of 30 minutes each collected over the course of 240 minutes. Fig. 2(c) shows the response of the Raman bands at 1153 and 1510 cm-1 corresponding to the major carotenoid to UVA radiation exposure at different time points. It is clearly seen that the resonance Raman signal resulting from the major carotenoid Deinoxanthin whose structure has 11 conjugated double bonds and a keto group in the ionone ring terminal is extremely photo stable throughout the exposure time. It is well known that D. radiodurans is one amongst the toughest bacterium on the planet and can resist extreme conditions due to its unique features of which its major carotenoid has a vital contribution. Fig. 2(b) shows the consequent Raman maps acquired from a bulk of cells depicting the relative intensity ratio distribution of the
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DOI: 10.1039/C5AN00438A
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carotenoids present at time points 30 and 240 minutes for D. radiodurans. The Raman maps were acquired from the same sample but from different areas; as we draw out 5 µl of the sample at different time points and dry the sample prior Raman analysis, there was no possibility of analysing the same area at a different time point. The maps are color coded from blue to yellow in the increasing order wherein blue regions represent minute or no presence of carotenoids and yellow represents a substantial presence. These time points for mapping were chosen based on the observations made in single cell measurements. The intention of acquiring a Raman map was solely to show that the photodegradation pattern is a collective response occurring in approximately all the cells and to detect if the carotenoid is stable or degraded at those respective time points in the bulk of cells. In general, the relative intensity ratio distribution of Raman signals 1153 and 1510 cm-1 corresponding to carotenoids observed in all the Raman maps quite clearly depicts the presence or absence of carotenoids at the respective time points, with the Raman map corresponding to D. radiodurans showing the presence of carotenoids even after 240 minutes of exposure to UVA radiation indicating its stability and resistance to photodegradation within the time points considered in this study. There wasn’t any definite pattern to indicate substantial degradation of carotenoids with time in this case as seen from both the single cell measurements and consequent Raman maps in Fig. 2. Fig. 2(c) shows the response of the Raman bands at 1153 and 1506 cm-1 corresponding to the major carotenoid to UVA radiation exposure at different time points. We also detected intense carotenoid signals in some of the cells at 240 minutes which matched the intensities of those observed at 30 minutes as shown in Fig. 2(c) clearly confirming that the carotenoid in D. radiodurans was overall unaffected during the time span of 240 minutes. A completely different photodegradation profile was observed in K. rosea. The major carotenoid bacterioruberin present in K. rosea has a Raman signal corresponding to ν(C=C) at 1506 cm-1 and an overall open chain structure consisting of 13 conjugated double bonds was completely photodegraded within a span of 150 minutes as shown in Fig. 3(a). However the samples were monitored for the complete duration of 240 minutes to confirm that there is no upregulation in carotenoid synthesis. The subsequent Raman maps shown in Fig. 3(b) acquired at 30 and 150 minutes illustrating the photodegradation of carotenoid in a large number of cells. Fig. 3(c) shows the response of the Raman bands at 1153 and 1506 cm-1 corresponding to the major carotenoid to UVA radiation exposure at different time points. This differential photodegradation of the carotenoids present in these two species of red pigmented bacteria towards UVA radiation clearly confirms the presence of different carotenoids irrespective of the proximities in their resonance Raman spectra. In general, it has been observed in previous studies via transient absorption spectroscopic techniques that a carotenoid with a keto group has a lower photodegradation quantum yield and is less reactive and more photostable.32 The rate constants of photobleaching have also been observed to be lower in a carotenoid that possesses a keto functional group in their structure.33 Our results as shown in Fig. 2(c) and Fig. 3(c) are in good correlation with these studies in that the carotenoid in D. radiodurans apparently is composed of such features and happens to be clearly
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more photostable displaying an extensively lower photodegradation level when compared to K. rosea. Fig. 4(a) illustrates the carotenoid photodegration profile of another red pigmented bacteria K. turfanensis analyzed in this study wherein the carotenoids are quite stable but photodegrade drastically post 180 minutes and are almost completely undetectable after 210 minutes of UVA exposure. The corresponding Raman maps in Fig. 4(b) at time points 30 and 210 minutes showing precise photodegradation of the carotenoids in the bulk of cells. Fig. 4(c) shows the response of the Raman bands at 1153 and 1514 cm-1 corresponding to the major carotenoid to UVA radiation exposure at different time points which clearly indicates a delayed degradation when compared to the carotenoid in K. rosea and highly less photostability when compared to the carotenoid in D. radiodurans. We can deduce from this pattern that the carotenoid present in this species could possibly be an open chain structure similar to that of the carotenoid present in K. rosea which is bacterioruberin. Such patterns have been observed previously wherein the carotenoids without ionone ring terminals and no carbonyl groups are degraded at higher rates when compared to their counterparts possessing these features.34,35 Such a pattern is also seen in Fig. 4(c) in comparison to the carotenoid in D. radiodurans as shown in Fig. 2(c). Although, it probably does have fewer numbers of double bonds in its structure when compared to bacterioruberin by taking into consideration also the position of the ν(C=C) Raman signal at 1514 cm-1 as mentioned previously. This could be one of the reasons associated with its delayed degradation when compared to the carotenoid in K. rosea. Nevertheless, the differential carotenoid photodegradation profile when compared to the other two red pigmented bacteria confirms the presence of a unique carotenoid in K. turfanensis. Similarly, the carotenoid photodegradation profile of two of the yellow pigmented bacteria M. luteus and K. salsicia were also analysed. The carotenoid degradation profiles of both these bacteria were very similar and the carotenoids were not photodegraded completely during the course of 240 minutes as seen from the single cell measurements and the consequent Raman maps shown in Fig. 5. And also the time dependent response of the marker band of carotenoids to UVA radiation confirms that they share an overall similar photodegradation pattern as shown in Fig. 6. Fig. 6 depicts that carotenoids in both these bacteria show a sharp decrease during the first 30 minutes of exposure and later is relatively stable until the 240 minutes of UVA radiation exposure. This pattern suggests that the carotenoids in these two bacteria have similar structures, however the position of the Raman signal corresponding to the conjugated chain length which appears at 1530 cm-1 for M. luteus and at 1523 cm-1 for K. salsicia respectively may be suggesting the presence of a difference in the conjugated double bond system of their structure which might be misleading. However, their similar photodegradation profile happens to signal that these two species of bacteria have similar carotenoids; the major carotenoid present in K. salsicia could possibly have a very similar structure as sarcinaxanthin which is the major carotenoid present in M. luteus as described previously. And the prominent red shift observed in the Raman signal corresponding to the conjugated chain length observed in their resonance Raman profile could be indeed resulting from an influence of the carotenoid interaction with
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other cell components as observed by Vanessa de Oliveira et al previously.14 This clearly advocates the need for reservations when using resonance Raman spectroscopy alone for decisive characterization of carotenoids which have not been studied previously. It must also be mentioned here that the substantial degradation of carotenoids observed in both these species when compared to the carotenoid in D. radiodurans suggests overall that D. radiodurans has the most photostable carotenoid amongst the five species analyzed in this study. Interestingly, the photodegradation pattern of the carotenoids we observed in the pigmented bacteria used in this study correlates well with a study based on UV B photodegradation of carotenoids present in human skin involving a closed chain β-carotene and open chain lycopene carotenoids.36 We could also deduce based on the photodegradation profile that carotenoids with ionone ring terminals and carbonyl groups, hydroxyl groups associated with ionone ring terminals and a lower conjugation chain length are possibly less susceptible to photodegradation than their counterparts as observed based on the profiles of D. radiodurans and M. luteus which both have carotenoids with such features. However, it should be noted that there is no link between the photodegradation profile and the antioxidant property of a carotenoid. A vast variety of bacteria and other microorganisms in general have evolved to upregulate the synthesis of carotenoids in the presence of ample nutrients when growing under stressful conditions like large doses of UV radiation which possibly nullifies the effect of possessing less photostable carotenoids.37,38 Additionally, to check the effect of UVA photodegradation of carotenoids on the other biomolecular components of the bacterial cells, we took the UVA exposed fraction of cells from all the samples after 240 minutes and spread plated this fraction in their respective media. Those species of bacteria wherein the carotenoids remained stable throughout the UVA exposure period i.e. in D. radiodurans, M. luteus and K. salsicia, were all viable and new colonies were formed. Whereas, in K. rosea and K. turfanensis wherein the carotenoids were degraded, there was no growth observed. And also, when the non pigmented bacterium M. lylae was exposed to UVA radiation, the UVA exposed fraction collected after just 30 minutes of exposure showed no growth, this is because of the obvious damages to DNA, protein etc induced by the UVA radiation which makes the bacteria non viable.39 This clearly demonstrated the protective role of the carotenoids against UVA radiation, its presence along with enzymatic antioxidant systems prove to be critical in the survival of bacteria against stressful conditions.40 In conclusion, our results precisely demonstrate the potential of resonance Raman spectroscopy in the detection and characterization of carotenoids from pure cultures of soil bacteria at a single cell resolution. Probing the photodegradation profile of the carotenoids against UVA radiation proves valuable as an important analysis to confirm the presence of different carotenoids amongst bacteria with similar colored pigments irrespective of the proximities in their resonance Raman profiles. A basic provisionary understanding of the structure of novel carotenoids can be possibly made by comparing their photodegration profile with carotenoids whose structures are well known. Anyhow, it must be mentioned that the separation of structurally very closely related carotenoids within a
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single and different systems is still not achievable using resonance Raman spectroscopy because of the overlap of the main Raman signals corresponding to carotenoids as may be seen in the case of M. luteus and K. salsicia and thereby must be supplemented with techniques like HPLC and LC-MS.
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In the present study, we report the characterization of carotenoids in five species of pure of cultures of pigmented soil bacteria. We demonstrate for the first time the influence of probing the photodegradation of carotenoids by UVA radiation in order to confirm the presence of different carotenoids irrespective of the proximities in their resonance Raman profiles. This method can be used to investigate if there is any influence of other cell components on the position of resonance Raman signals which is vital for the characterization of carotenoids in order to avoid missassignments. And it also has the potential to be applicable for gaining basic information about the overall structure of a novel carotenoid as shown in this study. However it is necessary to also analyze more pigmented bacteria preferably from those isolated from different environmental sources to validate further the utility of probing photodegradation patterns to assist in the characterization of carotenoids which will be addressed in our future studies. Nevertheless, an accurate and comprehensive prediction of a novel carotenoid would have to be supplemented with the analysis via gold standard techniques like HPLC and LC-MS.
AUTHOR INFORMATION 85
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Corresponding author * E-mail: [email protected], Phone: +49-3641-948320, +49-3641-206300, Fax: +49-3641-948302.
ACKNOWLEDGEMENTS This research is financially supported by the German Research Foundation (DFG) under the grant GRK 1257/2: “Alteration and element mobility at the microbe-mineral interface”.
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Notes The authors declare no competing financial interest. REFERENCES:
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41) T.R. Hata, T.A. Scholz, I.V. Ermakov, R.W. McClane, F. Khachik, W. Gellermann and L.K. Persching. J. Invest. Dermatol. 2000, 115, 441448.
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Fig 1. Resonance Raman spectra of carotenoids in (a) red pigmented K. rosea (b) red pigmented D. radiodurans (c) red pigmented K. turfanensis (d) yellow pigmented M. luteus (e) yellow pigmented K. salsicia (f) Integrated Raman intensity standard deviation of the two marker bands of carotenoids in all species (g) lycopene and canthaxanthin standards.
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Fig. 2
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Fig 2. (a) Single cell resonance Raman photodegradation profile of the carotenoid in D. radiodurans exposed to UVA radiation up to 240 minutes (b) Relative intensity ratio distribution of the Raman bands at 1510 and 1153 cm-1 representing carotenoids at time point 30 minutes (1) and 240 minutes (2); (c) Integrated peak area of the marker bands corresponding to the carotenoids as a function of UVA radiation exposure.
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Fig 3. (a) Single cell resonance Raman photodegradation profile of the carotenoid in K. rosea exposed to UVA radiation up to 240 minutes (b) Relative intensity ratio distribution of the Raman bands at 1506 and 1153 cm-1 representing carotenoids at time point 30 minutes (1) and 240 minutes (2); (c) Integrated peak area of the marker bands corresponding to the carotenoids as a function of UVA radiation exposure.
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Fig.4
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Fig 4. (a) Single cell resonance Raman photodegradation profile of the carotenoid in K. turfanensis exposed to UVA radiation up to 240 minutes (b) Relative intensity ratio distribution of the Raman bands at 1514 and 1153 cm-1 representing carotenoids at time point 30 minutes (1) and 240 minutes (2); (c) Integrated peak area of the marker bands corresponding to the carotenoids as a function of UVA radiation exposure.
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Fig 5. (a) Single cell resonance Raman photodegradation profile of the carotenoids in M. luteus exposed to UVA radiation up to 240 minutes. (b) Relative intensity ratio distribution of the Raman bands at 1530 and 1157 cm-1 representing carotenoids at time point 30 minutes (1) and 240 minutes (2). (c) Single cell resonance Raman photodegradation profile of the carotenoid in K. salsicia exposed to UVA radiation up to 240 minutes. (d) Relative intensity distribution of the Raman bands at 1523 and 1157 cm-1 representing carotenoids at time point 30 minutes (1) and 240 minutes (2).
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Fig 6. (a) Integrated peak area of the marker bands corresponding to the carotenoids in M. luteus as a function of UVA radiation exposure. (b) Integrated peak area of the marker bands corresponding to the carotenoids in K. salsicia as a function of UVA radiation exposure.
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