Use of Trifluoro-Acetate Derivatives for GC-MS and GC-MS/MS Quantification of Trace Amounts of Stera-3β,5α,6β-Triols (Tracers of Δ5-Sterol Autoxidation) in Environmental Samples

International audienceStera-3β,5α,6β-triols make useful tracers of the autoxidation of Δ5-sterols. These compounds are generally analyzed using gas chromatography–mass spectrometry (GC-MS) after silylation. Unfortunately, the 5α hydroxyl groups of these compounds, which are not derivatized by conventional silylation reagents, substantially alter the chromatographic properties of these derivatives, thus ruling out firm quantification of trace amounts. In this work, we developed a derivatization method (trifluoroacetylation) that enables derivatization of the three hydroxyl groups of 3β,5α,6β-steratriols. The derivatives thus formed present several advantages over silyl ethers: (i) better stability, (ii) shorter retention times, (iii) better chromatographic properties and (iv) mass spectra featuring specific ions or transitions that enable very low limits of detection in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) modes. This method, validated with cholesta-3β,5α,6β-triol, was applied to several environmental samples (desert dusts, marine sediments and particulate matter) and was able to quantify trace amounts of 3β,5α,6β-steratriols corresponding to several sterols: not only classical monounsaturated sterols (e.g., cholesterol, campesterol and sitosterol) but also, and for the first time, di-unsaturated sterols (e.g., stigmasterol, dehydrocholesterol and brassicasterol)

Cellular Damage of Bacteria Attached to Senescent Phytoplankton Cells as a Result of the Transfer of Photochemically Produced Singlet Oxygen: A Review

Several studies set out to explain the presence of high proportions of photooxidation products of cis-vaccenic acid (generally considered to be of bacterial origin) in marine environments. These studies show that these oxidation products result from the transfer of singlet oxygen from senescent phytoplankton cells to the bacteria attached to them in response to irradiation by sunlight. This paper summarizes and reviews the key findings of these studies, i.e., the demonstration of the process at work and the effect of different parameters (intensity of solar irradiance, presence of bacterial carotenoids, and presence of polar matrices such as silica, carbonate, and exopolymeric substances around phytoplankton cells) on this transfer. A large part of this review looks at how this type of alteration of bacteria can affect the preservation of algal material in the marine environment, especially in polar regions where conditions drive increased transfer of singlet oxygen from sympagic algae to bacteria

Use of Trifluoro-Acetate Derivatives for GC-MS and GC-MS/MS Quantification of Trace Amounts of Stera-3β,5α,6β-Triols (Tracers of Δ⁵-Sterol Autoxidation) in Environmental Samples

Stera-3β,5α,6β-triols make useful tracers of the autoxidation of Δ5-sterols. These compounds are generally analyzed using gas chromatography–mass spectrometry (GC-MS) after silylation. Unfortunately, the 5α hydroxyl groups of these compounds, which are not derivatized by conventional silylation reagents, substantially alter the chromatographic properties of these derivatives, thus ruling out firm quantification of trace amounts. In this work, we developed a derivatization method (trifluoroacetylation) that enables derivatization of the three hydroxyl groups of 3β,5α,6β-steratriols. The derivatives thus formed present several advantages over silyl ethers: (i) better stability, (ii) shorter retention times, (iii) better chromatographic properties and (iv) mass spectra featuring specific ions or transitions that enable very low limits of detection in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) modes. This method, validated with cholesta-3β,5α,6β-triol, was applied to several environmental samples (desert dusts, marine sediments and particulate matter) and was able to quantify trace amounts of 3β,5α,6β-steratriols corresponding to several sterols: not only classical monounsaturated sterols (e.g., cholesterol, campesterol and sitosterol) but also, and for the first time, di-unsaturated sterols (e.g., stigmasterol, dehydrocholesterol and brassicasterol)

Etude de l'effet des processus diagénétiques sur les alcénones (impact sur les estimations de paléotempératures)

Les alcénones constituent une classe de cétones insaturées à longue chaîne (C35 à C41)synthétisées par un nombre limité d haptophytes. La proportion des alcènones en C37 di et triinsaturéesvarie en fonction de la température de croissance de l haptophyte. A partir de cette caractéristique et de leur ubiquité dans l environnement marin, un indice nommé '37U K =[C37 :2] / ([C37 :2 + C37 :3]) est utilisé depuis la fin des années 1980s comme paléomarqueur des températures des eaux de surface.Des processus de dégradation biotique et abiotique sélectifs, jusqu alors partiellement ignorés,peuvent entraîner des biais significatifs (du fait de la perte préférentielle des alcénones les plus insaturées) dans les valeurs de paléotempératures estimées à partir de l '37U K . Ce travail a été entrepris dans le but d'étudier l'impact de ces processus diagénétiques sur les alcénones et d'évaluer leur importance dans l'environnement marin.Durant la première partie de ce travail, nous avons isolé et identifié diverses souches bactériennes à partir de cultures d Emiliania huxleyi, que nous avons testées pour leur capacité à dégrader les alcénones. La souche Dietzia maris s'est révélée capable de dégrader sélectivement les alcénones. Cette dégradation sélective fait intervenir une époxydation initiale des doubles liaisons des alcénones, qui est vraisemblablement induite par une monoxygénase ayant une plus grande affinité pour la double liaison en position w29 et peut conduire à une augmentation des valeurs de l' '37U K de l ordre de +0,10 (soit une surestimation des températures de +3C).L'impact de ces processus de dégradation (biotique et abiotique) in situ s'est révélé plus oumoins significatif selon les zones géographiques considérées. En mer Méditerranée,l'augmentation des valeurs de l' '37U K (0,43 à 0,55) s'explique essentiellement par une forte autoxydation des alcénones lors de leur sédimentation. La détection d'alcénones stéréomutées à la surface du sédiment nous a permis d'estimer que les processus de stéréomutatio npouvaient entraîner un biais dans les valeurs de l' '37U K de +0,05 dans cette région. En Alaskaainsi que dans le Pacifique équatorial, les biais observés résultent essentiellement d une dégradation bactérienne sélective des alcénones (+0,7 à +2,4C et +2C, respectivement).Lors de nos analyses de matériel particulaire provenant de l océan Pacifique équatorial, nos observations ont mis en évidence une relation entre l état de photo-oxydation des cellules phytoplanctoniques sénescentes et l état physiologique des bactéries qui leur sont associées.Un transfert d oxygène singulet (1O2) des phytodétritus aux bactéries entraînerait un déclin important de la croissance bactérienne et limiterait ainsi considérablement la biodégradation.Dans notre étude, ce transfert d 1O2 s est révélé plus efficace dans les particules en suspension(forte photo-oxydation des bactéries) que dans les particules prélevées par trappes (forte biodégradation du phytodétritus). Cette différence s expliquerait par l abondance de particules riches en silice dans les particules prélevées par trappes (dominées par des agglomérats de diatomées) dont le caractère polaire réduirait la durée de vie de l 1O2.Nos résultats confirment que les processus de (i) dégradation bactérienne sélective, (ii)d'autoxydation et (iii) de stéréomutation peuvent introduire des bais significatifs dans les reconstructions de paléotempératures et des moyens de corriger les biais résultant de cette diagenèse ont été proposés afin d'améliorer les reconstructions de paléotempératures basées sur cet outil.Alkenones constitute a class of long-chain unsaturated ketones (C35 to C41) synthesized by alimited number of haptophytes. The proportion of C37 di- and tri-unsaturated alkenones varies according to the growth temperature of the haptophytes. From this characteristic and theubiquity of alkenones in the marine environment, an index named '37U K = [C37: 2] / ([C37: 2 + C37:3]) is used since the late 1980s as paleomarker of sea surface temperatures. Selective biotic and abiotic degradation processes, previously ignored in the literature, canlead to significant biases (due to the preferential loss of the more unsaturated alkenones) in paleotemperature values estimated from the '37U K . This work was undertaken to estimate theimpact of diagenetic processes on alkenones in the marine environment.During the first part of this work, we isolated and identified various bacterial strains from cultures of Emiliania huxleyi, which were tested for their ability to degrade alkenones. Thestrain Dietzia maris sp. S1 appeared to be able to degrade selectively di- and tri-unsaturatedalkenones. This selective degradation involves an initial epoxidation of alkenone doublebonds, which is probably induced by a monooxygenase showing a greater affinity for the w29double bond and leads to increases of the '37U K values ranging from +0.05 to +0.10 units(corresponding to an overestimation of temperatures of 1.5 - 3C).The impact of these biotic and abiotic degradation processes in situ was more or lesssignificant depending on the area considered. In Mediterranean Sea, increasing values of '37U Kwith depth (0.43 to 0.55) seemed to mainly result from an intense autoxidation of alkenones.The detection of stereomutated alkenones in surface sediments also attested to the importanceof these processes in this region (increased in '37U K values of +0.05 units). In contrast, in Alaska and Equatorial Pacific, the biases observed (+0.7 to +2.4C and +2C, respectively) appeared to be mainly induced by selective bacterial degradation of alkenones.Analyses of particulate matter from the Equatorial Pacific Ocean, revealed a relationshipbetween the state of photo-oxidation of senescent phytoplankton cells and the physiologicalstate of associated bacteria. A transfer of singlet oxygen (1O2) from phytodetritus to thebacteria may induce damages in bacteria and thus significantly limit biodegradation. This transfer of 1O2 appeared to be more effective in suspended particles (high photo-oxidation ofbacteria and preservation of phytodetritus) than in the sinking particles (weak photo-oxidationof bacteria and high biodegradation of phytodetritus). These differences were attributed to theabundance of particles rich in silica in sinking particles (dominated by agglomerates ofdiatoms), whose the polar character could reduce lifetime of 1O2.Our results confirm that the process of (i) selective bacterial degradation, (ii) autoxidation and(iii) stereomutation may introduce significant biases in the reconstruction ofpaleotemperatures. Some tools were proposed to correct some of these biases and thus toimprove the paleotemperature reconstructions based on alkenonesAIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

Dégradation biotique et abiotique de la vitamine E dans l'environnement marin

La vitamine E, qui est un constituant important du système photoprotecteur de la plupart des organismes photosynthétiques, est fréquemment détectée dans les sédiments marins. Bien que la dégradation biotique et abiotique de ce composé puisse constituer une source potentielle de certains composés isoprénoïdes présents dans le milieu marin dont l origine n est pas encore connue, ces processus n ont jusqu ici jamais été pris en compte. Le but de cette thèse était donc d étudier l effet des différents processus, qui interviennent au cours de la diagenèse de la matière organique dans le milieu marin, sur la vitamine E. L étude de l autoxydation de la vitamine a montré que ce processus est très rapide dans le milieu naturel et qu il est une source de 4, 8,12,16 - tétraméthylheptadécan -4-olide, cette lactone a été proposée comme marqueur de sédimentation en conditions oxiques (chapitre III). A partir de sédiments marins et de tapis microbiens, nous avons pu isoler des souches bactériennes appartenant aux genres Halomonas, Idiomarina et bacillus présentant une grande efficacité de dégradation vis-à-vis de la vitamine E. Sur la base des métabolites identifiés nous avons pu proposer un mécanisme de dégradation faisant intervenir simultanément des processus biodégradatifs et autoxydatifs (chapitre IV). La photooxydation de la vitamine E induit la formation de nombreux composés isoprénoides et notamment de prist-1-ène résultant de la pyrolyse de produits d oxydation trimèriques de cette dernière (chapitre V). Dans la dernière partie de ce travail (chapitre VI) la dégradation anaérobie de la vitamine E a été étudiée dans les sédiments marins et les tapis microbiens. Une dégradation significative de notre substrat a pu être mise en évidence parallèlement à une production de pristane. Ces résultats très intéressants montrent que la vitamine E est un précurseur de pristane dans l environnement marin. La formation de cet alcane résulte de la combinaison de processus autoxydatifs (formation de trimères dont la dégradation dans les sédiments conduit à la formation de prist-1-ene) et biohydrogénation de ce dernier en pristane par les bactéries anaérobies.Vitamin E, which is an important constituent of the photoprotective system of main of photosynthetic organisms, is frequently detected in the marine sediments. Although the biotic and abiotic degradation of this compound can constitute a potential source of some isoprenoid compounds present in the marine environment, whose origin is not known, these processes were never taken into account. The aim of this thesis was thus to study the effect of the various processes undergoing during the diagenesis of organic matter in the marine environment, on vitamin E. The study of the autoxidation of vitamin E showed that this process is very fast in the natural environment and may be a source of 4,8,12,16-tetramethylheptadecan-4-olide. This isoprenoid lactone was proposed as tracer of sedimentation under oxic conditions (chapter III). We could isolate bacterial strains belonging to the genus Halomonas, Idiomarina and Bacillus from marine sediments and microbial mats. These strains present a great efficiency of degradation towards vitamin E. On the basis of the metabolites identified, we could propose degradation pathways involving simultaneously biodegradative and autoxidative processes (chapter IV). The photosensitized oxidation of vitamin E induces the formation of several isoprenoid compounds and notably of prist-1-ene. We could demonstrate that this isoprenoid alkene results from the pyrolysis of trimeric oxidation products of vitamin E (chapter V). In the last part of this work (chapter VI), anaerobic degradation of vitamin E was studied in marine sediments and microbial mats. A significant degradation of the substrate could be observed in parallel to a production of pristane. These very interesting results demonstrated that vitamin E constitutes a potential precursor of pristane in the marine environment. The formation of this isoprenoid alkane results from a combination of autoxidative processes (formation of trimers whose subsequent degradation in the sediments leads to the formation of prist-1-ene) and biohydrogenation of this isoprenoid alkene to pristane by anaerobic bacteria.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Devenir de la matière organique dans l'environnement marin (photodégradation des composants lipidiques des organismes photosynthétiques)

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

EIMS Fragmentation Pathways and MRM Quantification of 7α/β-Hydroxy-Dehydroabietic Acid TMS Derivatives

International audienceEI mass fragmentation pathways of TMS derivatives οf 7α/β-hydroxy-dehydroabietic acids resulting from NaBH4-reduction of oxidation products of dehydroabietic acid (a component of conifers) were investigated and deduced by a combination of (1) low energy CID-GC-MS/MS, (2) deuterium labeling, (3) different derivatization methods, and (4) GC-QTOF accurate mass measurements. Having identified the main fragmentation pathways, the TMS-derivatized 7α/β-hydroxy-dehydroabietic acids could be quantified in multiple reaction monitoring (MRM) mode in sea ice and sediment samples collected from the Arctic. These newly characterized transformation products of dehydroabietic acid constitute potential tracers of biotic and abiotic degradation of terrestrial higher plants in the environment

Use of Stress Signals of Their Attached Bacteria to Monitor Sympagic Algae Preservation in Canadian Arctic Sediments

Based on the strong aggregation of sympagic (ice-associated) algae and the high mortality or inactivity of bacteria attached to them, it was previously hypothesized that sympagic algae should be significant contributors to the export of carbon to Arctic sediments. In the present work, the lipid content of 30 sediment samples collected in the Canadian Arctic was investigated to test this hypothesis. The detection of high proportions of trans vaccenic fatty acid (resulting from cis-trans isomerase (CTI) activity of bacteria under hypersaline conditions) and 10S-hydroxyhexadec-8(trans)-enoic acid (resulting from 10S-DOX bacterial detoxification activity in the presence of deleterious free palmitoleic acid) confirmed: (i) the strong contribution of sympagic material to some Arctic sediments, and (ii) the impaired physiological status of its associated bacterial communities. Unlike terrestrial material, sympagic algae that had escaped zooplanktonic grazing appeared relatively preserved from biotic degradation in Arctic sediments. The expected reduction in sea ice cover resulting from global warming should cause a shift in the relative contributions of ice-associated vs. pelagic algae to the seafloor, and thus to a strong modification of the carbon cycl