The widespread diatom Pseudo-nitzschia can produce domoic acid (DA). DA is a compound with well described neurotoxic effects on vertebrates including humans known as amnesic shellfish poisoning (ASP) syndrome. It has also been suggested to serve as an organic ligand that binds to iron and copper. By binding these trace elements, DA may increase their solubility and bioavailability. In order to serve this function, DA has to be excreted and reabsorbed by the cells. Only few records of dissolved domoic acid (dDA) concentrations in the ocean exist. To accomplish quantification by ultra performance liquid chromatography (UPLC), samples have to be pre-concentrated and desalted using solid-phase extraction, a procedure commonly applied for dissolved organic matter. Our major goals were to quantify dDA in a basin-wide assessment in the East Atlantic Ocean, to determine extraction efficiencies for complexed and uncomplexed dDA, and to assess whether domoic acid is represented by its molecular formula in direct-infusion high resolution mass spectrometry. Our results showed that dDA was extracted almost quantitatively and occurred ubiquitously in the ocean surface but also in deeper (and older) water, indicating surprisingly high stability in seawater. The maximum concentration measured was 173 pmol L-1 and the average molar dDA carbon yield was 7.7 ppm. Both carbon yield and dDA concentration decreased with increasing water depth. Providing quantification of dDA in the water column, we seek to improve our understanding of toxic bloom dynamics and the mechanistic understanding of DA production.

RATIONALE:High-resolution mass spectrometry (HRMS) with high sample throughput has become an important analytical tool for the analysis of highly complex samples and data processing has become a major challenge for the user community. Evaluating direct-infusion HRMS data without automated tools for batch processing can be a time-consuming step in the analytical pipeline. Therefore, we developed a new browser-based software tool for processing HRMS data. METHODS:The software, named UltraMassExplorer (UME), was written in the R programming language using the shiny library to build the graphical user interface. The performance of the integrated formula library search algorithm was tested using HRMS data derived from analyses of up to 50 extracts of marine dissolved organic matter. RESULTS:The software supports the processing of lists of calibrated masses of neutral, protonated or deprotonated molecules, with masses of up to 700 Da and a mass accuracy <3 ppm. In the performance test, the number of assigned peaks per second increased with the number of submitted peaks and reached a maximum rate of 4745 assigned peaks per second. CONCLUSIONS:UME offers a complete data evaluation pipeline comprising a fast molecular formula assignment algorithm allowing for the swift reanalysis of complete datasets, advanced filter functions and the export of data, metadata and publication-quality graphics. Unique to UME is a fast and interactive connection between data and their visual representation. UME provides a new platform enabling an increased transparency, customization, documentation and comparability of datasets.

Conditioning films and biofilms forming on surfaces of solid materials exposed to aqueous media play a key role in in the interaction between the geo- and biospheres. In this study, time-of-flight secondary ion mass spectrometry and scanning electron microscopy were used to investigate the time scale, mode of formation, and chemistry of conditioning films and biofilms that formed on Si substrates exposed to aquifer water in the subsurface Äspö Hard Rock Laboratory, SE-Sweden. The detection of fragment ions of amino acids, carbohydrates, and carboxylic acids revealed that different types of organic compounds had adhered to the Si surface already after 10 min of exposure to the aquifer fluids, whereas the attachment of microbial cells was first observed after 1000 min. The organic compounds first formed isolated μm-sized accumulations and subsequently started to distribute on the wafer surface more homogenously. Simultaneously further microorganisms attached to the surface and formed biofilm-like cell accumulations after 3 months of exposure to aquifer water.

1Department of Earth and Planetary Sciences, Macquarie University, Sydney, NSW 2109, Australia; Email: tim.leefmann@mq.edu.au 2Department of Geobiology, Geoscience Centre, Georg-August University Gottingen, Goldschmidtstr. 3, 37077 Gottingen, Germany; Email: Volker.Thiel@geo.uni-goettingen.de 3Federal Institute for Geosciences and Natural Resources (BGR), Stilleweg 2, 30655 Hannover, Germany; Email: Martin.Blumenberg@bgr.de 4Department of Experimental and Applied Mineralogy, Geoscience Centre, Georg-August University Gottingen, Goldschmidtstr. 1, 37077 Gottingen, Germany

The taphonomic and diagenetic processes by which organic substances are preserved in animal remains are not completely known and the originality of putative metazoan biomolecules in fossil samples is a matter of scientific discussion. Here we report on biomarker information preserved in a fossil whale bone from an Oligocene phosphatic limestone (El Cien Fm., Mexico), with a focus on fatty acyl compounds. Extracts were quantitatively analysed using gas chromatography-mass spectrometry (GC-MS) and, to identify macromolecular-linked remains, demineralised extraction residues were subjected to catalytic hydropyrolysis (HyPy). To better recognise potential authentic (i.e. animal-derived) lipids, the data from the ancient bone were compared with those obtained from (i) the adjacent host sediment of the fossil and (ii) a recent whale (Phocoena phocoena) vertebra. In addition, the spatial distribution of organic and inorganic species was observed at the mu m level by imaging MS (time-of-flight-secondary ion mass spectrometry, ToF-SIMS). Our results revealed a rather even distribution of hydrocarbon-, O- and N-containing ions in the trabecular network of the ancient bone. A different, more patchy arrangement of organic compounds was evident in the former marrow cavities that were partly cemented by clotted micrites of putative microbial origin. The concentration of fatty acids (FAs) in the ancient bone was in the permil range of the amount extracted from the recent whale vertebra. Endogenous compounds, including monoenoic n-C-16 and n-C-18 as well as branched FAs, were identified in the fossil bone by comparison with the host sediment. Ca. 80% of the prevalent n-C-16 and n-C-18 moieties in the ancient bone were extractable as FAs, whereas ca. 20% were covalently bound in the non-saponifiable kerogen fraction. Ample pyrite precipitates, distinctive 10-methyl branched FAs and microbial microborings ("tunneling") indicate that sulfate reducers and collagen-degrading actinomycetes were central players in the microbial decomposition of the bone. Similarities with reported microbial FA patterns suggest that the FAs in the fossil bone were largely contributed by these microbial "last eaters". The results highlight some of the degradation and preservation mechanisms during marine FA diagenesis in the "natural laboratory" of bones, and therefore the processes that lead to either degradation, preservation, or introduction of these widespread biomolecules in the fossils of ancient marine animals. (C) 2013 Elsevier Ltd. All rights reserved.

An Oligocene cold-seep limestone (Lincoln Creek Formation, Washington State, USA) was studied for its lipid biomarker inventory. Biomarker analysis on minute amounts of sample (tens of mg) and complementary C-13(carbonate) analyses allowed us to link biogeochemical processes with individual, closely intertwined carbonate phases. The ancient seep deposit exhibits four major carbonate phases, according to the paragenetic sequence of (I) micrite, (II) yellow aragonite, (III) clear aragonite and (IV) equant calcite spar. For the micrite, varying but significant amounts of archaea-derived isoprenoids clearly indicate that the precipitation of this phase was induced by the microbial anaerobic oxidation of methane (AOM). However, water column-derived lipids present in this carbonate phase reflect the incorporation of organic matter from background sediment cemented by authigenic micrite. Yellow aragonite made up only a minor rock component (<10% vol.), but contained a major portion of lipid biomarkers indicative of AOM. Along with low delta C-13(carbonate) values (less than -30 parts per thousand Pee Dee Belemnite), this points to an intimate spatial association of AOM consortia with the precipitation of yellow aragonite. Clear aragonite showed similar delta C-13(carbonate) values but much lower, if any, contents of AOM biomarkers. This suggests that AOM-derived carbonate ions diffused over a greater distance to the site of precipitation compared with yellow aragonite. The latest phase, equant calcite spar, did not yield appreciable biomarkers, but showed a notable C-13(carbonate)-enrichment that is most likely caused by methanogenesis that prevailed in the sediments after AOM activity had ceased. A comparison of the ancient seep carbonates with modern counterparts from Hydrate Ridge (offshore Oregon, USA) revealed a remarkable coincidence of the respective mineral phases and their biomarker patterns. This suggests that the mechanisms of carbonate formation and the associated biogeochemical processes remained unchanged over geological times.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a surface sensitive MS technique that offers a new way of studying lipid biomarkers at the microscopic level, without the need to destroy the physical integrity of the sample by extraction. We applied ToF-SIMS to a cryosection of a microbial mat and compared the results with ToF-SIMS and gas chromatography-MS (GC-MS) analysis of extracts from the same material. A wide range of lipid biomarkers was identified with ToF-SIMS in the microbial mat cryosection. Spectra and ion images revealed that individual biomarkers, including fatty acids, mono-, di- and triacylglycerols, carotenoids and chlorophyll were localized with diatom cells identified as Planothidium lanceolatum using optical microscopy. This diatom species can thus be regarded as a major lipid source within the microbial mat system. The results underpin the idea that ToF-SIMS has the potential to become an important technique for future biomarker studies, in particular for the clear cut assignment of biomarkers to distinctive morphological structures and specific microorganisms within complex biogeochemical samples. (c) 2013 Elsevier Ltd. All rights reserved.

RATIONALE:Over the last decade, the high lateral resolution and imaging capabilities of time-of-flight secondary ion mass spectrometry (ToF-SIMS) have increasingly stimulated interest in studying organic molecules in complex environmental materials. However, unlike with the established mass spectrometric techniques, the use of ToF-SIMS in the biogeosciences is still hampered by a lack of reference spectra of the relevant biomarker compounds. Here we present and interpret ToF-SIMS reference spectra of ten different cyclic lipids that are frequently used as biological tracers in ecological, organic geochemical and geobiological studies.METHODS:Standard compounds of α,β,β-(20R,24S)-24-methylcholestane, (22E)-ergosta-5,7,22-trien-3β-ol, 17α(H),21β-(H)-30-norhopane, hope-17(21)-ene, hop-22(29)-ene, 17β(H),21β(H)-bacteriohopane-32,33,34,35-tetrol, 17β(H),21β(H)-35-aminobacteriohopane-32,33,34-triol, α-tocopherol, β,β-carotene, chlorophyll a, and cryosections of microbial mats and a fungus were analyzed using a ToF-SIMS instrument equipped with a Bi(3)(+) cluster ion source.RESULTS:The spectra obtained from the standard compounds showed peaks in the molecular weight range (molecular ions, protonated and deprotonated molecules, adduct ions) and diagnostic fragment ion peaks in both, positive and negative ion modes. For the cyclic hydrocarbons, however, the positive ion mode spectra typically showed more and stronger characteristic peaks than the negative ion mode spectra. Using real world samples the capability of ToF-SIMS to detect and image selected compounds in complex organic matrices was tested. 17β(H),21β(H)-35-Aminobacteriohopane-32,33,34-triol, carotene and chlorophyll a were successfully identified in cryosections of microbial mats, and the distribution of ergosterol was mapped at µm resolution in a cryosection of a fungus (Tuber uncinatum).CONCLUSIONS:This study further highlights the utility of ToF-SIMS for the identification and localization of lipids within environmental samples and as a technique for biomarker-related research in organic geochemistry and geobiology.

In recent years, time-of-flight secondary ion mass spectrometry (ToF-SIMS) with cluster ion sources has opened new perspectives for the analysis of lipid biomarkers in geobiology and organic geochemistry. However, published ToF-SIMS reference spectra of relevant compounds are still sparse, and the influence of the chemical environment (matrix) on the ionisation of molecules and their fragmentation is still not well explored. This study presents ToF-SIMS spectra of eight glycerolipids as common target compounds in biomarker studies, namely ester- and ether-bound phosphatidylethanolamine, ester- and ether-bound phosphatidylcholine, ester-bound phosphatidylglycerol, ester- and ether-bound diglycerides and archaeol, obtained with a Bi(3) (+) cluster ion source. For all of these compounds, the spectra obtained in positive and negative analytical modes showed characteristic fragments that could clearly be assigned to e.g. molecular ions, functional groups and alkyl chains. By comparison with the reference spectra, it was possible to track some of these lipids in a pre-characterised organic extract and in cryosections of microbial mats. The results highlight the potential of ToF-SIMS for the laterally resolved analysis of organic biomarkers in environmental materials. The identification of the target compounds, however, may be hampered by matrix effects (e.g. adduct formation) and often require careful consideration of all spectral features and taking advantage of the molecular imaging capability of ToF-SIMS.

Organic biomarkers may greatly aid in the interpretation of geobiological systems, but their significance is often restricted by the loss of spatial information during conventional sample work-up (solvent extraction). Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) is a quasi-non-destructive technique that simultaneously detects inorganic and organic molecules on solid surfaces, without the need to extract the sample in question (Fig. 1). Moreover, ToF-SIMS is capable of generating images displaying the intensities of any ion detected on a microscopic scale, thus exceeding by far the resolution achievable with the extract-based techniques routinely used in biomarker studies.

Methane-related carbonates from Hydrate Ridge typically show several macroscopically distinguishable mineral phases, namely whitish aragonite, lucent aragonite, and gray micrite. The relationship of these phases to particular microorganisms or biogeochemical processes is as yet unclear. We used a miniaturized biomarker technique on mg samples, combined with factor analysis and subsequent electron microprobe analysis, to study lipid biomarkers and chemical compositions of the individual phases. This allows us to identify particular mechanisms involved in the formation of the different carbonate precipitates. Our combined analysis of biomarkers and petrographic traits shows that most of the lipids related to the anaerobic oxidation of methane (> 90% by weight) are concentrated within only a minor compartment (similar to 20% by volume) of the Hydrate Ridge carbonates, the whitish aragonite. The patterns indicate that the whitish aragonite represents fossilized biofilms of methanotrophic consortia containing mainly archaea of the ANME-2 group and sulfate reducing bacteria, whereas the precipitation of the lucent aragonite may have lacked the immediate proximity of microorganisms during formation. By contrast, the gray micrite formed by incorporation of allochthonous organic and inorganic matter during carbonate precipitation induced by the anaerobic oxidation of methane involving ANME-1 archaea.

In this thesis ToF-SIMS was applied to the study of geobiological samples. Except for a few pioneering studies, ToF-SIMS has rarely been used in the field of geobiology and thus only a limited number of SIMS reference spectra for biogeochemical relevant compounds has as yet been published. One major goal of this thesis was to extend the spectral library by compounds particularly used as molecular markers in the analysis of geobiological systems. Consequently in the first study reference spectra of eight different glycerolipids functioning as important membrane constituents in eukaryotes, bacteria, and archaea, were presented and their detection in an extract and cryosections of microbial mats demonstrated. The analyses of the standard compounds showed that the detection of fragment ions, in particular from headgroups and alkyl chains, is particularly important for the reliable identification of glycerolipids in complex, environmental samples. As a follow-up study, ten cyclic lipid standards were analyzed by ToF-SIMS. These cyclic lipids are known as important membrane constituents or fulfill a protective function from oxidative stress in eukaryotes, bacteria, or archea. The results obtained showed that most of the compounds can reliably be analyzed in only one of the two polarity modes. In addition the study demonstrated, that not all fragment ions detected in the analyses of the standard compounds can be expected to be detectable in environmental samples containing this compound in complex mixtures with other organic molecules. The knowledge gained in the analysis of standard compounds was used to study a phototrophic microbial mat in detail for their lipid biomarker content by a combining ToF-SIMS, optical microscopy and GC-MS analyses. ToF-SIMS analyses of a cryosection of the microbial mat showed, that a wide range of lipids and pigments, e.g. acylglycerols, carotenoids and chlorophyll a, were present in the microbial mat. Testing the limits of the lateral resolution of the ToF-SIMS instrument, the burst alignment mode was used, to probe single cells for their biomarker content. By subsequent optical microscopy these cells were identified as the diatom species Planothidium lanceolatum and as a major source of lipids and pigments in the microbial mat. The results of the Tof-SIMS analyses were consistent with the diatom specific fatty acid pattern obtained from GC-MS analyses of the bulk microbial mat. This study demonstrated a potential future application of ToF-SIMS in biomarker related research, namely as a tool for the rapid, clear-cut assignment of biomarkers to specific microbial sources in complex environmental samples. This technique could be particularly useful for biomarker studies on the majority of microorganisms, which can as yet not be grown in pure cultures. However, it should be noted that most prokaryotic cells are by an order of magnitude smaller than the diatom cells analyzed in this study and thus more effort has to be spent on increasing the useful lateral resolution of ToF-SIMS imaging. In the last study ToF-SIMS was applied in combination with SEM to the analysis of thin organic films, i.e. conditioning films, forming on solid surfaces exposed to aquifer water prior to the attachment of microorganisms. The results showed that the initial conditioning film forming after a few minutes of exposure to aquifer water was inhomogenous and composed of amino acids, carbohydrates, and fatty acids. With increasing time, the film became more homogenous and microorganism had attached to the surface after a few hours. After 90 days biofilm like accumulations of cells enclosed in extracellular polymeric substances had formed on the wafer surface. This study gave an insight into the chemistry and growth rates of conditioning films and biofilms forming in aquifers in the subsurface and demonstrated the applicability of ToF-SIMS for the analysis of very low amounts of organic material in environmental samples. The studies comprised in this thesis showed that ToF-SIMS can offer unique analytical capabilities for the analyses of large organic ions in environmental samples.