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Marine sponges are critical components of marine benthic fauna assemblages, where their filter-feeding and reef-building capabilities provide bentho-pelagic coupling and crucial habitat. As potentially the oldest representation of a metazoan-microbe symbiosis, they also harbor dense, diverse, and species-specific communities of microbes, which are increasingly recognized for their contributions to dissolved organic matter (DOM) processing. Recent omics-based studies of marine sponge microbiomes have proposed numerous pathways of dissolved metabolite exchange between the host and symbionts within the context of the surrounding environment, but few studies have sought to experimentally interrogate these pathways. By using a combination of metaproteogenomics and laboratory incubations coupled with isotope-based functional assays, we showed that the dominant gammaproteobacterial symbiont, ‘Candidatus Taurinisymbion ianthellae’, residing in the marine sponge, Ianthella basta, expresses a pathway for the import and dissimilation of taurine, a ubiquitously occurring sulfonate metabolite in marine sponges. ‘Candidatus Taurinisymbion ianthellae’ incorporates taurine-derived carbon and nitrogen while, at the same time, oxidizing the dissimilated sulfite into sulfate for export. Furthermore, we found that taurine-derived ammonia is exported by the symbiont for immediate oxidation by the dominant ammonia-oxidizing thaumarchaeal symbiont, ‘Candidatus Nitrosospongia ianthellae’. Metaproteogenomic analyses also suggest that ‘Candidatus Taurinisymbion ianthellae’ imports DMSP and possesses both pathways for DMSP demethylation and cleavage, enabling it to use this compound as a carbon and sulfur source for biomass, as well as for energy conservation. These results highlight the important role of biogenic sulfur compounds in the interplay between Ianthella basta and its microbial symbionts.

Ectomycorrhizal plants trade plant-assimilated carbon for soil nutrients with their fungal partners. The underlying mechanisms, however, are not fully understood. Here we investigate the exchange of carbon for nitrogen in the ectomycorrhizal symbiosis of Fagus sylvatica across different spatial scales from the root system to the cellular level. We provided 15N-labelled nitrogen to mycorrhizal hyphae associated with one half of the root system of young beech trees, while exposing plants to a 13CO2 atmosphere. We analysed the short-term distribution of 13C and 15N in the root system with isotope-ratio mass spectrometry, and at the cellular scale within a mycorrhizal root tip with nanoscale secondary ion mass spectrometry (NanoSIMS). At the root system scale, plants did not allocate more 13C to root parts that received more 15N. Nanoscale secondary ion mass spectrometry imaging, however, revealed a highly heterogenous, and spatially significantly correlated distribution of 13C and 15N at the cellular scale. Our results indicate that, on a coarse scale, plants do not allocate a larger proportion of photoassimilated C to root parts associated with N-delivering ectomycorrhizal fungi. Within the ectomycorrhizal tissue, however, recently plant-assimilated C and fungus-delivered N were spatially strongly coupled. Here, NanoSIMS visualisation provides an initial insight into the regulation of ectomycorrhizal C and N exchange at the microscale.

Extracellular DNA is a major macromolecule in global element cycles, and is a particularly crucial phosphorus, nitrogen and carbon source for microorganisms in the seafloor. Nevertheless, the identities, ecophysiology and genetic features of DNA-foraging microorganisms in marine sediments are largely unknown. Here, we combined microcosm experiments, DNA stable isotope probing (SIP), single-cell SIP using nano-scale secondary isotope mass spectrometry (NanoSIMS) and genome-centric metagenomics to study microbial catabolism of DNA and its subcomponents in marine sediments. 13C-DNA added to sediment microcosms was largely degraded within 10 d and mineralized to 13CO2. SIP probing of DNA revealed diverse ‘Candidatus Izemoplasma’, Lutibacter, Shewanella and Fusibacteraceae incorporated DNA-derived 13C-carbon. NanoSIMS confirmed incorporation of 13C into individual bacterial cells of Fusibacteraceae sorted from microcosms. Genomes of the 13C-labelled taxa all encoded enzymatic repertoires for catabolism of DNA or subcomponents of DNA. Comparative genomics indicated that diverse ‘Candidatus Izemoplasmatales’ (former Tenericutes) are exceptional because they encode multiple (up to five) predicted extracellular nucleases and are probably specialized DNA-degraders. Analyses of additional sediment metagenomes revealed extracellular nuclease genes are prevalent among Bacteroidota at diverse sites. Together, our results reveal the identities and functional properties of microorganisms that may contribute to the key ecosystem function of degrading and recycling DNA in the seabed., Using microcosms, stable isotope probing, genome-resolved metagenomics and NanoSIMS, the authors identify diverse bacterial taxa that can degrade extracellular DNA in marine sediments, including ‘Candidatus Izemoplasma’, which encode numerous extracellular nucleases.

Marine sponges are critical components of marine benthic fauna assemblages where their filter-feeding and reef-building capabilities provide bentho-pelagic coupling and crucial habitat. As potentially the oldest representation of a metazoan-microbe symbiosis, they also harbor dense, diverse, and species-specific communities of microbes, which are increasingly recognized for their contributions to dissolved organic matter (DOM) processing. Recent omics-based studies of marine sponge microbiomes have proposed numerous pathways of dissolved metabolite exchange between the host and symbionts within the context of the surrounding environment, but few studies have sought to experimentally interrogate these pathways. By using a combination of metaproteogenomics and laboratory incubations coupled with isotope-based functional assays, we showed that the dominant gammaproteobacterial symbiont ‘CandidatusTaurinisymbion ianthellae’ residing in the marine sponge,Ianthella basta, expresses a pathway for the import and dissimilation of taurine, a ubiquitously occurring sulfonate metabolite in marine sponges. ‘CandidatusTaurinisymbion ianthellae’ incorporates taurine-derived carbon and nitrogen while, at the same time, oxidizing the dissimilated sulfite into sulfate for export. Furthermore, we found that taurine-derived ammonia is exported by the symbiont for immediate oxidation by the dominant ammonia-oxidizing thaumarchaeal symbiont ‘CandidatusNitrosospongia ianthellae’. Metaproteogenomic analyses also indicate that ‘CandidatusTaurinisymbion ianthellae’ likely imports DMSP and possesses both pathways for DMSP demethylation and cleavage, enabling it to use this compound as a carbon and sulfur source for biomass, as well as for energy conservation. These results highlight the important role of biogenic sulfur compounds in the interplay betweenIanthella bastaand its microbial symbionts.

The ruthenium‐based anticancer agent BOLD‐100/KP1339 has shown promising results in several in vitro and in vivo tumour models as well as in early clinical trials. However, its mode of action remains to be fully elucidated. Recent evidence identified stress induction in the endoplasmic reticulum (ER) and concomitant down‐modulation of HSPA5 (GRP78) as key drug effects. By exploiting the naturally formed adduct between BOLD‐100 and human serum albumin as an immobilization strategy, we were able to perform target‐profiling experiments that revealed the ribosomal proteins RPL10, RPL24, and the transcription factor GTF2I as potential interactors of this ruthenium(III) anticancer agent. Integrating these findings with proteomic profiling and transcriptomic experiments supported ribosomal disturbance and concomitant induction of ER stress. The formation of polyribosomes and ER swelling of treated cancer cells revealed by TEM validated this finding. Thus, the direct interaction of BOLD‐100 with ribosomal proteins seems to accompany ER stress‐induction and modulation of GRP78 in cancer cells., Multi‐omics analysis, including affinity proteomics, revealed that the anticancer drug candidate BOLD‐100/KP1339 may target the ribosomal proteins RPL10/RPL24 and GTF2I. Swelling of the endoplasmic reticulum (ER) and the observation of polyribosomes using imaging techniques indicate that BOLD‐100 treatment may not only inhibit proliferation, but also lead to a functional disturbance of the ER.

One of the biggest challenges in microbiome research in environmental and medical samples is to better understand functional properties of microbial community members at a single-cell level. Single-cell isotope probing has become a key tool for this purpose, but the current detection methods for determination of isotope incorporation into single cells do not allow high-throughput analyses. Here, we report on the development of an imaging-based approach termed stimulated Raman scattering–two-photon fluorescence in situ hybridization (SRS-FISH) for high-throughput metabolism and identity analyses of microbial communities with single-cell resolution. SRS-FISH offers an imaging speed of 10 to 100 ms per cell, which is two to three orders of magnitude faster than achievable by state-of-the-art methods. Using this technique, we delineated metabolic responses of 30,000 individual cells to various mucosal sugars in the human gut microbiome via incorporation of deuterium from heavy water as an activity marker. Application of SRS-FISH to investigate the utilization of host-derived nutrients by two major human gut microbiome taxa revealed that response to mucosal sugars tends to be dominated by Bacteroidales, with an unexpected finding that Clostridia can outperform Bacteroidales at foraging fucose. With high sensitivity and speed, SRS-FISH will enable researchers to probe the fine-scale temporal, spatial, and individual activity patterns of microbial cells in complex communities with unprecedented detail.

Significance Increasing atmospheric methane concentrations contribute significantly to global warming. The only known biological sink for atmospheric methane is oxidation by methane oxidizing bacteria (MOB). Due to the lack of pure cultures, the physiology and metabolic potential of MOB that oxidize atmospheric methane remains a mystery. Here, we report on isolation and characterization of a MOB that can grow on air and utilizes methane at its atmospheric trace concentration as a carbon and energy source. Furthermore, this strain has the potential to utilize five additional atmospheric gases, carbon dioxide, carbon monoxide, hydrogen, nitrogen, and oxygen to supply its metabolism. This metabolic versatility might be the key to life on air and this discovery is essential for studying the biological methane sink., The global atmospheric level of methane (CH4), the second most important greenhouse gas, is currently increasing by ∼10 million tons per year. Microbial oxidation in unsaturated soils is the only known biological process that removes CH4 from the atmosphere, but so far, bacteria that can grow on atmospheric CH4 have eluded all cultivation efforts. In this study, we have isolated a pure culture of a bacterium, strain MG08 that grows on air at atmospheric concentrations of CH4 [1.86 parts per million volume (p.p.m.v.)]. This organism, named Methylocapsa gorgona, is globally distributed in soils and closely related to uncultured members of the upland soil cluster α. CH4 oxidation experiments and 13C-single cell isotope analyses demonstrated that it oxidizes atmospheric CH4 aerobically and assimilates carbon from both CH4 and CO2. Its estimated specific affinity for CH4 (a0s) is the highest for any cultivated methanotroph. However, growth on ambient air was also confirmed for Methylocapsa acidiphila and Methylocapsa aurea, close relatives with a lower specific affinity for CH4, suggesting that the ability to utilize atmospheric CH4 for growth is more widespread than previously believed. The closed genome of M. gorgona MG08 encodes a single particulate methane monooxygenase, the serine cycle for assimilation of carbon from CH4 and CO2, and CO2 fixation via the recently postulated reductive glycine pathway. It also fixes dinitrogen and expresses the genes for a high-affinity hydrogenase and carbon monoxide dehydrogenase, suggesting that atmospheric CH4 oxidizers harvest additional energy from oxidation of the atmospheric trace gases carbon monoxide (0.2 p.p.m.v.) and hydrogen (0.5 p.p.m.v.).

The selective oxidation underneath the scale layer of an industrially hot rolled Fe-1.8Mn-0.8Cr steel at temperatures between 600 and 700 °C has been investigated. The spatial distribution and composition of formed precipitates has been studied by high-resolution topochemical analysis via TEM-EELS and NanoSIMS and revealed heterogeneities in chemical composition, especially along grain boundaries. It could be shown that grain boundary oxides are predominantly composed of aluminium, chromium or silicon oxides/nitrides, surrounded by manganese-rich oxides. Experimental results of phase stability have been compared to numerical simulations, considering the distribution of more than 40 potentially stable oxide-, nitride- and carbide phases, and differences are critically discussed.

Oxaliplatin is a very potent platinum(ii) drug which is frequently used in poly-chemotherapy schemes against advanced colorectal cancer. However, its benefit is limited by severe adverse effects as well as resistance development. Based on their higher tolerability, platinum(iv) prodrugs came into focus of interest. However, comparable to their platinum(ii) counterparts they lack tumor specificity and are frequently prematurely activated in the blood circulation. With the aim to exploit the enhanced albumin consumption and accumulation in the malignant tissue, we have recently developed a new albumin-targeted prodrug, which supposed to release oxaliplatin in a highly tumor-specific manner. In more detail, we designed a platinum(iv) complex containing two maleimide moieties in the axial position (KP2156), which allows selective binding to the cysteine 34. In the present study, diverse cell biological and analytical tools such as laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS), isotope labeling, and nano-scale secondary ion mass spectrometry (NanoSIMS) were employed to better understand the in vivo distribution and activation process of KP2156 (in comparison to free oxaliplatin and a non-albumin-binding succinimide analogue). KP2156 forms very stable albumin adducts in the bloodstream resulting in a superior pharmacological profile, such as distinctly prolonged terminal excretion half-life and enhanced effective platinum dose (measured by ICP-MS). The albumin-bound drug is accumulating in the malignant tissue, where it enters the cancer cells via clathrin- and caveolin-dependent endocytosis, and is activated by reduction to release oxaliplatin. This results in profound, long-lasting anticancer activity of KP2156 against CT26 colon cancer tumors in vivo based on cell cycle arrest and apoptotic cell death. Summarizing, albumin-binding of platinum(iv) complexes potently enhances the efficacy of oxaliplatin therapy and should be further developed towards clinical phase I trials., Albumin-targeting of a maleimide-containing oxaliplatin-releasing platinum(iv) prodrug results in tumor-specific drug delivery and activity as shown by LA-ICP-MS, isotope-labeling and NanoSIMS in cell culture and in vivo.

Oxaliplatin shows a superior clinical activity in colorectal cancer compared to cisplatin. Nevertheless, the knowledge about its cellular distribution and the mechanisms responsible for the different range of oxaliplatin-responsive tumors is far from complete. In this study, we combined highly sensitive element specific and isotope selective imaging by nanometer-scale secondary ion mass spectrometry (NanoSIMS) with transmission electron microscopy to investigate the subcellular accumulation of oxaliplatin in three human colon cancer cell lines (SW480, HCT116 wt, HCT116 OxR). Oxaliplatin bearing dual stable isotope labeled moieties, i.e.2H-labeled diaminocyclohexane (DACH) and 13C-labeled oxalate, were applied for comparative analysis of the subcellular distribution patterns of the central metal and the ligands. In all the investigated cell lines, oxaliplatin was found to have a pronounced tendency for cytoplasmic aggregation in single membrane bound organelles, presumably related to various stages of the endocytic pathway. Moreover, nuclear structures, heterochromatin and in particular nucleoli, were affected by platinum-drug exposure. In order to explore the consequences of oxaliplatin resistance, subcellular drug distribution patterns were investigated in a pair of isogenic malignant cell lines with distinct levels of drug sensitivity (HCT116 wt and HCT116 OxR, the latter with acquired resistance to oxaliplatin). The subcellular platinum distribution was found to be similar in both cell lines, with only slightly higher accumulation in the sensitive HCT116 wt cells which is inconsistent with the resistance factor of more than 20-fold. Instead, the isotopic analysis revealed a disproportionally high accumulation of the oxalate ligand in the resistant cell line.

Stable isotope probing (SIP) is a key tool for identifying the microorganisms catalyzing the turnover of specific substrates in the environment and to quantify their relative contributions to biogeochemical processes. However, SIP-based studies are subject to the uncertainties posed by cross-feeding, where microorganisms release isotopically labeled products, which are then used by other microorganisms, instead of incorporating the added tracer directly. Here, we introduce a SIP approach that has the potential to strongly reduce cross-feeding in complex microbial communities. In this approach, the microbial cells are exposed on a membrane filter to a continuous flow of medium containing isotopically labeled substrate. Thereby, metabolites and degradation products are constantly removed, preventing consumption of these secondary substrates. A nanoSIMS-based proof-of-concept experiment using nitrifiers in activated sludge and 13C-bicarbonate as an activity tracer showed that Flow-SIP significantly reduces cross-feeding and thus allows distinguishing primary consumers from other members of microbial food webs.

34 Pags.- 9 Figs. The definitive version is available at: https://onlinelibrary.wiley.com/journal/13653040, Legume–rhizobia symbioses play a major role in food production for an ever growing human population. In this symbiosis, dinitrogen is reduced (“fixed”) to ammonia by the rhizobial nitrogenase enzyme complex and is secreted to the plant host cells, whereas dicarboxylic acids derived from photosynthetically produced sucrose are transported into the symbiosomes and serve as respiratory substrates for the bacteroids. The symbiosome membrane contains high levels of SST1 protein, a sulfate transporter. Sulfate is an essential nutrient for all living organisms, but its importance for symbiotic nitrogen fixation and nodule metabolism has long been underestimated. Using chemical imaging, we demonstrate that the bacteroids take up 20‐fold more sulfate than the nodule host cells. Furthermore, we show that nitrogenase biosynthesis relies on high levels of imported sulfate, making sulfur as essential as carbon for the regulation and functioning of symbiotic nitrogen fixation. Our findings thus establish the importance of sulfate and its active transport for the plant–microbe interaction that is most relevant for agriculture and soil fertility., This work was funded by the Austrian Science Fund (DK plus, W 1257‐820) and COST action FA1306 to S.W. and by grant AGL2017‐85775‐R from Ministerio de Economía y Competitividad‐Fondos Europeos de Desarrollo Regional (Spain) to M.B.

Resource limitation is a fundamental factor governing the composition and function of ecological communities. However, the role of resource supply in structuring the intestinal microbiome has not been established and represents a challenge for mammals that rely on microbial symbionts for digestion: too little supply might starve the microbiome while too much supply might starve the host. Here, we present evidence that microbiota occupy a habitat limited in total nitrogen supply within the large intestines of 30 mammal species. Furthermore, lowering dietary protein levels in mice reduced bacterial fecal concentrations. A gradient of stoichiometry along the length of the gut was consistent with the hypothesis that intestinal nitrogen limitation results from host absorption of dietary nutrients. Nitrogen availability though is also likely shaped by host-microbe interactions: levels of host-secreted nitrogen were altered in germfree mice and when bacterial loads were reduced via experimental antibiotic treatment. Single-cell spectrometry revealed that members of the phylum Bacteroidetes consumed nitrogen in the large intestine more readily than other commensal taxa. Collectively, our findings support a model where nitrogen limitation arises from preferential host utilization of dietary nutrients, and we speculate that this resource limitation could enable hosts to regulate microbial communities in the large intestine. Furthermore, commensal microbiota may have adapted to nitrogen-limited settings, suggesting why excess dietary protein has been associated with degraded gut microbial ecosystems.

Our current understanding of microbial hotspots such as the rhizosphere mainly stems from observations through measurements at the macroscopic scale, integrating a multitude of microbial cells and taxa into a few measured variables. Consequently, we still lack an understanding of the individual participants that actively contribute to processes. Identifying microorganisms and relating their activity to these processes within the soil-plant interface on a microscopic scale represent a missing link in understanding nutrient flux in agriculturally important ecosystems such as rice cultivation.I will present a novel workflow for single-cell isotope imaging in the rhizosphere that combines fluorescence in situ hybridization, gold-targeted secondary electron microscopy, and nano-scale secondary ion mass spectrometry. Based on correlative microscopy and hotspot detection, this approach now allows to (i) identify single bacteria on root surfaces that actively incorporate stable isotopes, (ii) quantify their contribution to processes of interest within a given population, and (iii) potentially trace nutrient fluxes among plants and bacteria on a microscale.Illuminating plant-microorganism interactions on a microscale provides the potential to evaluate the actual impact of bio-inoculants applied as fertilizers and to engineer plant-microorganism associations which may be essential to increase the production of major staple crops for a growing world population.

Chemosynthetic symbioses occur worldwide in marine habitats, but comprehensive physiological studies of chemoautotrophic bacteria thriving on animals are scarce. Stilbonematinae are coated by monocultures of thiotrophic Gammaproteobacteria. As these nematodes migrate through the redox zone, their ectosymbionts experience varying oxygen concentrations. However, nothing is known about how these variations affect their physiology or metabolism. Here, by applying omics, Raman microspectroscopy and stable isotope labelling, we investigated the effect of oxygen on Candidatus Thiosymbion oneisti. Unexpectedly, sulfur oxidation genes were upregulated in anoxic relative to oxic conditions, but carbon fixation genes and incorporation of 13C-labeled bicarbonate were not. Instead, several genes involved in carbon fixation were upregulated in oxic conditions, together with genes involved in organic carbon assimilation, polyhydroxyalkanoate (PHA) biosynthesis, nitrogen fixation and urea utilization. Furthermore, in the presence of oxygen, stress-related genes were upregulated together with vitamin biosynthesis genes likely necessary to withstand its deleterious effects, and the symbiont appeared to proliferate less. Based on its physiological response to oxygen, we propose that Ca. T. oneisti may exploit anaerobic sulfur oxidation coupled to denitrification to proliferate in anoxic sand. However, the ectosymbiont would still profit from the oxygen available in superficial sand, as the energy-efficient aerobic respiration would facilitate carbon and nitrogen assimilation by the ectosymbiont.IMPORTANCEChemoautotrophic endosymbionts are famous for exploiting sulfur oxidization to feed marine organisms with fixed carbon. However, the physiology of thiotrophic bacteria thriving on the surface of animals (ectosymbionts) is less understood. One long standing hypothesis posits that attachment to animals that migrate between reduced and oxic environments would boost sulfur oxidation, as the ectosymbionts would alternatively access sulfide and oxygen, the most favorable electron acceptor. Here, we investigated the effect of oxygen on the physiology of Candidatus Thiosymbion oneisti, a Gammaproteobacterium which lives attached to marine nematodes inhabiting shallow water sand. Surprisingly, sulfur oxidation genes were upregulated in anoxic relative to oxic conditions. Furthermore, under anoxia, the ectosymbiont appeared to be less stressed and to proliferate more. We propose that animal-mediated access to oxygen, rather than enhancing sulfur oxidation, would facilitate assimilation of carbon and nitrogen by the ectosymbiont.

Extracellular DNA is a major macromolecule in global element cycles, and is a particularly crucial phosphorus, nitrogen and carbon source for microorganisms in the seafloor. Nevertheless, the identities, ecophysiology and genetic features of DNA-foraging microorganisms in marine sediments are largely unknown. Here, we combined microcosm experiments, DNA stable isotope probing (SIP), single-cell SIP using nano-scale secondary isotope mass spectrometry (NanoSIMS) and genome-centric metagenomics to study microbial catabolism of DNA and its subcomponents in marine sediments.

Stable isotope labeling of agricultural polyesters enables demonstration of their microbial utilization in soils., Plastic materials are widely used in agricultural applications to achieve food security for the growing world population. The use of biodegradable instead of nonbiodegradable polymers in single-use agricultural applications, including plastic mulching, promises to reduce plastic accumulation in the environment. We present a novel approach that allows tracking of carbon from biodegradable polymers into CO2 and microbial biomass. The approach is based on 13C-labeled polymers and on isotope-specific analytical methods, including nanoscale secondary ion mass spectrometry (NanoSIMS). Our results unequivocally demonstrate the biodegradability of poly(butylene adipate-co-terephthalate) (PBAT), an important polyester used in agriculture, in soil. Carbon from each monomer unit of PBAT was used by soil microorganisms, including filamentous fungi, to gain energy and to form biomass. This work advances both our conceptual understanding of polymer biodegradation and the methodological capabilities to assess this process in natural and engineered environments.

A NanoSIMS, LA-ICP-MS and TEM combinatorial approach was applied to analyze the distribution of platinum(iv) prodrugs in murine tissue samples., Nano-scale secondary ion mass spectrometry (NanoSIMS) enables trace element and isotope analyses with high spatial resolution. This unique capability has recently been exploited in several studies analyzing the subcellular distribution of Au and Pt anticancer compounds. However, these studies were restricted to cell culture systems. To explore the applicability to the in vivo setting, we developed a combined imaging approach consisting of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), NanoSIMS and transmission electron microscopy (TEM) suitable for multi-scale detection of the platinum distribution in tissues. Applying this approach to kidney and tumor samples upon administration of selected platinum(iv) anticancer prodrugs revealed uneven platinum distributions on both the organ and subcellular scales. Spatial platinum accumulation patterns were quantitatively assessed by LA-ICP-MS in histologically heterogeneous organs (e.g., higher platinum accumulation in kidney cortex than in medulla) and used to select regions of interest for subcellular-scale imaging with NanoSIMS. These analyses revealed cytoplasmic sulfur-rich organelles accumulating platinum in both kidney and malignant cells. Those in the tumor were subsequently identified as organelles of lysosomal origin, demonstrating the potential of the combinatorial approach for investigating therapeutically relevant drug concentrations on a submicrometer scale.

Nutritional interactions between corals and symbiotic dinoflagellate algae lie at the heart of the structural foundation of coral reefs. Whilst the genetic diversity of Symbiodinium has attracted particular interest because of its contribution to the sensitivity of corals to environmental changes and bleaching (i.e. disruption of coral-dinoflagellate symbiosis), very little is known about the in hospite metabolic capabilities of different Symbiodinium types. Using a combination of stable isotopic labelling and nanoscale secondary ion mass spectrometry (NanoSIMS), we investigated the ability of the intact symbiosis between the reef-building coral Isopora palifera, and SymbiodiniumC or D types, to assimilate dissolved inorganic carbon (via photosynthesis) and nitrogen (as ammonium). Our results indicate that Symbiodinium types from two clades naturally associated with I.palifera possess different metabolic capabilities. The SymbiodiniumC type fixed and passed significantly more carbon and nitrogen to its coral host than the D type. This study provides further insights into the metabolic plasticity among different Symbiodinium types in hospite and strengthens the evidence that the more temperature-tolerant SymbiodiniumD type may be less metabolically beneficial for its coral host under non-stressful conditions.

In this contribution 18O tracer diffusion experiments were carried out on electroceramics and analysed by time-of-flight secondary-ion mass spectrometry. For the analysis, different operation modes of the Bi liquid metal ion gun were compared with respect to signal intensity, lateral resolution and mass resolution. Procedures for obtaining a basic estimation of the 18O fraction are given. It is shown that high target current, i.e. high number of primary ions, increases the accuracy and the precision of the determination of the 18O fraction if the observed count rate of the 16O signal is lower than 0.95 cts/shot. As an example, 18O diffusion profiles at the surface of a ZrO2:9.5mol%Y2O3 single crystals are shown. Copyright © 2012 John Wiley & Sons, Ltd.

Information on the active and resistive zones involved in the oxygen reduction reaction at SOFC cathodes is often deduced indirectly, for example from interpretations of impedance spectra or from geometry dependent polarization resistance measurements. A more direct means for obtaining information on the decisive zones is voltage driven tracer incorporation and subsequent spatially resolved analysis of tracer concentrations by secondary ion mass spectrometry. In this contribution it is first discussed under which conditions quantitative data on the width of three phase boundaries (TPB) can be determined. The calculations are then exemplified by measurements on the TPB width of Pt electrodes on YSZ. Moreover, it is shown for Sr-doped LaCoO3-δ cathodes that also resistive regions of oxygen reduction can be visualized by voltage driven tracer studies.

A study of grain boundary diffusion of oxygen in hot-rolled steel sheets is carried out by means of time-of-flight secondary-ion-mass-spectrometry (ToF-SIMS). This involves polishing of the sample surface prior to the oxygen exposure. A nickel layer deposited after exposure ensures a homogeneous extraction field for ToF-SIMS measurements at the Ni-steel interface. The sample is bevelled at an angle of 11.5° to spread up the diffusion pathway by a factor of 5. The oxygen distribution is then acquired via ToF-SIMS in imaging mode from which diffusion parameters are calculated according to the Whipple-Le Claire's approach.

High power impulse magnetron sputtering (HIPIMS) has gained increasing scientific and industrial attention as it allows high plasma densities without the drawback of droplet formation. Recently, we showed that by a combination of HIPIMS with dc magnetron sputtering the properties of the coatings are comparable to those prepared solely with HIPIMS, but with the advantage of increased deposition rate. Here, we show that for CrN HIPIMS /TiN DCMS multilayered coatings the friction coefficient µ decreases from 0.7 to 0.35 (with an almost constant hardness H around 25 GPa, and modulus of indentation around 375 GPa) when decreasing the bilayer period λ from 7.8 to 6.4 nm, while keeping the CrN HIPIMS layer thickness constant at 3.2 nm. A further reduction of the friction coefficient at room temperature dry-sliding testing to ∼ 0.25 or 0.05 is obtained when an additional HIPIMS cathode equipped with a Cr or Ti target material, respectively, is added to the process. Contact angle measurements of distilled water drops on as deposited film surfaces were carried out to investigate their wettability. The measurements show, that with increasing contact angle from 70° to 90°, for the individual coatings prepared, also their friction coefficient increases from ∼ 0.05 to ∼ 0.8. The depositions of all coatings were achieved with two- and threefold substrate rotation, which meet the industrial requirements of uniform deposition on complex shaped specimens.

In this work the electrochemical oxygen exchange reaction on the system platinum/yttria stabilized zirconia (YSZ) was investigated by means of impedance spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS) on well-defined dense platinum thin film electrodes. From impedance measurements on differently sized electrodes it was possible to show that the resistance caused by the rate determining step scales with the inverse length of the three phase boundary (TPB). To reveal information on the properties of the electrochemically active zone - i.e. the zone where oxygen is incorporated into the electrolyte - 18O tracer experiments combined with ToF-SIMS analysis were carried out. The extension of the active zone was found to depend on the overpotential η. In addition, the ToF-SIMS investigations revealed an enrichment of impurities in the vicinity of the TPB after dc load. The influence of sample pretreatment on the electrode performance is discussed.

Total parenteral nutrition (TPN) leads to a shift in small intestinal microbiota with a characteristic dominance of Proteobacteria. This study examined how metabolomic changes within the small bowel support an altered microbial community in enterally deprived mice. C57BL/6 mice were given TPN or enteral chow. Metabolomic analysis of jejunal contents was performed by liquid chromatography/mass spectrometry (LC/MS). In some experiments, leucine in TPN was partly substituted with [13C]leucine. Additionally, jejunal contents from TPN-dependent and enterally fed mice were gavaged into germ-free mice to reveal whether the TPN phenotype was transferrable. Small bowel contents of TPN mice maintained an amino acid composition similar to that of the TPN solution. Mass spectrometry analysis of small bowel contents of TPN-dependent mice showed increased concentration of 13C compared with fed mice receiving saline enriched with [13C]leucine. [13C]leucine added to the serosal side of Ussing chambers showed rapid permeation across TPN-dependent jejunum, suggesting increased transmucosal passage. Single-cell analysis by fluorescence in situ hybridization (FISH)-NanoSIMS demonstrated uptake of [13C]leucine by TPN-associated bacteria, with preferential uptake by Enterobacteriaceae. Gavage of small bowel effluent from TPN mice into germ-free, fed mice resulted in a trend toward the proinflammatory TPN phenotype with loss of epithelial barrier function. TPN dependence leads to increased permeation of TPN-derived nutrients into the small intestinal lumen, where they are predominately utilized by Enterobacteriaceae. The altered metabolomic composition of the intestinal lumen during TPN promotes dysbiosis.

Oxidizing hydrogen in place of nitrite Microorganisms are important drivers of Earth's nitrogen cycle. Many of the organisms responsible for mediating the reactions of one phase of nitrogen to another are thought to be ecologic specialists. Using a combination of genomic and experimental analyses, Koch et al. show that Nitrospira moscoviensis , a member of a widely distributed genus of nitrite-oxidizing bacteria, can oxidize hydrogen instead of nitrite to support growth when oxygen is present. Not only does this ecologic flexibility suggest a broader distribution of these organisms in natural settings, but they may be important in engineered environments as well. Science , this issue p. 1052

Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscaleresolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Ramanbased cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/ or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for singlecell genomics.

International audience; Subsurface microbial life contributes significantly to biogeochemical cycling, yet it remains largely uncharacterized, especially its archaeal members. This 'microbial dark matter' has been explored by recent studies that were, however, mostly based on DNA sequence information only. Here, we use diverse techniques including ultrastuctural analyses to link genomics to biology for the SM1 Euryarchaeon lineage, an uncultivated group of subsurface archaea. Phylogenomic analyses reveal this lineage to belong to a widespread group of archaea that we propose to classify as a new euryarchaeal order ('Candidatus Altiarchaeales'). The representative, double-membraned species 'Candidatus Altiarchaeum hamiconexum' has an autotrophic metabolism that uses a not-yet-reported Factor 420-free reductive acetyl-CoA pathway, confirmed by stable carbon isotopic measurements of archaeal lipids. Our results indicate that this lineage has evolved specific metabolic and structural features like nano-grappling hooks empowering this widely distributed archaeon to predominate anaerobic groundwater, where it may represent an important carbon dioxide sink.

Photosynthetic microbial mats are complex, stratified ecosystems in which high rates of primary production create a demand for nitrogen, met partially by N₂ fixation. Dinitrogenase reductase (nifH) genes and transcripts from Cyanobacteria and heterotrophic bacteria (for example, Deltaproteobacteria) were detected in these mats, yet their contribution to N2 fixation is poorly understood. We used a combined approach of manipulation experiments with inhibitors, nifH sequencing and single-cell isotope analysis to investigate the active diazotrophic community in intertidal microbial mats at Laguna Ojo de Liebre near Guerrero Negro, Mexico. Acetylene reduction assays with specific metabolic inhibitors suggested that both sulfate reducers and members of the Cyanobacteria contributed to N₂ fixation, whereas (15)N₂ tracer experiments at the bulk level only supported a contribution of Cyanobacteria. Cyanobacterial and nifH Cluster III (including deltaproteobacterial sulfate reducers) sequences dominated the nifH gene pool, whereas the nifH transcript pool was dominated by sequences related to Lyngbya spp. Single-cell isotope analysis of (15)N₂-incubated mat samples via high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that Cyanobacteria were enriched in (15)N, with the highest enrichment being detected in Lyngbya spp. filaments (on average 4.4 at% (15)N), whereas the Deltaproteobacteria (identified by CARD-FISH) were not significantly enriched. We investigated the potential dilution effect from CARD-FISH on the isotopic composition and concluded that the dilution bias was not substantial enough to influence our conclusions. Our combined data provide evidence that members of the Cyanobacteria, especially Lyngbya spp., actively contributed to N₂ fixation in the intertidal mats, whereas support for significant N₂ fixation activity of the targeted deltaproteobacterial sulfate reducers could not be found.

Multi-elemental, isotope selective nano-scale secondary ion mass spectrometry (NanoSIMS) combined with confocal laser-scanning microscopy was used to characterize the subcellular distribution of 15N-labeled cisplatin in human colon cancer cells. These analyses indicated predominant cisplatin colocalisation with sulfur-rich structures in both the nucleus and cytoplasm. Furthermore, colocalisation of platinum with phosphorus-rich chromatin regions was observed, which is consistent with its binding affinity to DNA as the generally accepted crucial target of the drug. Application of 15N-labeled cisplatin and subsequent measurement of the nitrogen isotopic composition and determination of the relative intensities of platinum and nitrogen associated secondary ion signals in different cellular compartments with NanoSIMS suggested partial dissociation of Pt–N bonds during the accumulation process, in particular within nucleoli at elevated cisplatin concentrations. This finding raises the question as to whether the observed intracellular dissociation of the drug has implications for the mechanism of action of cisplatin. Within the cytoplasm, platinum mainly accumulated in acidic organelles, as demonstrated by a direct combination of specific fluorescent staining, confocal laser scanning microscopy and NanoSIMS. Different processing of platinum drugs in acidic organelles might be relevant for their detoxification, as well as for their mode of action.

Nutritional interactions between corals and symbiotic dinoflagellate algae lie at the heart of the structural foundation of coral reefs. Whilst the genetic diversity of Symbiodinium has attracted particular interest because of its contribution to the sensitivity of corals to environmental changes and bleaching (i.e. disruption of coral-dinoflagellate symbiosis), very little is known about the in hospite metabolic capabilities of different Symbiodinium types. Using a combination of stable isotopic labelling and nanoscale secondary ion mass spectrometry (NanoSIMS), we investigated the ability of the intact symbiosis between the reef-building coral Isopora palifera, and Symbiodinium C or D types, to assimilate dissolved inorganic carbon (via photosynthesis) and nitrogen (as ammonium). Our results indicate that Symbiodinium types from two clades naturally associated with I. palifera possess different metabolic capabilities. The Symbiodinium C type fixed and passed significantly more carbon and nitrogen to its coral host than the D type. This study provides further insights into the metabolic plasticity among different Symbiodinium types in hospite and strengthens the evidence that the more temperature-tolerant Symbiodinium D type may be less metabolically beneficial for its coral host under non-stressful conditions.

Genes of archaea encoding homologues of ammonia monooxygenases have been found on a widespread basis and in large amounts in almost all terrestrial and marine environments, indicating that ammonia oxidizing archaea (AOA) might play a major role in nitrification on Earth. However, only one pure isolate of this group from a marine environment has so far been obtained, demonstrating archaeal ammonia oxidation coupled with autotrophic growth similar to the bacterial counterparts. Here we describe the cultivation and isolation of an AOA from soil. It grows on ammonia or urea as an energy source and is capable of using higher ammonia concentrations than the marine isolate, Nitrosopumilus maritimus . Surprisingly, although it is able to grow chemolithoautotrophically, considerable growth rates of this strain are obtained only upon addition of low amounts of pyruvate or when grown in coculture with bacteria. Our findings expand the recognized metabolic spectrum of AOA and help explain controversial results obtained in the past on the activity and carbon assimilation of these globally distributed organisms.

In this study the electrochemically active region of oxygen incorporation into yttria stabilized zirconia (YSZ) was visualized by means of (18)O tracer incorporation experiments on dense Pt thin film microelectrodes combined with ToF-SIMS analysis. The localization and the shape of the incorporation zone were found to strongly depend on the polarization of the electrode. In case of lower overpotentials the active zone next to the three phase boundary (TPB) was frame-shaped and located beneath the Pt electrode. Increases in polarization led to an extension of the incorporation zone along the free YSZ surface. Owing to the low temperature of 300-330 °C a profile-broadening caused by diffusion in YSZ could be minimized and quantitatively separated from the measured profiles. The TPB-width (i.e. the decay length of electrochemical activity) was determined to be approximately 1.0-1.3 μm at these temperatures.

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In this paper we present a method to measure oxygen tracer diffusion coefficients in thin ion conducting films without being limited by slow oxygen incorporation kinetics. The method is based on a two step process. In the first step a substantial amount of 18O tracer is locally incorporated for example into an yttria stabilized zirconia (YSZ) layer at low temperatures with the aid of an electric current, thus overcoming slow thermal oxygen exchange while still limiting lateral diffusion to a minimum. In the second step controlled diffusion takes place at elevated temperatures in ultra high vacuum (UHV) to impede loss of tracer due to oxygen exchange at the film surface. In this second step the surface of the thin film may additionally be modified compared to the oxygen incorporation step. This allows to easily investigate effects of interfaces on ion transport. The achieved in-plane concentration profiles are then measured by secondary ion mass spectrometry (SIMS). Comparison with electrical measurements on YSZ thin films proves the applicability of the method.

Refractory metals are primarily characterized by a high melting point combined with a rather poor corrosion resistance under oxidizing atmosphere and therefore are mainly used in high temperature processes under reducing atmosphere or in vacuum. Exposure to an atmosphere of high humidity can lead to oxidation of the material even at room temperature. Different methods of surface pre-treatment have been applied to investigate their influence on the oxidation behavior of high-purity molybdenum. Within the scope of this work molybdenum foils and molybdenum discs consisting of the same base material were investigated. Since lateral surface structure as well as the in-depth distribution of contaminants are expected to play an important role in the oxidation process, both the in-depth distribution of the constituents within the oxide layer and the lateral distribution at the surface level of the oxide have been analyzed. Scanning SIMS has been engaged to analyze the uppermost structures of the oxide layer. In order to achieve maximum detection power and to gain the in-depth information, stigmatic SIMS has been applied to investigate the in-depth distribution of the interesting specimen constituents.