Anaerobic oxidation of ethane by archaea from a marine hydrocarbon seep.

Ethane is the second most abundant component of natural gas in addition to methane, and-similar to methane-is chemically unreactive. The biological consumption of ethane under anoxic conditions was suggested by geochemical profiles at marine hydrocarbon seeps1-3, and through ethane-dependent sulfate reduction in slurries4-7. Nevertheless, the microorganisms and reactions that catalyse this process have to date remained unknown8. Here we describe ethane-oxidizing archaea that were obtained by specific enrichment over ten years, and analyse these archaea using phylogeny-based fluorescence analyses, proteogenomics and metabolite studies. The co-culture, which oxidized ethane completely while reducing sulfate to sulfide, was dominated by an archaeon that we name 'Candidatus Argoarchaeum ethanivorans'; other members were sulfate-reducing Deltaproteobacteria. The genome of Ca. Argoarchaeum contains all of the genes that are necessary for a functional methyl-coenzyme M reductase, and all subunits were detected in protein extracts. Accordingly, ethyl-coenzyme M (ethyl-CoM) was identified as an intermediate by liquid chromatography-tandem mass spectrometry. This indicated that Ca. Argoarchaeum initiates ethane oxidation by ethyl-CoM formation, analogous to the recently described butane activation by 'Candidatus Syntrophoarchaeum'9. Proteogenomics further suggests that oxidation of intermediary acetyl-CoA to CO2 occurs through the oxidative Wood-Ljungdahl pathway. The identification of an archaeon that uses ethane (C2H6) fills a gap in our knowledge of microorganisms that specifically oxidize members of the homologous alkane series (CnH2n+2) without oxygen. Detection of phylogenetic and functional gene markers related to those of Ca. Argoarchaeum at deep-sea gas seeps10-12 suggests that archaea that are able to oxidize ethane through ethyl-CoM are widespread members of the local communities fostered by venting gaseous alkanes around these seeps.

Detection and Exclusion of False-Positive Molecular Formula Assignments via Mass Error Distributions in UHR Mass Spectra of Natural Organic Matter.

Ultrahigh resolution mass spectrometry (UHRMS) routinely detects and identifies thousands of mass peaks in complex mixtures, such as natural organic matter (NOM) and petroleum. The assignment of several chemically plausible molecular formulas (MFs) for a single accurate mass still poses a major problem for the reliable interpretation of NOM composition in a biogeochemical context. Applying sensible chemical rules for MF validation is often insufficient to eliminate multiple assignments (MultiAs)─especially for mass peaks with low abundance or if ample heteroatoms or isotopes are included - and requires manual inspection or expert judgment. Here, we present a new approach based on mass error distributions for the identification of true and false assignments among MultiAs. To this end, we used the mass error in millidalton (mDa), which was superior to the commonly used relative mass error in ppm. We developed an automatic workflow to group MultiAs based on their shared formula units and Kendrick mass defect values and to evaluate the mass error distribution. In this way, the number of valid assignments of chlorinated disinfection byproducts was increased by 8-fold as compared to only applying 37Cl/35Cl isotope ratio filters. Likewise, phosphorus-containing MFs can be differentiated against chlorine-containing MFs with high confidence. Further, false assignments of highly aromatic sulfur-containing MFs ("black sulfur") to sodium adducts in negative ionization mode can be excluded by applying our approach. Overall, MFs for mass peaks that are close to the detection limit or where naturally occurring isotopes are rare (e.g., 15N) or absent (e.g., P and F) can now be validated, substantially increasing the reliability of MF assignments and broadening the applicability of UHRMS analysis to even more complex samples and processes.

Post column infusion of an internal standard into LC-FT-ICR MS enables semi-quantitative comparison of dissolved organic matter in original samples.

Ultrahigh resolution mass spectrometry hyphenated with liquid chromatography (LC) is an emerging tool to explore the isomeric composition of dissolved organic matter (DOM). However, matrix effects limit the potential for semi-quantitative comparison of DOM molecule abundances across samples. We introduce a post-column infused internal standard (PCI-IS) for reversed-phase LC-FT-ICR MS measurements of DOM and systematically evaluate matrix effects, detector linearity and the precision of mass peak intensities. Matrix effects for model compounds spiked into freshwater DOM samples ranging from a headwater stream to a major river were reduced by 5-10% for PCI-IS corrected mass peak intensities as compared to raw (i.e., untransformed) intensities. A linear regression of PCI-IS corrected DOM mass peak intensities across a typical DOM concentration range (2-15 mg dissolved organic carbon L-1) in original, non-extracted freshwater samples demonstrates excellent linearity of the detector response (r2 > 0.9 for 98% of detected molecular formulas across retention times). Importantly, PCI-IS could compensate for 80% of matrix effects across an environmental gradient of DOM composition from groundwater to surface water. This enabled studying the ionization efficiency of DOM isomers and linking the observed differences to the biogeochemical sources. With PCI-IS original, non-extracted DOM samples can be analysed by LC-FT-ICR MS without carbon load adjustment, and mass peak intensities can be reliably used to semi-quantitatively compare isomer abundances between compositionally similar DOM samples.

Direct Analysis of Marine Dissolved Organic Matter Using LC-FT-ICR MS.

Marine dissolved organic matter (DOM) is an important component of the global carbon cycle, yet its intricate composition and the sea salt matrix pose major challenges for chemical analysis. We introduce a direct injection, reversed-phase liquid chromatography ultrahigh resolution mass spectrometry approach to analyze marine DOM without the need for solid-phase extraction. Effective separation of salt and DOM is achieved with a large chromatographic column and an extended isocratic aqueous step. Postcolumn dilution of the sample flow with buffer-free solvents and implementing a counter gradient reduced salt buildup in the ion source and resulted in excellent repeatability. With this method, over 5,500 unique molecular formulas were detected from just 5.5 nmol carbon in 100 µL of filtered Arctic Ocean seawater. We observed a highly linear detector response for variable sample carbon concentrations and a high robustness against the salt matrix. Compared to solid-phase extracted DOM, our direct injection method demonstrated superior sensitivity for heteroatom-containing DOM. The direct analysis of seawater offers fast and simple sample preparation and avoids fractionation introduced by extraction. The method facilitates studies in environments, where only minimal sample volume is available e.g. in marine sediment pore water, ice cores, or permafrost soil solution. The small volume requirement also supports higher spatial (e.g., in soils) or temporal sample resolution (e.g., in culture experiments). Chromatographic separation adds further chemical information to molecular formulas, enhancing our understanding of marine biogeochemistry, chemodiversity, and ecological processes.

Combining Advanced Analytical Methodologies to Uncover Suspect PFAS and Fluorinated Pharmaceutical Contributions to Extractable Organic Fluorine in Human Serum (Tromsø Study).

A growing number of studies have reported that routinely monitored per- and polyfluoroalkyl substances (PFAS) are not sufficient to explain the extractable organic fluorine (EOF) measured in human blood. In this study, we address this gap by screening pooled human serum collected over 3 decades (1986-2015) in Tromsø (Norway) for >5000 PFAS and >300 fluorinated pharmaceuticals. We combined multiple analytical techniques (direct infusion Fourier transform ion cyclotron resonance mass spectrometry, liquid chromatography-Orbitrap-high-resolution mass spectrometry, and total oxidizable precursors assay) in a three-step suspect screening process which aimed at unequivocal suspect identification. This approach uncovered the presence of one PFAS and eight fluorinated pharmaceuticals (including some metabolites) in human serum. While the PFAS suspect only accounted for 2-4% of the EOF, fluorinated pharmaceuticals accounted for 0-63% of the EOF, and their contribution increased in recent years. Although fluorinated pharmaceuticals often contain only 1-3 fluorine atoms, our results indicate that they can contribute significantly to the EOF. Indeed, the contribution from fluorinated pharmaceuticals allowed us to close the organofluorine mass balance in pooled serum from 2015, indicating a good understanding of organofluorine compounds in humans. However, a portion of the EOF in human serum from 1986 and 2007 still remained unexplained.

A Temporal Graph Model to Predict Chemical Transformations in Complex Dissolved Organic Matter.

Dissolved organic matter (DOM) is a complex mixture of thousands of natural molecules that undergo constant transformation in the environment, such as sunlight induced photochemical reactions. Despite molecular level resolution from ultrahigh resolution mass spectrometry (UHRMS), trends of mass peak intensities are currently the only way to follow photochemically induced molecular changes in DOM. Many real-world relationships and temporal processes can be intuitively modeled using graph data structures (networks). Graphs enhance the potential and value of AI applications by adding context and interconnections allowing the uncovering of hidden or unknown relationships in data sets. We use a temporal graph model and link prediction to identify transformations of DOM molecules in a photo-oxidation experiment. Our link prediction algorithm simultaneously considers educt removal and product formation for molecules linked by predefined transformation units (oxidation, decarboxylation, etc.). The transformations are further weighted by the extent of intensity change and clustered on the graph structure to identify groups of similar reactivity. The temporal graph is capable of identifying relevant molecules subject to similar reactions and enabling to study their time course. Our approach overcomes previous data evaluation limitations for mechanistic studies of DOM and leverages the potential of temporal graphs to study DOM reactivity by UHRMS.

The effect of root hairs on exudate composition: a comparative non-targeted metabolomics approach.

Root exudation is a major pathway of organic carbon input into soils. It affects soil physical properties, element solubility as well as speciation, and impacts the microbial community in the rhizosphere. Root exudates contain a large number of primary and secondary plant metabolites, and the amount and composition are highly variable depending on plant species and developmental stage. Detailed information about exudate composition will allow for a better understanding of exudate-driven rhizosphere processes and their feedback loops. Although non-targeted metabolomics by high-resolution mass spectrometry is an established tool to characterize root exudate composition, the extent and depth of the information obtained depends strongly on the analytical approach applied. Here, two genotypes of Zea mays L., differing in root hair development, were used to compare six mass spectrometric approaches for the analysis of root exudates. Reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography combined with time-of-flight mass spectrometry (LC-TOF-MS), as well as direct infusion Fourier-transform ion cyclotron resonance mass spectrometry (DI-FT-ICR-MS), were applied with positive and negative ionization mode. By using the same statistical workflow, the six approaches resulted in different numbers of detected molecular features, ranging from 176 to 889, with a fraction of 48 to 69% of significant features (fold change between the two genotypes of > 2 and p-value < 0.05). All approaches revealed the same trend between genotypes, namely up-regulation of most metabolites in the root hair defective mutant (rth3). These results were in agreement with the higher total carbon and nitrogen exudation rate of the rth3-mutant as compared to the corresponding wild-type maize (WT). However, only a small fraction of features were commonly found across the different analytical approaches (20-79 features, 13-31% of the rth3-mutant up-regulated molecular formulas), highlighting the need for different mass spectrometric approaches to obtain a more comprehensive view into the composition of root exudates. In summary, 111 rth3-mutant up-regulated compounds (92 different molecular formulas) were detected with at least two different analytical approaches, while no WT up-regulated compound was found by both, LC-TOF-MS and DI-FT-ICR-MS. Zea mays L. exudate features obtained with multiple analytical approaches in our study were matched against the metabolome database of Zea mays L. (KEGG) and revealed 49 putative metabolites based on their molecular formula.

Zebrafish Oatp1d1 Acts as a Cellular Efflux Transporter of the Anionic Herbicide Bromoxynil.

Toxicokinetics (TK) of ionic compounds in the toxico-/pharmacological model zebrafish embryo (Danio rerio) depend on absorption, distribution, metabolism, and elimination (ADME) processes. Previous research indicated involvement of transport proteins in the TK of the anionic pesticide bromoxynil in zebrafish embryos. We here explored the interaction of bromoxynil with the organic anion-transporting polypeptide zebrafish Oatp1d1. Mass spectrometry imaging revealed accumulation of bromoxynil in the gastrointestinal tract of zebrafish embryos, a tissue known to express Oatp1d1. In contrast to the Oatp1d1 reference substrate bromosulfophthalein (BSP), which is actively taken up by transfected HEK293 cells overexpressing zebrafish Oatp1d1, those cells accumulated less bromoxynil than empty vector-transfected control cells. This indicates cellular efflux of bromoxynil by Oatp1d1. This was also seen for diclofenac but not for carbamazepine, examined for comparison. Correspondingly, internal concentrations of bromoxynil and diclofenac in the zebrafish embryo were increased when coexposed with BSP, inhibiting the activities of various transporter proteins, including Oatp1d1. The effect of BSP on accumulation of bromoxynil and diclofenac was enhanced in further advanced embryo stages, indicating increased efflux activity in those stages. An action of Oatp1d1 as an efflux transporter of ionic environmental compounds in zebrafish embryos should be considered in future TK assessments.

Discovery of Polar Ozonation Byproducts via Direct Injection of Effluent Organic Matter with Online LC-FT-ICR-MS.

Effluent organic matter (EfOM), a major ozone consumer during wastewater ozonation, is a complex mixture of natural and anthropogenic organic molecules. Ozonation of EfOM adds to molecular complexity by introducing polar and potentially mobile ozonation byproducts (OBPs). Currently, nontargeted direct infusion (DI) ultrahigh resolution mass spectrometry (e.g. FT-ICR-MS) is used to study OBPs but requires sample extraction, limiting the accessible polarity range of OBPs. To better understand the impact of ozonation on EfOM and the formation of polar OBPs, nonextracted effluent was analyzed by direct injection onto a reversed-phase liquid chromatography system (RP-LC) online hyphenated with an FT-ICR-MS. Over four times more OBPs were detected in nonextracted EfOM compared to effluent extracted with solid phase extraction and measured with DI-FT-ICR-MS (13817 vs 3075). Over 1500 highly oxygenated OBPs were detected exclusively in early eluting fractions of nonextracted EfOM, indicating polar OBPs. Oxygenation of these newly discovered OBPs is higher than previously found, with an average molecular DBE-O value of -3.3 and O/C ratio of 0.84 in the earliest eluting OBP fractions. These polar OBPs are consistently lost during extraction but may play an important role in understanding the environmental impact of ozonated EfOM. Moreover, 316 molecular formulas classified as nonreactive to ozone in DI-FT-ICR-MS can be identified with LC-FT-ICR-MS as isomers with varying degrees of reactivity, providing for the first time experimental evidence of differential reactivity of complex organic matter isomers with ozone.

Aromaticity Index with Improved Estimation of Carboxyl Group Contribution for Biogeochemical Studies.

Natural organic matter (NOM) components measured with ultrahigh-resolution mass spectrometry (UHRMS) are often assessed by molecular formula-based indices, particularly related to their aromaticity, which are further used as proxies to explain biogeochemical reactivity. An aromaticity index (AI) is calculated mostly with respect to carboxylic groups abundant in NOM. Here, we propose a new constrained AIcon based on the measured distribution of carboxylic groups among individual NOM components obtained by deuteromethylation and UHRMS. Applied to samples from diverse sources (coal, marine, peat, permafrost, blackwater river, and soil), the method revealed that the most probable number of carboxylic groups was two, which enabled to set a reference point n = 2 for carboxyl-accounted AIcon calculation. The examination of the proposed AIcon showed the smallest deviation to the experimentally determined index for all NOM samples under study as well as for individual natural compounds obtained from the Coconut database. In particular, AIcon performed better than AImod for all compound classes in which aromatic moieties are expected: aromatics, condensed aromatics, and unsaturated compounds. Therefore, AIcon referenced with two carboxyl groups is preferred over conventional AI and AImod for biogeochemical studies where the aromaticity of compounds is important to understand the transformations and fate of NOM compounds.

Legacy Effects of Sorption Determine the Formation Efficiency of Mineral-Associated Soil Organic Matter.

Sorption of dissolved organic matter (DOM) is one major pathway in the formation of mineral-associated organic matter (MOM), but there is little information on how previous sorption events feedback to later ones by leaving their imprint on mineral surfaces and solutions ("legacy effect"). In order to conceptualize the role of legacy effects in MOM formation, we conducted sequential sorption experiments with kaolinite and gibbsite as minerals and DOM derived from forest floor materials. The MOM formation efficiency leveled off upon repeated addition of identical DOM solutions to minerals due to the retention of highly sorptive organic molecules (primarily aromatic, nitrogen-poor, hydrogen-poor, and oxygen-rich molecules), which decreased the sorption site availability and simultaneously modified the mineral surface charge. Organic-organic interactions as postulated in multilayer models played a negligible role in MOM formation. Continued exchange between DOM and MOM molecules upon repeated sorption altered the DOM composition but not the MOM formation efficiencies. Sorption-induced depletion of high-affinity compounds from solutions further decreased the MOM formation efficiencies to pristine minerals. Overall, the interplay between the differential sorptivities of DOM components and the mineral surface chemistry explains the legacy effects that contribute to the regulation of fluxes and the distribution of organic matter in the soil.

Thermophilic archaea activate butane via alkyl-coenzyme M formation.

The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C1-compounds. Here we show that an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria apparently uses partly similar pathways to oxidize the C4 hydrocarbon butane. The archaea, proposed genus 'Candidatus Syntrophoarchaeum', show the characteristic autofluorescence of methanogens, and contain highly expressed genes encoding enzymes similar to methyl-coenzyme M reductase. We detect butyl-coenzyme M, indicating archaeal butane activation analogous to the first step in anaerobic methane oxidation. In addition, Ca. Syntrophoarchaeum expresses the genes encoding ß-oxidation enzymes, carbon monoxide dehydrogenase and reversible C1 methanogenesis enzymes. This allows for the complete oxidation of butane. Reducing equivalents are seemingly channelled to HotSeep-1, a thermophilic sulfate-reducing partner bacterium known from the anaerobic oxidation of methane. Genes encoding 16S rRNA and methyl-coenzyme M reductase similar to those identifying Ca. Syntrophoarchaeum were repeatedly retrieved from marine subsurface sediments, suggesting that the presented activation mechanism is naturally widespread in the anaerobic oxidation of short-chain hydrocarbons.

Online Counter Gradient LC-FT-ICR-MS Enables Detection of Highly Polar Natural Organic Matter Fractions.

Natural organic matter (NOM) is a highly complex mixture of natural organic molecules. The recent developments in NOM molecular characterization methods have shown that ESI-FT-ICR hyphenated with liquid chromatography (LC) is a promising approach to also obtain chemical information (such as polarity and molecular size) about NOM molecules. However, due to changing solvent composition during gradient elution in LC-FT-ICR-MS, ionization conditions also change throughout the chromatographic separation process. In this study, we applied a post-LC column counter gradient (CG) to ensure stable solvent conditions for transient ESI-MS signals. Suwanee River Fulvic Acid (SRFA) standard and a peat pore water were used as representative dissolved NOM samples for method development and validation. Our results show that in polar NOM fractions (which elute with <50% methanol) the TIC intensity and number of assigned molecular formulas were increased by 48% and 20%, as compared to the standard gradient (SG) method. Further application of a Q-isolation and selective ion accumulation for low abundance fractions revealed over 3 times more molecular formulas (especially for CHNO, CHOS, CHNOS formula classes) than in full scan mode. The number of detected highly polar NOM compounds (with elemental ratios H/C < 1, O/C > 0.6) were more than 20 times larger for CG-LC mode as compared to direct infusion (DI) (5715 vs 266 MF). We conclude that the application of a postcolumn counter gradient in LC-FT-ICR-MS analyses of NOM offers novel insight into the most polar fractions of NOM which are inaccessible in conventional DI measurements.

Conditioning Film and Early Biofilm Succession on Plastic Surfaces.

In the context of environmental plastic pollution, it is still under debate if and how the "plastisphere", a plastic-specific microbial community, emerges. In this study, we tested the hypothesis that the first conditioning film of dissolved organic matter (DOM) sorbs selectively to polymer substrates and that microbial attachment is governed in a substrate-dependent manner. We investigated the adsorption of stream water-derived DOM to polyethylene terephthalate (PET), polystyrene (PS), and glass (as control) including UV-weathered surfaces by Fourier-transform ion cyclotron mass spectrometry. Generally, the saturated, high-molecular mass and thus more hydrophobic fraction of the original stream water DOM preferentially adsorbed to the substrates. The UV-weathered polymers adsorbed more polar, hydrophilic OM as compared to the dark controls. The amplicon sequencing data of the initial microbial colonization process revealed a tendency of substrate specificity for biofilm attachment after 24 h and a clear convergence of the communities after 72 h of incubation. Conclusively, the adsorbed OM layer developed depending on the materials' surface properties and increased the water contact angles, indicating higher surface hydrophobicity as compared to pristine surfaces. This study improves our understanding of molecular and biological interactions at the polymer/water interface that are relevant to understand the ecological impact of plastic pollution on a community level.

Refinement of Compound Aromaticity in Complex Organic Mixtures by Stable Isotope Label Assisted Ultrahigh-Resolution Mass Spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) provides a unique opportunity for molecular analysis of natural complex mixtures. In many geochemical and environmental studies structure-propertry relations are based solely on the elemental compositional information. Several calculated parameters were proposed to increase reliability of structural attribution, among which aromaticity indices (AI and AImod) are widely used. Herein, we applied a combination of selective labeling reactions in order to obtain direct structural information on the individual components of lignin-derived polyphenolic material. Carboxylic (COOH), carbonyl (C═O), and hydroxyl (OH) groups were enumerated by esterification, reducing, and acetylation reactions, respectively, followed by FTICR MS analyses. Obtained information was enabled to constrain aromaticity accounting for the carbon skeleton only. We found that actual aromaticity of components may be both higher or lower than approximated values depending on the abundance of COOH, C═O, and OH groups. The results are of importance for the geochemical community studying terrestrial NOM with structural gradients.

Improved Understanding of Dissolved Organic Matter Processing in Freshwater Using Complementary Experimental and Machine Learning Approaches.

Dissolved organic matter plays an important role in aquatic ecosystems and poses a major problem for drinking water production. However, our understanding of DOM reactivity in natural systems is hampered by its complex molecular composition. Here, we used Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and data from two independent studies to disentangle DOM reactivity based on photochemical and microbial-induced transformations. Robust correlations of FT-ICR-MS peak intensities with chlorophyll a and solar irradiation were used to define 9 reactivity classes for 1277 common molecular formulas. Germany's largest drinking water reservoir was sampled for 1 year, and DOM processing in stratified surface waters could be attributed to photochemical transformations during summer months. Microbial DOM alterations could be distinguished based on correlation coefficients with chlorophyll a and often shared molecular features (elemental ratios and mass) with photoreactive compounds. In particular, many photoproducts and some microbial products were identified as potential precursors of disinfection byproducts. Molecular DOM features were used to further predict molecular reactivity for the remaining compounds in the data set based on a random forest model. Our method offers an expandable classification approach to integrate the reactivity of DOM from specific environments and link it to molecular properties and chemistry.

Exploring the potential of laser desorption ionisation time-of-flight mass spectrometry to analyse organic capping agents on inorganic nanoparticle surfaces.

Analytical techniques are in high demand for the determination of organic capping agents on surfaces of metallic nanoparticles (NPs) such as gold (Au) and silver (Ag). In this study, the potential of laser desorption ionisation time-of-flight mass spectrometry (LDI-ToF-MS) as a technique fit for this purpose is demonstrated. First, a collection of reference spectra of most commonly used organic capping agents, including small molecules and polymers was established. Second, the robustness of the method was tested towards parameters like NP core material and NP size. In a third step, the quantitative capabilities of LDI-ToF-MS were determined. Finally, the potential to detect chemical alterations of the organic capping agent was evaluated. LDI-ToF-MS is able to detect capping agents ranging from small molecules (citric acid, tannic acid, lipoic acid) to large polymers (polyvinylpyrrolidone, branched polyethylenimine and methoxy polyethylene glycol sulfhydryl) on Au and Ag NPs based on characteristic signals for each capping agent. Small molecules showed characteristic fragment ions with low intensities, whereas polymers showed intense signals of the monomeric subunit. The NP concentration range comprises about two orders of magnitude with lowest detection limits of 5 mg/L or a capping agent concentration in the lower nM range. Changes in capping agent composition are detectable at NP concentrations in the g/L range. Thus, LDI-ToF-MS is particularly suitable for characterisation of polymer-capped NPs with high NP concentrations. This may be the case for quality control as part of the material synthesis and testing. Graphical abstract.

Quantifying a Biocatalytic Product from a Few Living Microbial Cells Using Microfluidic Cultivation Coupled to FT-ICR-MS.

The in vivo quantification of metabolic products from microbial single cells is one of the last grand challenges in (bio)analytical chemistry. To date, no label-free analytical concept exists that is powerful enough to detect or even quantify the minute amounts of secreted low molecular weight compounds produced by living and isolated single bacteria or yeast cells. Coupling microfluidic cultivation systems with ultrahigh resolution electrospray-ionization mass spectrometry with its exquisite sensitivity and specificity offers the prospect of single-cell product analysis and quantification, but has not been successfully implemented yet. We report an analytical framework that interfaces noninvasive microfluidic trapping and cultivation of a few bacterial single cells with the analysis of their catalytic products by Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Cell trapping was performed with the microfluidic Envirostat platform for cultivating bacterial cells under continuous perfusion via negative dielectrophoresis (nDEP). A total of 1.5 µL of product-containing cell supernatant was sampled into microcapillaries using a dead volume-reduced world-to-chip interface. The samples were analyzed with a nanoESI ion source coupled to a FT-ICR-MS (limit of detection for lysine: 0.5 pg). As a biocatalytic model system, we analyzed few Corynebacterium glutamicum DM 1919 pSenLys cells that synthesized l-lysine from d-glucose. Secreted lysine was quantified from a few cells (down to 19). Single-cell specific lysine productivities were 2 and 10 fmol/cell/h. This demonstrates that coupling microfluidics and mass spectrometry (SIC-MS) now enables the quantification of catalytic products and extracellular metabolites from only a few living microbial cells.

2D Liquid Chromatographic Fractionation with Ultra-high Resolution MS Analysis Resolves a Vast Molecular Diversity of Tropospheric Particle Organics.

A 2D-liquid chromatographic fractionation method was combined with direct infusion electrospray ionization Fourier transform-ion cyclotron resonance mass spectrometry to better resolve the high complexity of the organic material in atmospheric particles. The number of assigned molecular formulas increased by a factor of 2.3 for the fractionated sample (18 144) compared to a bulk sample analysis without fractionation (7819), while simultaneously allowing the identification of 71 240 isomeric compounds. Accounting for these isomers has an impact on the means and distributions of different descriptive sample parameters. More than 15 000 compounds were exclusively identified in the fractionated sample providing insights regarding the formation of organosulfates, reduced N-containing compounds, and polyaromatic compounds. Further, a new method for assigning organonitrates and poly-organonitrates based on Kendrick mass defect analysis is presented. The current study implicates that analytical separation leads to much more detailed insights into particle organics composition, while more commonly applied direct infusion MS studies can strongly underestimate composition complexity and lead to biased assignments of bulk organic properties. Overall, the particle organics composition is far more complex than previously shown, while separation through better chromatographic techniques helps to understand formation processes of atmospheric particle constituents.

Photochemically Induced Bound Residue Formation of Carbamazepine with Dissolved Organic Matter.

More than 400 new nitrogen containing products were detected upon experimental sunlight photolysis of the pharmaceutical carbamazepine (CBZ) in the presence of dissolved organic matter (DOM) by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). These products were presumably formed through covalent binding of CBZ phototransformation products with DOM molecules. About 50% of these newly formed bound residues contained one nitrogen atom and had a molecular mass between 375 and 525 Da, which was 150 to 200 Da higher than for an average DOM molecule. In addition, a previously unknown CBZ phototransformation product, 3-quinolinecarboxylic acid (3-QCA), was identified by liquid chromatography high resolution tandem mass spectrometry (LC-HRMS/MS). 3-QCA was likely formed through oxidative ring cleavage and subsequent decarboxylation of acridine, a well-known phototransformation product of CBZ. Collision induced dissociation experiments and Kendrick mass defect analyses corroborated that about 160 of the new products were formed via covalent binding of 3-QCA with DOM molecules of above-average O/C and H/C ratios. Experiments at lower CBZ concentration suggested that the importance of bound residue formation increases with increasing DOM/CBZ ratios. Photochemically induced bound residue formation of polar contaminants with DOM in the aqueous phase is thus a disregarded pathway along which contaminants can be transformed in the environment. The method presented here offers a new possibility to study the formation of bound residues, which may be of relevance also for other transformation processes in natural waters where radical intermediates are generated.