Extreme enrichment in atmospheric 15N15N.

Molecular nitrogen (N2) comprises three-quarters of Earth's atmosphere and significant portions of other planetary atmospheres. We report a 19 per mil (‰) excess of 15N15N in air relative to a random distribution of nitrogen isotopes, an enrichment that is 10 times larger than what isotopic equilibration in the atmosphere allows. Biological experiments show that the main sources and sinks of N2 yield much smaller proportions of 15N15N in N2. Electrical discharge experiments, however, establish 15N15N excesses of up to +23‰. We argue that 15N15N accumulates in the atmosphere because of gas-phase chemistry in the thermosphere (>100 km altitude) on time scales comparable to those of biological cycling. The atmospheric 15N15N excess therefore reflects a planetary-scale balance of biogeochemical and atmospheric nitrogen chemistry, one that may also exist on other planets

Engineering soil organic matter quality: Biodiesel Co-Product (BCP) stimulates exudation of nitrogenous microbial biopolymers

Biodiesel Co-Product (BCP) is a complex organic material formed during the transesterification of lipids. We investigated the effect of BCP on the extracellular microbial matrix or ‘extracellular polymeric substance’ (EPS) in soil which is suspected to be a highly influential fraction of soil organic matter (SOM). It was hypothesised that more N would be transferred to EPS in soil given BCP compared to soil given glycerol. An arable soil was amended with BCP produced from either 1) waste vegetable oils or 2) pure oilseed rape oil, and compared with soil amended with 99% pure glycerol; all were provided with 15N labelled KNO3. We compared transfer of microbially assimilated 15N into the extracellular amino acid pool, and measured concomitant production of exopolysaccharide. Following incubation, the 15N enrichment of total hydrolysable amino acids (THAAs) indicated that intracellular anabolic products had incorporated the labelled N primarily as glutamine and glutamate. A greater proportion of the amino acids in EPS were found to contain 15N than those in the THAA pool, indicating that the increase in EPS was comprised of bioproducts synthesised de novo. Moreover, BCP had increased the EPS production efficiency of the soil microbial community (μg EPS per unit ATP) up to approximately double that of glycerol, and caused transfer of 21% more 15N from soil solution into EPS-amino acids. Given the suspected value of EPS in agricultural soils, the use of BCP to stimulate exudation is an interesting tool to consider in the theme of delivering sustainable intensification

HIL real-time simulation of a digital fractional order PI controller for time delay processes

Fractional order control has been used extensively in the last decade for controlling various types of processes. Several design approaches have been proposed so far, the closed loop performance results obtained being tested using different simulation conditions. The hardware-in-the-loop (HIL) real-time simulation offers a more reliable method for evaluating the closed loop performance of such controllers prior to their actual implementation on the real processes, such HIL simulation being highly suitable especially for complex, hazardous processes in which human and equipment errors should be avoided. The present paper proposes a hardware-in-the-loop real-time simulation setting for a digital fractional order PI controller in a Smith Predictor structure. The designed control strategy and fractional order controller is then tested under nominal and uncertain conditions, considering a time delay process

Control of a train of high purity distillation columns for efficient production of 13C isotopes

It is well-known that high-purity distillation columns are difficult to control due to their ill-conditioned and strongly nonlinear behaviour. The fact that these processes are operated over a wide range of feed compositions and flow rates makes the control design even more challenging. This paper proposes the most suitable control strategies applicable to a series of cascaded distillation column processes. The conditions for control and input-output relations are discusssed in view of the global control strategy. The increase in complexity with increased number of series cascaded distillation column processes is tackled. Uncertainty in the model parameters is discussed with respect to the dynamics of the global train distillation process. The main outcome of this work is insight into the possible control methodologies for this particular class of distillation processes

Isotope ratio - Mass spectrometry

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/32166This article summarizes the configurations involving isotope ratio mass spectrometry (IRMS) technology available at the CCiTUB and the wide range of possible applications. Some examples of these applications are shown

Comparative Metabolomics of Early Development of the Parasitic Plants Phelipanche aegyptiaca and Triphysaria versicolor.

Parasitic weeds of the family Orobanchaceae attach to the roots of host plants via haustoria capable of drawing nutrients from host vascular tissue. The connection of the haustorium to the host marks a shift in parasite metabolism from autotrophy to at least partial heterotrophy, depending on the level of parasite dependence. Species within the family Orobanchaceae span the spectrum of host nutrient dependency, yet the diversity of parasitic plant metabolism remains poorly understood, particularly during the key metabolic shift surrounding haustorial attachment. Comparative profiling of major metabolites in the obligate holoparasite Phelipanche aegyptiaca and the facultative hemiparasite Triphysaria versicolor before and after attachment to the hosts revealed several metabolic shifts implicating remodeling of energy and amino acid metabolism. After attachment, both parasites showed metabolite profiles that were different from their respective hosts. In P. aegyptiaca, prominent changes in metabolite profiles were also associated with transitioning between different tissue types before and after attachment, with aspartate levels increasing significantly after the attachment. Based on the results from 15N labeling experiments, asparagine and/or aspartate-rich proteins were enriched in host-derived nitrogen in T. versicolor. These results point to the importance of aspartate and/or asparagine in the early stages of attachment in these plant parasites and provide a rationale for targeting aspartate-family amino acid biosynthesis for disrupting the growth of parasitic weeds

Development of Alditol Acetate Derivatives for the Determination of 15N-Enriched Amino Sugars by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry

Amino sugars can be used as indices to evaluate the role of soil microorganisms in active nitrogen (N) cycling in soil. This paper details the assessment of the suitability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the analysis of ¹⁵N-enriched amino sugars as alditol acetate derivatives prior to application of a novel ¹⁵N stable isotope probing (SIP) approach to amino sugars. The efficient derivatization and cleanup of alditol acetate derivatives for GC was achieved using commercially available amino sugars, including glucosamine, mannosamine, galactosamine, and muramic acid, as laboratory standards. A VF-23ms stationary phase was found to produce optimal separations of all four compounds. The structure of the alditol acetate derivatives was confirmed using gas chromatography/mass spectrometry (GC/MS). For GC–C–IRMS determinations, implementation of a two-point normalization confirmed the optimal carrier gas flow rate to be 1.7 mL min^–1. Linearity of δ¹⁵N value determinations up to δ¹⁵N_t of 469 ± 3.1‰ (where δ¹⁵N_t is the independently measured δ¹⁵N value) was confirmed when 30 nmol N was injected on-column, with the direction of deviation from δ¹⁵N_t at low sample amount dependent on the ¹⁵N abundance of the analyte. Observed between- and within-run memory effects were significant (<i>P</i> < 0.007) when a highly enriched standard (469 ± 3.1‰) was run; therefore, analytical run order and variation in ¹⁵N enrichment of analytes within the same sample must be considered. The investigated parameters have confirmed the isotopic robustness of alditol acetate derivatives of amino sugars for the GC–C–IRMS analysis of ¹⁵N-enriched amino sugars in terms of linearity over an enrichment range (natural abundance to 469 ± 3.1‰) with on-column analyte amount over 30 nmol N

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS

Post-transcriptional RNA modifications play an important role in cellular metabolism with homoeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukaemia, and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography-mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9-min sample-to-sample run. To validate the performance and versatility of our method, we analysed tRNA modification patterns of representative microorganisms from each domain of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae), and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homoeostasis studies of RNA modification in complex biological samples.Peer reviewe

Sensitive Isotope Analysis of Micropollutants in Complex Sample Matrices

In den letzten Jahrzehnten wurden zunehmend Berichte über die Kontamination von Gewässern durch Spurenschadstoffe veröffentlicht. Dabei stellen persistente und polare Spurenschadstoffe ein besonders großes Risiko dar, da sie in das Grundwasser sickern können und somit die Haupt-Trinkwasserquelle vieler europäischer Länder verunreinigen können. Aus diesem Grund ist es im Interesse der Umweltbehörden und der Forschung das Verhalten dieser Spurenschadstoffe in der Umwelt zu untersuchen. Herkömmliche Methoden zur Einschätzung des Umweltverhaltens basieren auf Konzentrationsmessungen eines Schadstoffes, sowie dessen Abbauprodukt. Wird ein Abbauprodukt allerdings nicht nur gebildet, sondern dieses ebenfalls weiter transformiert, können die Resultate über das Abbauverhalten der Muttersubstanz mehrdeutig sein. Neben dem weiteren Abbau des Metaboliten kann es durch unterschiedliche Mobilitäten des Metaboliten und des Ausgangsstoffes, sowie durch wiederholte Remobilisierungen aus dem Boden zu Fehleinschätzungen bezüglich des Abbaus kommen, was eine Risikobeurteilung erschwert. Eine alternative Herangehensweise zur Identifikation von Abbauprozessen ist die substanzspezifische Stabil-Isotopen-Analytik (compound-specific stable isotope analysis, CSIA). Bei dieser Methode wird die natürliche Verteilung der Isotopenhäufigkeit eines Elements (z.B. Kohlenstoff, Stickstoff) analysiert. Bisher war die substanzspezifische Stabil- Isotopen-Analytik allerdings auf die Analyse von Schadstoffen im unteren μg/l Konzentrationsbereich beschränkt. Daher war ein Ziel dieser Arbeit den Anwendungsbereich der substanzspezifischen Stabil-Isotopen-Analytik zu erweitern, um robuste Analysen für umweltrelevante Konzentrationen zu bewerkstelligen. Realisiert wurde dies durch die Entwicklung analytischer Methoden für polare Spurenschadstoffe in Umweltproben, sowie deren Anwendung auf systematische Feldstudien. Zudem wurden die Limitierungen der substanzspezifischen Stabil-Isotopen-Analytik im untern ng/l Konzentrationsbereich untersucht. Dafür wurden indikativ die häufig detektierten Spurenschadstoffe Desphenylchloridazon (DPC), 2,6-Dichlorbenzamid (BAM), Atrazin (ATZ) und Desethylatrazin (DEA) als Modellsubstanzen verwendet. Im zweiten Kapitel dieser Arbeit wurden Methoden zur Kohlen- und Stickstoffisotopenanalyse (δ13C und δ15N) polarer Spurenschadstoffe am Beispiel von DPC entwickelt. Zur Bestimmung der Kohlenstoffisotopenverhältnisse wurde Flüssigchromatographie mit einem Isotopenmassenspektrometer (LC-IRMS) gekoppelt, während für die Bestimmung der Stickstoffisotopenverhältnisse eine Methode mittels Derivatisierung und Gaschromatographie-Isotopenmassenspektrometrie entwickelt wurde. Beide Methoden zeigten reproduzierbare und akkurate δ13C und δ15N Isotopenwerte mit einer Präzisionsgrenze im μg/l Konzentrationsbereich. Dabei waren 996 ng an DPC auf der GC- Säule (on-column) ausreichend für die Kohlenstoffisotopenanalyse. Für die Stickstoffisotopenanalyse musste das DPC zunächst mit einem 160-fachen Überschuss an Trimethylsilyldiazomethan (TMSD) derivatisiert werden. Dabei wurde eine Präzisionsgrenze von 1200 ng DPC auf der GC-Säule bestimmt. Da Spurenschadstoffe in der Umwelt allerdings in einem geringeren Konzentrationsbereich vorkommen (ng/l bis μg/l), war eine Optimierung dieser Methode hinsichtlich ihrer Sensitivität notwendig. Durch die Probeninjektion direkt auf die GC-Säule (on-column Injektion) statt der bisherigen Splitless-Injektionstechnik, konnte für die Bestimmung der Stickstoffisotopenverhältnisse eine Präzisionsgrenze von 100 ng DPC auf der GC-Säule erreicht werden. Danach wurde die Eignung beider Methoden für die Messung von niedrig konzentrierten Umweltproben geprüft. Dafür wurden die Stickstoffisotopenverhältnisse von DPC in mit DPC kontaminiertem Sickerwasser analysiert. Zusätzlich wurde das Sickerwasser mit Chloridazon (CLZ) versetzt, welches sich nach und nach zu DPC abgebaut hat. Die Analyse der Stickstoffisotopenverhältnisse von DPC zeigte Unterschiede in den Isotopensignaturen, was die Differenzierung zwischen unterschiedlichen Eintragungsquellen des DPCs ermöglicht. Nachdem die Methoden zur CSIA polarer Spurenschadstoffe am Beispiel des DPCs in Kapitel 2 entwickelt worden war, wurde eine systematische Feldstudie zum Umweltverhalten des DPC und seiner Ausgangsverbindung CLZ in Lysimetern durchgeführt. Die in Kapitel 3 beschriebene Studie lieferte neue Erkenntnisse über den Abbau von DPC. Dabei wurde der Aspekt der zeitgleichen Bildung und Transformation des Metaboliten betrachtet. Die Erkenntnisse wurden mithilfe zweier analytischer Ansätze ermittelt: der bereits etablierten Methode basierend auf den Konzentrationsverhältnissen von Metabolit zu Ausgangsstoff, und der seit kurzem verfügbaren Kohlenstoff- und Stickstoffisotopenanalytik. Es zeigte sich, dass: (i) DPC in allen Lysimetern mit einer signifikanten 13C und 15N Anreicherung von bis zu +4 ‰ bzw. +3 ‰ transformiert wurde und (ii) das gebildete DPC, welches noch nicht transformiert worden war, den gleichen Stickstoffisotopenwert wie sein Ausgangsstoff CLZ hatte. Nachdem es allerdings weiter abgebaut wurde, konnte eine signifikante Kohlenstoff- und Stickstoffisotopenfraktionierung beobachtet werden. Das Ausmaß der Isotopenfraktionierung wurde teilweise durch die Remobilisierung von nicht-transformiertem DPC abgeschwächt. II Zusammenfassung Zudem zeigte sich, dass das Ausmaß der Isotopenfraktionierung in Abhängigkeit von der Art der Anwendung des Herbizides und des Metaboliten variierte. Dies impliziert den Einfluss von Pflanzen und der präferentiellen Flüsse auf die Bildung und den Abbau von DPC. Zusätzlich konnte gezeigt werden, dass (iii) bei einer Transformation von DPC die Isotopensignatur als Indikator für den Abbau zuverlässiger war, als das Verhältnis von Metabolit zu Ausgangsverbindung. Daher diente CSIA als Indikator für die DPC- Transformation, vorausgesetzt, es findet keine gleichzeitige Bildung und Transformation von DPC statt. Sobald jedoch die DPC-Bildung dominierte, war der Nachweis des DPC-Abbaus durch CSIA nicht mehr eindeutig, da die Änderungen der Isotopenwerte durch den erneuten Eintrag von DPC verringert wurden. Dabei erreichten die Metabolit-zu-Ausgangsstoff- Verhältnisse ein Maximum und konnten somit den Nachweis für die DPC-Bildung erbringen. Das bedeutet, dass sich beide Methoden ergänzen, insbesondere, wenn nur ein teilweiser Abbau des Herbizids stattfindet, denn während das Metabolit-zu-Ausgangsstoff-Verhältnis Informationen über die Remobilisierung eines Analyten liefert, zeigt CSIA die Entwicklung des Abbaus einer Verbindung. Das vierte Kapitel dieser Arbeit befasst sich mit den Herausforderungen der CSIA polarer und persistenter Spurenschadstoffe im natürlichen System Grundwasser. Zur Identifikation und Bewertung der Herausforderungen wurde Grundwasser mit ATZ, DEA und BAM versetzt und die Modellsubstanzen aus großen Volumina extrahiert. Im Gegensatz zu den vorangegangenen Laborversuchen, bei denen die Analyten in Leitungswasser gelöst waren, führte die Extraktion der Substanzen aus dem Grundwasser zu kleinen und nicht reproduzierbaren Wiederfindungsraten. Als Grund für die unvollständige Wiederfindung der Analyten wird der Einfluss der Grundwassermatrix bei der Extraktion angenommen. So können organische Bestandteile des Grundwassers wie z.B. Humin- und Fulvinsäuren mit den Modellsubstanzen ATZ und DEA sogenannte Analyt-Fulvinsäure-Komplexe bilden. Diese Komplexe werden vor allem unter sauren pH-Bedingungen gebildet und das Einstellen eines niedrigen pH- Wertes war Teil dieser Methode. Neben den unvollständigen Wiederfindungsraten wurde bei der großvolumigen Probenanreicherung (Extraktion von bis zu 100 L pro Probe) eine starke Isotopenfraktionierung beobachtet. Die Fraktionierung entsteht durch den Einfluss der Matrix, welche ebenfalls bei der Festphasenextraktion angereichert wurde. Dabei beeinträchtigt die Isotopensignatur der organischen Bestandteile des Grundwassers die Isotopensignatur des Analyten und kann, falls nicht identifiziert, zu einer Fehleinschätzung in der Quantifizierung des Schadstoffabbaus führen. Die Prüfung der in diesem Kapitel vorgestellten Methode zeigt die Notwendigkeit einer kritischen Begutachtung und Identifikation von Fehlerquellen im Vorfeld von künftigen Methodenentwicklungen mit besonderem Augenmerk auch auf Matrixeffekte. Ziel zukünftiger Studien wird es sein, die in diesem Kapitel identifizierten Limitierungen der substanzspezifischen Stabil-Isotopen-Analytik durch die Weiterentwicklung und Optimierung von Methoden und Analysegeräten zu eliminieren.Reports of the contamination of natural water bodies with micropollutants have increased in the last decades. Most importantly, persistent and polar micropollutants are of major concern as they may leach into groundwater, the main source of drinking water in many countries within the European Union. Consequently, for environmental authorities and researchers, it is important to investigate the environmental fate of such micropollutants. Conventional assessment approaches rely on changes in concentrations of the contaminant and its metabolite. This, however, is often inconclusive as the simultaneous formation and transformation of the metabolite, differences in the mobility between parent compound and metabolite, or repeated mobilization may lead to erroneous interpretations. Compound- specific stable isotope analysis (CSIA) is a complementary approach to identify transformation processes based on the analysis of natural isotope abundances of an element (e.g. carbon, nitrogen). As CSIA has so far been limited to the analysis of pollutants in the sub-μg/L range, this thesis aims to broaden the application of CSIA by developing analytical methods for polar micropollutants in environmental samples, applying these for systematic field studies and testing the limits of CSIA in concentrations in the low ng/L range. To this end, the frequently detected micropollutants desphenylchloridazon (DPC), 2,6- dichlorobenzamide (BAM), atrazine (ATZ) and desethylatrazine (DEA) were used as model compounds. In the second chapter of this thesis, methods for carbon- and nitrogen-isotope analysis (δ13C and δ15N) of polar micropollutants were developed using liquid chromatography-isotope-ratio mass spectrometry (LC-IRMS) and derivatization gas chromatography-IRMS (GC-IRMS). DPC was used as a representative compound for polar contaminants during method development. Both methods resulted in reproducible and accurate δ13C and δ15N analysis of DPC with a limit of precise isotope analysis in the μg/L concentration range. For carbon isotope analysis 996 ng of DPC on-column were sufficient. Nitrogen isotope analysis was achieved by derivatization of DPC with a 160-fold excess of (trimethylsilyl)diazomethane. To enable the application of CSIA to environmental samples, where micropollutants are present in a concentration range of ng/L to sub-μg/L, more sensitive methods were required. Thus, the nitrogen isotope analysis was optimized using on-column injection, which resulted in accurate δ15N analysis for amounts greater than 100 ng DPC on-column. The feasibility of both methods was proven by measuring the isotopic composition of DPC in DPC-containing environmental-seepage water spiked with chloridazon (CLZ). The analysis indicated that it is possible to distinguish DPC containing different isotopic signatures. After the feasibility of CSIA for polar micropollutants such as DPC was shown, a systematic field study of the DPC and its parent compound CLZ was carried out as detailed in Chapter 3. This study gave new insights into DPC degradation pinpointing the influence of simultaneous formation and transformation of the metabolite using two analytical approaches—the well- established metabolite-to-parent compound ratio and the recently available carbon and nitrogen CSIA. We found that (i) DPC was transformed in all lysimeters, showing a significant enrichment in 13C and 15N by approximately +4 ‰ and +3 ‰, respectively. (ii) Formed DPC, which had not been subject to further transformation yet, showed the same nitrogen isotope value as its precursor CLZ. As further transformation took place, significant carbon and nitrogen isotope fractionation was observed that was partially attenuated when mixing with freshly mobilized DPC from the vadose zone took place. The extent of isotope fractionation varied depending on the method of application of the parent herbicide and metabolite, implying the influence of plants, and the preferential flow on the formation and degradation of DPC. Additionally, we demonstrated that (iii) when DPC was further transformed, the isotopic signature, as an integrated signal of DPC degradation, was more reliable as an indicator of degradation than the metabolite-to-parent-compound ratio. Hence, this study enables the application of CSIA as an indication of DPC transformation, provided that there is no simultaneous formation and transformation of DPC. On the other hand, when DPC formation dominated and evidence from CSIA was not conclusive because changes in isotope values were reduced by the fresh input, metabolite-to-parent-ratios reached a maximum and could provide evidence of DPC formation. This leads to the conclusion that both methods are complementary, in particular when only partial degradation of the herbicide is occurring. While the metabolite-to-parent ratio provides information about the re-mobilization of a compound, CSIA shows the evolution of a compound’s degradation. In Chapter 4 of this thesis, challenges in CSIA of polar and persistent micropollutants in groundwater were identified and critically discussed by evaluating a large volume extraction method of up to 100 L groundwater using ATZ, DEA and BAM as model compounds in low ng/L concentration ranges. It was found that, in contrast to previous laboratory experiments, where tap water was used, extracts from environmental groundwater resulted in low and non- reproducible recoveries. Since groundwater contains organic matter such as humic and fulvic acids, and as acidification was part of the extraction procedure, it is assumed that the change in pH prior to solid-phase extraction (SPE) may favor the formation of analyte-fulvic acid complexes leading to the low recoveries observed. In addition to unsatisfactory recoveries, the extensive sample enrichment also resulted in an extensive isotope fractionation as the isotopic signature of the organic matter interfered with the carbon isotope ratio of the target analytes. Such an interference would lead to an overestimation in the quantification of degradation, if unidentified. Thus, it is essential for future analytical method developments to critically evaluate each method and to include the investigation about a possible influence of sample matrix on the analysis already in the pre-tests. As this study has shown the limitations of CSIA of polar micropollutants in complex sample matrices, future studies may use this as a starting point towards more sensitive isotope analysis by methodological and instrumental advances