Asymmetrically substituted 5,5 `-bistriazoles - nitrogen-rich materials with various energetic functionalities

In this contribution the synthesis and full structural and spectroscopic characterization of three asymmetrically substituted bis-1,2,4-triazoles, along with different energetic moieties like amino, nitro, nitrimino and azido moieties, is presented. Additionally, selected nitrogen-rich ionic derivatives have been prepared and characterized. This comparative study on the influence of these energetic moieties on structural and energetic properties constitutes a complete characterization including IR, Raman and multinuclear NMR spectroscopy. Single crystal X-ray crystallographic measurements were performed and provide insight into structural characteristics as well as inter-and intramolecular interactions. The standard enthalpies of formation were calculated for all compounds at the CBS-4M level of theory, revealing highly positive heats of formation for all compounds. The detonation parameters were calculated using the EXPLO5 program and compared to the common secondary explosive RDX as well as recently published symmetric bistriazoles. As expected, the measured sensitivities to mechanical stimuli and decomposition temperatures strongly depend on the energetic moiety of the triazole ring. All compounds were characterized in terms of sensitivities (impact, friction, electrostatic) and thermal stabilities, the ionic derivatives were found to be thermally stable, insensitive compounds

Turnover of soil monosaccharides: Recycling versus Stabilization

Soil organic matter (SOM) represents a mixture of differently degradable compounds. Each of these compounds are characterised by different dynamics due to different chemical recalcitrance, transformation or stabilisation processes in soil. Carbohydrates represent one of these compounds and contribute up to 25 % to the soil organic matter. Vascular plants are the main source of pentose sugars (Arabinose and Xylose), whereas hexoses (Galactose and Mannose) are primarily produced by microorganisms. Several studies suggest that the mean turnover times of the carbon in soil sugars are similar to the turnover dynamics of the bulk carbon in soil. The aim of the study is to characterise the influence of stabilisation and turnover of soil carbohydrates. Soil samples are collected from (i)a continuous maize cropping experiment (“Höhere Landbauschule” Rotthalmünster, Bavaria) established 1979 on a Stagnic Luvisol and (ii) from a continuous wheat cropping, established 1969, as reference site. The effect of stabilisation is estimated by the comparison of turnover times of microbial and plant derived soil carbohydrates. As the dynamics of plant derived carbohydrate are solely influenced by stabilisation processes, whereas the dynamics of microbial derived carbohydrates are affected by recycling of organic carbon compounds derived from C3 plant substrate as well as stabilisation processes. The compound specific isotopic analysis (CSIA) of soil carbohydrates was performed using a HPLC/o/IRMS system. The chromatographic and mass spectrometric subunits were coupled with a LC–Isolink interface. Soil sugars were extracted after mild hydrolysis using 4 M trifluoroacetic acid (TFA)

Effects of climate and land use on carbon and nutrients cycles control soil organic matter pools at Mount Kilimanjaro

Ecosystem functions of tropical mountain ecosystems and their ability to provide ecosystem services are particularly threatened by the combined impact of climate and land-use change. Soils, as the linkage between abiotic and biotic components of an ecosystem, are strongly affected by these changes. To understand impacts of climate and land use changes on biodiversity and accompanying ecosystem stability and services at Mt. Kilimanjaro, detailed understanding and description of the current biotic and abiotic controls on ecosystem Carbon (C) and nutrient fluxes are needed. Therefore, we quantitatively described cycles of C and major nutrients (N, P, K, Ca, Mg, Mn, Na, S) on pedon and stand level scale along a 3500 m elevation gradient and in up to three stages of land-use intensification. Qualitative indicators (composition of soil organic matter and microbial communities) were used to relate pool changes to underlying processes. Annual pattern of litterfall and decomposition were closely related to rainfall seasonality and temperature. Several factors, such as decomposition rate, C & N contents, microbial biomass (MBC) and leaf litter quality, increased at mid elevation. This was reflected in shifts of soil organic matter composition and microbial communities controlling soil C stability. Land-use intensification led to 40-80% losses in topsoil C and MBC contents as well as an increased turnover through higher microbial demand for new C sources. In ecosystems with strong seasonal variations (savanna and alpine helichrysum cushion) the effectiveness of C storage and N turnover was strongly affected by spatial vegetation heterogeneity. Ecosystems at mid elevation (~2000 m) represent the interception zone of optimal moisture and temperature conditions. High inputs and fast turnover control the C sequestration in these ecosystems, while climatic restrains on input and decomposition limit the C turnover in soils at lower and higher elevation. Land-use intensification increases C and nutrient cycling, decreases stabilization from new C inputs through increased microbial C demand and thus decreases soil C storage

Biochemistry of hexose and pentose transformations in soil analyzed by position-specific labeling and 13C-PLFA

© 2014 Elsevier Ltd. Microbial transformations are key processes of soil organic matter (SOM) formation, stabilization and decomposition. Combination of position-specific 13C labeling with compound-specific 13C-PLFA analysis is a novel tool to trace metabolic pathways. This combination was used to analyze short-term transformations (3 and 10 days after tracer application) of two key monosaccharides: glucose and ribose in soil under field conditions. Transformations of sugars were quantified by the incorporation of 13C from individual molecule positions in bulk soil, microbial biomass (by CFE) and in cell membranes of microbial groups classified by 13C-PLFA.The 13C incorporation in the Gram negative bacteria was higher by one order of magnitude compared to all other microbial groups. All of the 13C recovered in soil on day 3 was allocated in microbial biomass. On day 10 however, a part of the 13C was recovered in non-extractable microbial cell components or microbial excretions. As sugars are not absorbed by mineral particles due to a lack of charged functional groups, their quick mineralization from soil solution is generally expected. However, microorganisms transformed sugars to metabolites with a slower turnover. The 13C incorporation from the individual glucose positions into soil and microbial biomass showed that the two main glucose utilizing pathways in organisms - glycolysis and the pentose phosphate pathway - exist in soils in parallel. However, the pattern of 13C incorporation from individual glucose positions into PLFAs showed intensive recycling of the added 13C via gluconeogenesis and a mixing of both glucose utilizing pathways. The pattern of position-specific incorporation of ribose C also shows initial utilization in the pentose phosphate pathway but is overprinted on day 10, again due to intensive recycling and mixing. This shows that glucose and ribose - as ubiquitous substrates - are used in various metabolic pathways and their C is intensively recycled in microbial biomass.Analyzing the fate of individual C atoms by position-specific labeling deeply improves our understanding of the pathways of microbial utilization of sugars (and other compounds) by microbial groups and so, of soil C fluxes

Turnover of microbial groups and cell components in soil: ¹³C analysis of cellular biomarkers

© 2017 The Author(s).Microorganisms regulate the carbon (C) cycle in soil, controlling the utilization and recycling of organic substances. To reveal the contribution of particular microbial groups to C utilization and turnover within the microbial cells, the fate of 13C-labelled glucose was studied under field conditions. Glucose-derived 13C was traced in cytosol, amino sugars and phospholipid fatty acid (PLFA) pools at intervals of 3, 10 and 50 days after glucose addition into the soil. 13C enrichment in PLFAs (∼1.5% of PLFA C at day 3) was an order of magnitude greater than in cytosol, showing the importance of cell membranes for initial C utilization. The 13C enrichment in amino sugars of living microorganisms at day 3 accounted for 0.57% of total C pool; as a result, we infer that the replacement of C in cell wall components is 3 times slower than that of cell membranes. The C turnover time in the cytosol (150 days) was 3 times longer than in PLFAs (47 days). Consequently, even though the cytosol pool has the fastest processing rates compared to other cellular compartments, intensive recycling of components here leads to a long C turnover time. Both PLFA and amino-sugar profiles indicated that bacteria dominated in glucose utilization. 13C enrichment decreased with time for bacterial cell membrane components, but it remained constant or even increased for filamentous microorganisms. 13C enrichment of muramic acid was the 3.5 times greater than for galactosamine, showing a more rapid turnover of bacterial cell wall components compared to fungal. Thus, bacteria utilize a greater proportion of low-molecular-weight organic substances, whereas filamentous microorganisms are responsible for further C transformations. Thus, tracing 13C in cellular compounds with contrasting turnover rates elucidated the role of microbial groups and their cellular compartments in C utilization and recycling in soil. The results also reflect that microbial C turnover is not restricted to the death or growth of new cells. Indeed, even within living cells, highly polymeric cell compounds are constantly replaced and renewed. This is especially important for assessing C fluxes in soil and the contribution of C from microbial residues to soil organic matter

Fate of low molecular weight organic substances in an arable soil: From microbial uptake to utilisation and stabilisation

Microbial uptake and utilisation are the main transformation pathways of low molecular weight organic substances (LMWOS) in soil, but details on transformations are strongly limited. As various LMWOS classes enter biochemical cycles at different steps, we hypothesize that the percentage of their carbon (C) incorporation into microbial biomass and consequently stabilisation in soil are different. Representatives of the three main groups of LMWOS: amino acids (alanine, glutamate), sugars (glucose, ribose) and carboxylic acids (acetate, palmitate) - were applied at naturally-occurring concentrations into a loamy arable Luvisol in a field experiment. Incorporation of 13C from these LMWOS into extractable microbial biomass (EMB) and into phospholipid fatty acids (PLFAs) was investigated 3d and 10d after application. The microbial utilisation of LMWOS for cell membrane construction was estimated by replacement of PLFA-C with 13C.35-80% of initially applied LMWOS-13C was still present in the composition of soil organic matter after 10 days of experiment, with 10-24% of 13C incorporation into EMB at day three and 1-15% at day 10. Maximal incorporation of 13C into EMB was observed from sugars and the least from amino acids. Strong differences in microbial utilisation between LMWOS were observed mainly at day 10. Thus, despite similar initial rapid uptake by microorganisms, further metabolism within microbial cells accounts for the specific fate of C from various LMWOS in soils.13C from each LMWOS was incorporated into each PLFA. This reflects the ubiquitous utilisation of all LMWOS by all functional microbial groups. The preferential incorporation of palmitate into PLFAs reflects its role as a direct precursor for fatty acids. Higher 13C incorporation from alanine and glucose into specific PLFAs compared to glutamate, ribose and acetate reflects the preferential use of glycolysis-derived substances in the fatty acids synthesis.Gram-negative bacteria (16:1ω7c and 18:1ω7c) were the most abundant and active in LMWOS utilisation. Their high activity corresponds to a high demand for anabolic products, e.g. to dominance of pentose-phosphate pathway, i.e. incorporation of ribose-C into PLFAs. The 13C incorporation from sugars and amino acids into filamentous microorganisms was lower than into all prokaryotic groups. However, for carboxylic acids, the incorporation was in the same range (0.1-0.2% of the applied carboxylic acid 13C) as that of gram-positive bacteria. This may reflect the dominance of fungi and other filamentous microorganisms for utilisation of acidic and complex organics.Thus, we showed that despite similar initial uptake, C from individual LMWOS follows deviating metabolic pathways which accounts for the individual fate of LMWOS-C over 10 days. Consequently, stabilisation of C in soil is mainly connected with its incorporation into microbial compounds of various stability and not with its initial microbial uptake. © 2014 Elsevier Ltd

Detection of the DCC gene product in normal and malignant colorectal tissues and its relation to a codon 201 mutation.

Protein expression of the putative tumour-suppressor gene DCC on chromosome 18q was evaluated in a panel of 16 matched colorectal cancer and normal colonic tissue samples together with DCC mRNA expression and allelic deletions (loss of heterozygosity, LOH). Determined by a polymerase chain reaction (PCR)-LOH assay, 12 of the 16 (75%) cases were informative with LOH occurring in 2 of the 12 cases. For DCC mRNA, transcripts could be detected in all analysed normal tissues (eight out of eight) by RT-PCR, whereas 6 of the 15 tumours were negative. DCC protein expression, investigated by immunohistochemistry using the monoclonal antibody 15041 A directed against the intracellular domain, was homogeneously positive in all normal tissue samples. In tumour tissues, no DCC protein was seen in 11 out of 16 samples (69%). For the DCC codon 201, we found a loss of a wild-type codon sequence caused by mutation or LOH in at least 8 out of 15 cases (53%) compared with the corresponding normal tissue. DCC protein expression was undetectable in eight of the nine tumours missing both wild-type codons. Only one of the five tumours with retained DCC protein expression had no detectable wild-type codon 201. In addition, 9 out of 15 normal tissue specimens were mutated in codon 201. In two out of three cases with homozygous wild-type codons in peripheral blood lymphocyte (PBL) DNA, mutations were already observed in the tumour adjacent normal colonic mucosa. We conclude that DCC immunostaining should be introduced in the clinicopathological routine because of its strong correlation with the known prognostic markers 18q LOH and mutation of codon 201

Wie beeinflusst Sorption die Metabolisierung von Alanin durch mikrobielle Gruppen?

Mikrobieller Abbau ist ein wesentlicher Transformationsprozess der organischen Bodensubstanz. Sorption von niedermolekularen organischen Substanzen wie Alanin an Mineraloberflächen kann diese vor dem mikrobiellen Abbau und Mineralisierung schützen. Somit wäre Sorption ein entscheidender Stabilisierungsprozess für die organische Bodensubstanz. Dennoch ist es für Mikroorganismen möglich, niedermolekulare organische Substanzen von Mineraloberflächen zu desorbieren. Das exakte Prozessgefüge dieser Desorption und mikrobiellen Verwertung, aber auch die involvierten mikrobiellen Gruppen sind jedoch weitestgehend unbekannt. Wir verwendeten positionsspezifisch C-13-markiertes Alanin, dessen Mineralisation zu CO2 und dessen Einbau in Phospholipid-Fettsäuren (PLFAs) quantifiziert wurde, um die mikrobielle Verwendung von sorbiertem und nicht-sorbiertem Alanin im Boden zu unterscheiden. Um die durch die Sorption bedingten Veränderungen der Verfügbarkeit und Metabolisierung des Alanins zu erfassen, wurden einheitlich- und positionsspezifisch C-13- und C-14-markiertes Alanin in einem Ap-Horizont einer lehmigen Parabraunerde über 10 Tage inkubiert. Das CO2 aus der Respiration wurde in NaOH-Fallen erfasst und dessen C-14-Aktivität bestimmt. Die Verwendung der funktionellen Gruppen des Alanins durch die verschiedenen mikrobiellen Gruppen wurde mittels C-13-PLFA Analyse ermittelt. Keine der mikrobiellen Gruppen bevorzugte das unsorbierte Alanin gegenüber dem sorbierten – der Großteil der Gruppen inkorporierte die gleiche Menge in ihre PLFAs. So zeigten beispielsweise Gram negative Bakterien eine hohe Wettbewerbsfähigkeit hinsichtlich des Alanins, allerdings keine Präferenz bezüglich des Unsorbierten. Nur die Gruppe der Pilze inkorporierten signifikant mehr sorbiertes Alanin: Sie sind prädestiniert dafür, Mineraloberfläche zu umwachsen und daran sorbierte Substanzen mit ihren Hyphen aufzunehmen. Diese Präferenz ist mit einem veränderten Abbauweg, der über Glukoneogenese und den Pentose-Phosphat-Weg führt, verbunden. Sorption kann zu einer kurzfristigen Verzögerung des Abbaus kleiner geladener Moleküle führen, jedoch ist kein langfristiger Stabilisierungseffekt erkennbar. Die meisten mikrobiellen Gruppen nehmen sowohl sorbiertes und nicht-sorbiertes Alanin im gleichen Maße auf und nur einzelne Gruppen, wie Pilze, haben sich auf die präferentielle Aufnahme sorbierter Substanzen spezialisiert

Microbial metabolism in soil at subzero temperatures: Adaptation mechanisms revealed by position-specific ¹³C labeling

© 2017 Bore, Apostel, Halicki, Kuzyakov and Dippold.Although biogeochemical models designed to simulate carbon (C) and nitrogen (N) dynamics in high-latitude ecosystems incorporate extracellular parameters, molecular and biochemical adaptations of microorganisms to freezing remain unclear. This knowledge gap hampers estimations of the C balance and ecosystem feedback in high-latitude regions. To analyze microbial metabolism at subzero temperatures, soils were incubated with isotopomers of position-specifically 13C-labeled glucose at three temperatures: +5 (control), -5, and -20°C. 13C was quantified in CO2, bulk soil, microbial biomass, and dissolved organic carbon (DOC) after 1, 3, and 10 days and also after 30 days for samples at -20°C. Compared to +5°C, CO2 decreased 3- and 10-fold at -5 and -20°C, respectively. High 13C recovery in CO2 from the C-1 position indicates dominance of the pentose phosphate pathway at +5°C. In contrast, increased oxidation of the C-4 position at subzero temperatures implies a switch to glycolysis. A threefold higher 13C recovery in microbial biomass at -5 than +5°C points to synthesis of intracellular compounds such as glycerol and ethanol in response to freezing. Less than 0.4% of 13C was recovered in DOC after 1 day, demonstrating complete glucose uptake by microorganisms even at -20°C. Consequently, we attribute the fivefold higher extracellular 13C in soil than in microbial biomass to secreted antifreeze compounds. This suggests that with decreasing temperature, intracellular antifreeze protection is complemented by extracellular mechanisms to avoid cellular damage by crystallizing water. The knowledge of sustained metabolism at subzero temperatures will not only be useful for modeling global C dynamics in ecosystems with periodically or permanently frozen soils, but will also be important in understanding and controlling the adaptive mechanisms of food spoilage organisms

Die unterschätzte Rolle des Recyclings intakter Metabolite für die Umsatzraten der organischen Bodensubstanz

Obwohl Sorption an Mineraloberflächen als dominierender Prozess zur Erklärung des langsamen Umsatzes mineralassoziierter organischer Bodensubstanz (OBS) dient, widerspricht diese Idee einer zunehmenden Zahl an Inkubationsstudien, die zeigen, dass für niedermolekulare Substanzen nicht nur die mikrobielle Aufnahme kompetitivier als die Sorption ist, sondern auch sorbierte Substanzen in hohem Maße desorbiert und mikrobiell verwertet werden können. Dabei wurde gezeigt, dass sich die Verstoffwechselung desorbierter Substanzen zugunsten eines erhöhten Recyclings verschiebt. Dies wirft die Frage auf, ob Recycling von intakten Metaboliten, d.h. unter Erhalt des Kohlenstoffgerüstes, generell ein bisher stark unterschätzter Prozess ist, der die relativ hohen 14C Alter der OBS teilweise erklären kann. Nach Applikation hoher Toxindosen konnte nachgewiesen werden, dass die nachfolgende Reetablierung der mikrobiellen Gemeinschaft zu großem Anteil auf Recycling der Nekromasskomponenten der Vorgeneration basiert. Intaktes Metabolitrecycling ist jedoch unter steady-state Bedingungen nur äußerst schwierig von der direkten Stabiliserung zu unterscheiden. Um diesen Prozess im Fließgleichgewicht nachzuweisen muss 1) ein Biomolekül untersucht werden, dessen Biosyntheseweg so aufwändig ist, dass Recycling einen deutlichen Vorteil für die Zelle im Vergleich zur Neusynthese darstellt, 2) dieses Biomolkül in lebenden Zellen in einer anderen Form gebunden sein, als die zu recycelnde Einheit in der Bodenlösung, so dass beide Zustandsformen unterschieden werden können und 3) dieses Biomolekül positionsspezifisch isotopenmarkiert zugegeben werden, so dass über einen identischen Einbau der Positionen die Intaktheit des Kohlenstoffgerüstes nachgewiesen werden kann. Am Beispiel der Alkylketten von Fettsäuren, die in mikrobiellen Zellen primär als Phospholipide in den Membranen gebunden sind, soll dieses Prinzip veranschaulicht werden. Eine erste Abschätzung des intakten Recyclings von Alkylketten durch Mikroorganismen in Böden zeigt, dass von den 0.03% der basierend auf Alkyl-Kohlenstoff neugebildeten PLFA mehr als 75% aus intaktem Recycling dieser Ketten hervor gingen. Obwohl der Beitrag des Recyclings intakter Metabolite zur Umsatzzeit der gesamten OBS aufgrund der geringen Anzahl bisher untersuchter Metabolite noch nicht final quantifiziert werden kann, untermauern die hier vorgestellten Ergebnisse jedoch die hohe Relevanz dieses Prozesses für die Dynamik der OBS