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

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

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

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

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

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

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

An unanticipated prolonged baseline ACT during cardiac surgery due to factor XII deficiency

Factor XII (FXII) deficiency is a congenital disorder inherited as an autosomal recessive condition. In his heterozygous form, it is relatively common in the general population. However, a total absence of FXII as seen in homozygous patients, is rare, with an incidence of approximately 1/1,000,000 individuals. Surprisingly, FXII deficiency is rather associated with thromboembolic complications. Patients do not experience a higher risk of surgical bleeding despite a markedly prolonged activated partial thromboplastin time. Given its low incidence in the general population, the finding of an unknown FXII deficiency is rare during cardiac surgery. This unique case describes a patient with an unanticipated prolonged baseline activated clotting time (ACT) during cardiac surgery in which his bleeding history and rotational thromboelastometry tracings lead us to the diagnosis of a FXII deficiency. The finding of a hypocoagulable INTEM tracing and a concurrent normal EXTEM tracing in a sample of a patient with prolonged ACT and adverse anamnestic bleeding history should prompt clinicians to consider a FXII deficiency. It may help clinicians in further perioperative management where there is not enough time to wait for the results of individual coagulation factor testing