Analysis of the central carbon metabolism of the unicellular cyanobacterium Synechocystis sp. PCC 6803 in photomixotrophic and heterotrophic growth mode using 13C metabolic flux analysis

Cyanobacteria are a promising host for sustainable production of a wide variety of biotechnological products. One of these specimens is the unicellular cyanobacterium Synechocystis sp. PCC 6803 that has been extensively studied and became a model organism for photosynthesis. Recently two novel pathways have been identified: Gens for two phosphoketolases and the Entner-Doudoroff pathway (ED). Resolution of the core metabolism of Synechocystis sp. PCC 6803 was encumbered by model topology and the unique demands for cultivation techniques. Identification of optimum tracer and analysis setups for generation of a data basis with sufficient resolution power was achieved by in silico experiments and aided by detailed analysis of cultivation methods. All optimization efforts were finalised in complete and precise resolution of the core metabolism of Synechocystis sp. PCC 6803 under photomixotrophic and heterotrophic growth regime. Photomixotrophic growth is dominated by high Calvin-Benson-Basham cycle activity that is boosted with glucose from medium, whereas heterotrophic metabolism is dominated by the oxidative branch of the pentose phosphate pathway. Tricarboxylic acid cycle was providing biomass building blocks in both growth modes. Novel pathways were found inactive during both tested conditions. This work demonstrated the impact of cultivation parameters and experimental setup on physiology.Cyanobakterien sind eine vielversprechende Gruppe von Organismen, die für eine diverse Palette biotechnologischer Produkte genutzt werden könnten. Kürzlich wurden zwei neue Stoffwechselwege identifiziert: Zwei Phosphoketolasen und der Entner-Doudoroff-Weg. Die Auflösung des Zentralstoffwechsels wurde maßgebliche durch die einzigartigen Anforderungen an Kultivierungsmethoden und Modellierung erschwert. Detaillierte Analyse der Kultivierungssystemen und in silico basiertes experimentelles Design hat schlussendlich ermöglicht qualitativ hochwertige Markierungsdaten für metabolischen Steady-State zu generieren. Alle Optimierungs-Anstrengungen erlaubten die detaillierte Auflösung des Zentralstoffwechsel von Synechocystis sp. PCC 6803 unter photomixotrophen und heterotrophen Wachstumsbedingungen. Dabei war photomixotrophischer Metabolismus vom Calvin-Benson- Basham-Zyklus dominiert, in den Glucose aus dem Medium als zusätzliches Substrat eingeflossen ist. Unter heterotrophen Bedingungen war vor allem der oxidative Teil des Pentosephosphatweg aktiv. Unter beiden Bedingungen stellte der Tricarbonsäurezyklus Biomasse-Bausteine bereit. Neue Stoffwechselwege waren inaktiv, unabhängig vom Wachstumsregime. Diese Arbeit demonstriert den Einfluss von Kultivierungsparametern auf die Physiologie von Mikroorganismen.Deutsche Forschungsgemeinschaft: INST 256/418-1, WI 1796/3-1, GU 1522/2-1; Horizon 2020 Research Infrastructure: 730976 (HIGHFLUX); Agence Nationale de la Recherche: MetaboHUB-ANR-11-INBS-001

Worker Perception of Quality Assurance Mechanisms in Crowdsourcing and Human Computation Markets

Many human computation systems utilize crowdsourcing marketplaces to recruit workers. Because of the open nature of these marketplaces, requesters need to use appropriate quality assurance mechanisms to guarantee high quality results. Previous research has mostly focused on the statistical aspects of quality assurance. Instead, we analyze the worker perception of five quality assurance mechanisms (Qualification Test, Qualification Restriction, Gold Standard, Majority Vote, Validating Review) according to subjective (fairness, offense, benefit) and objective (necessity, accuracy, cost) criteria. Based on theory from related areas like labor psychology, we develop a conceptual model and test it with a survey on Mechanical Turk. Our results show big differences in perception, especially with respect to Majority Vote which is rated low by workers. On the basis of these results, we show implications for theory and give requesters on crowdsourcing markets the advice to integrate the worker view when selecting an appropriate quality assurance mechanism

Livestock grazing reduces sediment deposition and accretion rates on a highly anthropogenically altered marsh island in the Wadden Sea

Coastal salt marshes and their provided ecosystem services are threatened by rising sea levels all over the world. In the Northern Wadden Sea region, a sea-level rise of 4 mm y−1 was recorded for recent years. Identifying and understanding factors that affect sediment deposition and determine vertical accretion of salt marshes is crucial for the management of these ecosystems. Even though major processes contributing to sedimentation and accretion have already been identified, the influence of reduced canopy heights due to livestock grazing is still debated. On a highly anthropogenically altered marsh island in the Wadden Sea, sediment deposition, accretion and suspended sediment concentration was analyzed on grazed and adjacent ungrazed plots both at the marsh edge and at the marsh interior. Due to a low seawall (a so-called ‘summer dike’), flooding frequency on the island is reduced and flooding mainly takes place during storm surges. After five flooding events within a year, mean sediment deposition and accretion were found to be up to seven times higher on ungrazed plots compared to grazed plots, but only at the marsh edges. This result was not explained by the overmarsh suspended sediment concentration (SSC), which was found to be twice as high on grazed plots compared to ungrazed plots. It is concluded that grazing has a negative effect on sediment deposition and accretion on Wadden Sea marsh islands and areas with similar conditions (e.g. presence of a summer dike) by reducing the sediment trapping capacity of those marshes. Overall, vertical marsh accretion ranged from 0.11 ± 0.09 mm y−1 on a grazed plot at the marsh edge to 1.12 ± 0.71 mm y−1 on an ungrazed plot at the marsh edge. By increasing the discrepancy between accretion and sea-level rise, livestock grazing can lead to higher inundation levels and in turn to increased hydrodynamic forces acting on these anthropogenically altered marshes

Laboratory Measurements to Image Endobenthos and Bioturbation with a High-Frequency 3D Seismic Lander

The presented 3D seismic system operates three transducers (130 kHz) from a stationary lander and allows non-destructive imaging of small-scale objects within the top decimeters of silty sediments, covering a surface area of 0.2 m2. In laboratory experiments, samples such as shells, stones, and gummy worms of varied sizes (down to approx. 1 cm diameter) could be located in the 3D seismic cube to a depth of more than 20 cm and differentiated by a reflected amplitude intensity and spatial orientation. In addition, simulated bioturbation structures could be imaged. In a practical application, the system allows to determine the abundance of endobenthos and its dynamic in muddy deposits in-situ and thus identify the intensity of local bioturbation

Effects of small-scale patterns of vegetation structure on suspended sediment concentration and sediment deposition in a salt marsh

Salt marshes contribute to coastal protection by attenuating waves and reducing flow velocities. Nevertheless, coastal salt marshes are threatened by rising sea levels. In order to keep pace with rising sea levels, salt marshes need to grow vertically by sediment input. Although major processes contributing to sediment deposition in salt marshes are known, there is still a lack of understanding of the influence of canopy height and biomass on suspended sediment concentration and sediment deposition and on the spatial scale beyond which an influence of vegetation on sediment deposition comes into effect. Furthermore, vegetation can be heterogenous and little is known on the role of small-scale patterns of vegetation structure on suspended sediment concentration and sediment deposition. We investigated the effects of small-scale patterns of vegetation on suspended sediment concentration and sediment deposition in a field experiment with two vegetation types (i.e. Spartina anglica in the low marsh and Elymus athericus in the high marsh). Partial mowing of the vegetation resulted in a pattern of mown subplots and control subplots with a size of 4 m2 in various combinations adjacent to a creek. Based on the results, it can be concluded that on the spatial scale of 4 m2, there is no effect of the vegetation on water flow as the sediment deposition between mown and control subplots did not differ in both the high and the low marsh. Furthermore, a mown or a control subplot next to the creek had no influence on the sediment deposition on a mown or control subplot behind. In summary, based on the results of our study, it can be concluded that the presence of salt marsh vegetation not automatically leads to higher sediment deposition on vegetated patches compared to mown patches in both the low and high marsh

A Parallel Perifusion Slide From Glass for the Functional and Morphological Analysis of Pancreatic Islets.

An islet-on-chip system in the form of a completely transparent microscope slide optically accessible from both sides was developed. It is made from laser-structured borosilicate glass and enables the parallel perifusion of five microchannels, each containing one islet precisely immobilized in a pyramidal well. The islets can be in inserted via separate loading windows above each pyramidal well. This design enables a gentle, fast and targeted insertion of the islets and a reliable retention in the well while at the same time permitting a sufficiently fast exchange of the media. In addition to the measurement of the hormone content in the fractionated efflux, parallel live cell imaging of the islet is possible. By programmable movement of the microscopic stage imaging of five wells can be performed. The current chip design ensures sufficient time resolution to characterize typical parameters of stimulus-secretion coupling. This was demonstrated by measuring the reaction of the islets to stimulation by glucose and potassium depolarization. After the perifusion experiment islets can be removed for further analysis. The live-dead assay of the removed islets confirmed that the process of insertion and removal was not detrimental to islet structure and viability. In conclusion, the present islet-on-chip design permits the practical implementation of parallel perifusion experiments on a single and easy to load glass slide. For each immobilized islet the correlation between secretion, signal transduction and morphology is possible. The slide concept allows the scale-up to even higher degrees of parallelization

Impact of Sparse Benthic Life on Seafloor Roughness and High-Frequency Acoustic Scatter

Quantitative acoustic marine habitat mapping needs to consider the impact of macrobenthic organisms on backscatter data. However, the sensitivity of hydroacoustic systems to epibenthic life is poorly constrained. This study explores the impact of a benthic community with sparse abundance on seafloor microroughness and acoustic backscatter at a sandy seafloor in the German North Sea. A multibeam echo sounder survey was ground-truthed by lander measurements combining a laser line scanner with sub-mm resolution and broad-band acoustic transducers. Biotic and abiotic features and spatial roughness parameters were determined by the laser line scanner. At the same locations, acoustic backscatter was measured and compared with an acoustic scatter model utilizing the small-roughness perturbation approximation. Results of the lander experiments show that a coverage with epibenthic features of 1.6% increases seafloor roughness at spatial wavelengths between 0.005–0.03 m, increasing both spectral slope and intercept. Despite the fact that a strong impact on backscatter was predicted by the acoustic model based on measured roughness parameters, only a minor (1.1 dB) change of backscatter was actually observed during both the lander experiments and the ship-based acoustic survey. The results of this study indicate that benthic coverage of less than 1.6% is insufficient to be detected by current acoustic remote sensing

Glycolytic Shunts Replenish the Calvin-Benson-Bassham Cycle as Anaplerotic Reactions in Cyanobacteria

The recent discovery of the Entner-Doudoroff (ED) pathway as a third glycolytic route beside Embden-Meyerhof-Parnas (EMP) and oxidative pentose phosphate (OPP) pathway in oxygenic photoautotrophs requires a revision of their central carbohydrate metabolism. In this study, unexpectedly, we observed that deletion of the ED pathway alone, and even more pronounced in combination with other glycolytic routes, diminished photoautotrophic growth in continuous light in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, we found that the ED pathway is required for optimal glycogen catabolism in parallel to an operating Calvin–Benson–Bassham (CBB) cycle. It is counter-intuitive that glycolytic routes, which are a reverse to the CBB cycle and do not provide any additional biosynthetic intermediates, are important under photoautotrophic conditions. However, observations on the ability to reactivate an arrested CBB cycle revealed that they form glycolytic shunts that tap the cellular carbohydrate reservoir to replenish the cycle. Taken together, our results suggest that the classical view of the CBB cycle as an autocatalytic, completely autonomous cycle that exclusively relies on its own enzymes and CO2 fixation to regenerate ribulose-1,5-bisphosphate for Rubisco is an oversimplification. We propose that in common with other known autocatalytic cycles, the CBB cycle likewise relies on anaplerotic reactions to compensate for the depletion of intermediates, particularly in transition states and under fluctuating light conditions that are common in nature

Seasonal and spatial within-marsh differences of biophysical plant properties: implications for wave attenuation capacity of salt marshes

Salt marshes attenuate waves and thus have an important function for coastal protection. Biophysical properties of salt-marsh plants play a key role in the process of wave attenuation and can be differentiated by morphological properties such as stem density, vegetation height and aboveground biomass as well as by biomechanical properties related to stem flexibility. Numerical or physical scale models predicting wave attenuation over vegetated surfaces need to include biophysical properties. However, few studies have quantified morphological and biomechanical properties of salt-marsh plants and fewer have considered seasonal and within-marsh spatial variability of biomechanical properties. The aim of this study was to quantify biophysical properties of the common salt-marsh grasses Spartina anglica and Elymus athericus, including stem flexibility and density as well as aboveground biomass, temporally and spatially. Samples were collected in spring and in summer 2014 at a study site located in the Northern German Wadden Sea. Aboveground biomass was harvested in plots of 50 × 50 cm, stem density was determined by counting and flexibility of plant stems was determined with three-point bending tests. Biophysical properties of both species varied significantly between seasons with plant stem stiffness being 5.0 (S. anglica) and 2.9 times (E. athericus) higher and aboveground biomass being 2.1 (S. anglica) and 1.3 times (E. athericus) higher in summer than in spring. Small-scale spatial differences for those biophysical plant properties were found for S. anglica with plant stem stiffness being 4.0 (spring) and 2.8 times (summer) higher and aboveground biomass being 1.6 (spring) and 1.5 times (summer) higher in a landward than in a seaward-located zone. Small-scale spatial differences of biophysical properties were not found in E. athericus. We conclude that variability in biophysical properties should be considered in models and experiments especially for S. anglica when predicting and quantifying marsh wave attenuation capacity

Electron dynamics in planar radio frequency magnetron plasmas: I. The mechanism of Hall heating and the {\mu}-mode

The electron dynamics and the mechanisms of power absorption in radio-frequency (RF) driven, magnetically enhanced capacitively coupled plasmas (MECCPs) at low pressure are investigated. The device in focus is a geometrically asymmetric cylindrical magnetron with a radially nonuniform magnetic field in axial direction and an electric field in radial direction. The dynamics is studied analytically using the cold plasma model and a single-particle formalism, and numerically with the inhouse energy and charge conserving particle-in-cell/Monte Carlo collisions code ECCOPIC1S-M. It is found that the dynamics differs significantly from that of an unmagnetized reference discharge. In the magnetized region in front of the powered electrode, an enhanced electric field arises during sheath expansion and a reversed electric field during sheath collapse. Both fields are needed to ensure discharge sustaining electron transport against the confining effect of the magnetic field. The corresponding azimuthal ExB-drift can accelerate electrons into the inelastic energy range which gives rise to a new mechanism of RF power dissipation. It is related to the Hall current and is different in nature from Ohmic heating, as which it has been classified in previous literature. The new heating is expected to be dominant in many magnetized capacitively coupled discharges. It is proposed to term it the "{\mu}-mode" to separate it from other heating modes