Ligand cross-feeding resolves bacterial vitamin B12 auxotrophies.

Cobalamin (vitamin B12, herein referred to as B12) is an essential cofactor for most marine prokaryotes and eukaryotes1,2. Synthesized by a limited number of prokaryotes, its scarcity affects microbial interactions and community dynamics2-4. Here we show that two bacterial B12 auxotrophs can salvage different B12 building blocks and cooperate to synthesize B12. A Colwellia sp. synthesizes and releases the activated lower ligand α-ribazole, which is used by another B12 auxotroph, a Roseovarius sp., to produce the corrin ring and synthesize B12. Release of B12 by Roseovarius sp. happens only in co-culture with Colwellia sp. and only coincidently with the induction of a prophage encoded in Roseovarius sp. Subsequent growth of Colwellia sp. in these conditions may be due to the provision of B12 by lysed cells of Roseovarius sp. Further evidence is required to support a causative role for prophage induction in the release of B12. These complex microbial interactions of ligand cross-feeding and joint B12 biosynthesis seem to be widespread in marine pelagic ecosystems. In the western and northern tropical Atlantic Ocean, bacteria predicted to be capable of salvaging cobinamide and synthesizing only the activated lower ligand outnumber B12 producers. These findings add new players to our understanding of B12 supply to auxotrophic microorganisms in the ocean and possibly in other ecosystems.

A-Si/SiO2 nanolaminates for tuning the complex refractive index and band gap in optical interference coatings.

A-S i/S i O 2 nanolaminates are deposited by magnetron sputtering and show a decreasing absorption when the a-Si single-layer thickness is reduced from 2.4n m to 0.7n m. Moreover, an increase of the Tauc band gap by 0.18e V is measured. Experimental Tauc band gaps are compared to calculated effective band gaps, utilizing a numerical Schrödinger solver. Further, it is demonstrated that the refractive index can be controlled by adjusting the a-Si and S i O 2 single-layer thicknesses in the nanolaminates. The nanolaminates are optically characterized by spectroscopic ellipsometry, transmittance, and reflectance measurements. Additionally, TEM images reveal uniform, well-separated layers, and EDX measurements show the silicon and oxygen distribution in the nanolaminates.

Improving optical thickness monitoring by including systematic and process-influenced transmittance deviations.

During optical monitoring using broadband transmittance measurement, the accuracy depends on how both the substrate and the optical path are aligned. We present a correction procedure to improve the accuracy of the monitoring, even if the substrate has features such as absorption or if there is misalignment of the optical path. The substrate in this case can either be a test glass or a product. The algorithm is proven by experimental coatings which were produced with and without the correction. Additionally, the optical monitoring system was also used to perform an in situ quality check. The system allows a detailed spectral analysis of all substrates with a high position resolution. Both plasma and the temperature effects on the central wavelength of a filter are identified. This knowledge enables the optimization of the following runs.

On the material dependency of peri-implant morphology and stability in healing bone.

The microstructural architecture of remodeled bone in the peri-implant region of screw implants plays a vital role in the distribution of strain energy and implant stability. We present a study in which screw implants made from titanium, polyetheretherketone and biodegradable magnesium-gadolinium alloys were implanted into rat tibia and subjected to a push-out test four, eight and twelve weeks after implantation. Screws were 4 mm in length and with an M2 thread. The loading experiment was accompanied by simultaneous three-dimensional imaging using synchrotron-radiation microcomputed tomography at 5 µm resolution. Bone deformation and strains were tracked by applying optical flow-based digital volume correlation to the recorded image sequences. Implant stabilities measured for screws of biodegradable alloys were comparable to pins whereas non-degradable biomaterials experienced additional mechanical stabilization. Peri-implant bone morphology and strain transfer from the loaded implant site depended heavily on the biomaterial utilized. Titanium implants stimulated rapid callus formation displaying a consistent monomodal strain profile whereas the bone volume fraction in the vicinity of magnesium-gadolinium alloys exhibited a minimum close to the interface of the implant and less ordered strain transfer. Correlations in our data suggest that implant stability benefits from disparate bone morphological properties depending on the biomaterial utilized. This leaves the choice of biomaterial as situational depending on local tissue properties.

Vitamin B12 is not shared by all marine prototrophic bacteria with their environment.

Vitamin B12 (cobalamin, herein B12) is an essential cofactor involved in amino acid synthesis and carbon resupply to the TCA cycle for most prokaryotes, eukaryotic microorganisms, and animals. Despite being required by most, B12 is produced by only a minor fraction of prokaryotes and therefore leads to complex interaction between prototrophs and auxotrophs. However, it is unknown how B12 is provided by prototrophs to auxotrophs. In this study, 33 B12 prototrophic alphaproteobacterial strains were grown in co-culture with Thalassiosira pseudonana, a B12 auxotrophic diatom, to determine the bacterial ability to support the growth of the diatom by sharing B12. Among these strains, 18 were identified to share B12 with the diatom, while nine were identified to retain B12 and not support growth of the diatom. The other bacteria either shared B12 with the diatom only with the addition of substrate or inhibited the growth of the diatom. Extracellular B12 measurements of B12-provider and B12-retainer strains confirmed that the cofactor could only be detected in the environment of the tested B12-provider strains. Intracellular B12 was measured by LC-MS and showed that the concentrations of the different B12-provider as well as B12-retainer strains differed substantially. Although B12 is essential for the vast majority of microorganisms, mechanisms that export this essential cofactor are still unknown. Our results suggest that a large proportion of bacteria that can synthesise B12 de novo cannot share the cofactor with their environment.

A Novel Coenzyme A Analogue in the Anaerobic, Sulfate-Reducing, Marine Bacterium Desulfobacula toluolica Tol2T.

Coenzyme A (CoA) thioesters are formed during anabolic and catabolic reactions in every organism. Degradation pathways of growth-supporting substrates in bacteria can be predicted by differential proteogenomic studies. Direct detection of proposed metabolites such as CoA thioesters by high-performance liquid chromatography coupled with high-resolution mass spectrometry can confirm the reaction sequence and demonstrate the activity of these degradation pathways. In the metabolomes of the anaerobic sulfate-reducing bacterium Desulfobacula toluolica Tol2T grown with different substrates various CoA thioesters, derived from amino acid, fatty acid or alcohol metabolism, have been detected. Additionally, the cell extracts of this bacterium revealed a number of CoA analogues with molecular masses increased by 1 dalton. By comparing the chromatographic and mass spectrometric properties of synthetic reference standards with those of compounds detected in cell extracts of D. toluolica Tol2T and by performing co-injection experiments, these analogues were identified as inosino-CoAs. These CoA thioesters contain inosine instead of adenosine as the nucleoside. To the best of our knowledge, this finding represents the first detection of naturally occurring inosino-CoA analogues.

Simultaneous quantification of all B vitamins and selected biosynthetic precursors in seawater and bacteria by means of different mass spectrometric approaches.

B vitamins have high microbiological relevance in the marine environment, but their very low concentrations and the chemical heterogeneity of the individual vitamins make their analysis challenging. Mass spectrometric analysis of B vitamins in environmental samples at trace levels has mainly been performed using triple quadrupole mass spectrometers operated in targeted analysis mode. The development of such a method can be laborious and error prone. Additionally, high-resolution mass spectrometers can be used to measure a sample in full scan mode and subsequently search the total ion current chromatogram for extracted ion chromatograms of targeted vitamins. Three different analytical approaches for trace analysis of all B vitamins and some of their biosynthetic precursors were optimized and compared on two different mass spectrometers. A triple quadrupole mass spectrometer in selected reaction monitoring mode, and a high-resolution orbitrap mass spectrometer in parallel reaction monitoring, as well as in full scan mode were employed. Detection limits down to 10 ng/L were achieved with all three techniques. The methods were applied to a marine water sample from the North Sea and to the cell extract of a bacterial culture of Phaeobacter inhibens. Most vitamins and precursors were found in the bacterial cell extract and the seawater sample with all three measuring methods. The results of this study emphasize that, in addition to tandem mass spectrometry, high-resolution full scan mass spectrometry is a promising technique for the simultaneous detection of structurally diverse B vitamins in complex natural samples. This enables highly sensitive measurements without loss of detailed mass spectrometric information, which is inevitable when using a triple quadrupole system in MS/MS mode.

The overlooked role of a biotin precursor for marine bacteria - desthiobiotin as an escape route for biotin auxotrophy.

Biotin (vitamin B7) is involved in a wide range of essential biochemical reactions and a crucial micronutrient that is vital for many pro- and eukaryotic organisms. The few biotin measurements in the world's oceans show that availability is subject to strong fluctuations. Numerous marine microorganisms exhibit biotin auxotrophy and therefore rely on supply by other organisms. Desthiobiotin is the primary precursor of biotin and has recently been detected at concentrations similar to biotin in seawater. The last enzymatic reaction in the biotin biosynthetic pathway converts desthiobiotin to biotin via the biotin synthase (BioB). The role of desthiobiotin as a precursor of biotin synthesis in microbial systems, however, is largely unknown. Here we demonstrate experimentally that bacteria can overcome biotin auxotrophy if they retain the bioB gene and desthiobiotin is available. A genomic search of 1068 bacteria predicts that the biotin biosynthetic potential varies greatly among different phylogenetic groups and that 20% encode solely bioB and thus can potentially overcome biotin auxotrophy. Many Actino- and Alphaproteobacteria cannot synthesize biotin de novo, but some possess solely bioB, whereas the vast majority of Gammaproteobacteria and Flavobacteriia exhibit the last four crucial biotin synthesis genes. We detected high intra- and extracellular concentrations of the precursor relative to biotin in the prototrophic bacterium, Vibrio campbellii, with extracellular desthiobiotin reaching up to 1.09 ± 0.15*106 molecules per cell during exponential growth. Our results provide evidence for the ecological role of desthiobiotin as an escape route to overcome biotin auxotrophy for bacteria in the ocean and presumably in other ecosystems.

Machine learning denoising of high-resolution X-ray nanotomography data.

High-resolution X-ray nanotomography is a quantitative tool for investigating specimens from a wide range of research areas. However, the quality of the reconstructed tomogram is often obscured by noise and therefore not suitable for automatic segmentation. Filtering methods are often required for a detailed quantitative analysis. However, most filters induce blurring in the reconstructed tomograms. Here, machine learning (ML) techniques offer a powerful alternative to conventional filtering methods. In this article, we verify that a self-supervised denoising ML technique can be used in a very efficient way for eliminating noise from nanotomography data. The technique presented is applied to high-resolution nanotomography data and compared to conventional filters, such as a median filter and a nonlocal means filter, optimized for tomographic data sets. The ML approach proves to be a very powerful tool that outperforms conventional filters by eliminating noise without blurring relevant structural features, thus enabling efficient quantitative analysis in different scientific fields.

Improved dynamic imaging of multiphase flow by constrained tomographic reconstruction.

Dynamic tomography has become an important technique to study fluid flow processes in porous media. The use of laboratory X-ray tomography instruments is, however, limited by their low X-ray brilliance. The prolonged exposure times, in turn, greatly limit temporal resolution. We have developed a tomographic reconstruction algorithm that maintains high image quality, despite reducing the exposure time and the number of projections significantly. Our approach, based on the Simultaneous Iterative Reconstruction Technique, mitigates the problem of few and noisy exposures by utilising a high-quality scan of the system before the dynamic process is started. We use the high-quality scan to initialise the first time step of the dynamic reconstruction. We further constrain regions of the dynamic reconstruction with a segmentation of the static system. We test the performance of the algorithm by reconstructing the dynamics of fluid separation in a multiphase system. The algorithm is compared quantitatively and qualitatively with several other reconstruction algorithms and we show that it can maintain high image quality using only a fraction of the normally required number of projections and with a substantially larger noise level. By robustly allowing fewer projections and shorter exposure, our algorithm enables the study of faster flow processes using laboratory tomography instrumentation but it can also be used to improve the reconstruction quality of dynamic synchrotron experiments.

Direct evidence for eoarchean iron metabolism?

Metasedimentary rocks from Isua, West Greenland (> 3,700 million years old) contain carbonaceous compounds, compatible with a biogenic origin (Hassenkam, Andersson, Dalby, Mackenzie, & Rosing, 2017; Ohtomo, Kakegawa, Ishida, Nagase, & Rosing, 2014; Rosing, 1999). The metamorphic mineral assemblage with garnet and quartz intergrowths contains layers of carbonaceous inclusions contiguous with carbon-rich sedimentary beds in the host rock. Previous studies (Hassenkam et al., 2017; Ohtomo et al., 2014; Rosing, 1999) on Isua rocks focused on testing the biogenic origin of the carbonaceous material, but here we searched for evidence which could provide new insights into the nature of the life that generated this carbonaceous material. We studied material trapped in inclusions armoured within quartz grains inside garnet porphyroblasts by non-destructive ptychographic X-ray nanotomography (PXCT). The 3D electron density maps generated by PXCT were correlated with maps from X-ray fluorescence tomography and micro-Raman spectroscopy. We found that the material trapped inside inclusions in the quartz grains consist of disordered carbon material encasing domains of iron-rich carbonaceous material. These results corroborate earlier claims (Hassenkam et al., 2017; Ohtomo et al., 2014; Rosing, 1999) for biogenic origins and are compatible with relics of metamorphosed biological material originally containing high iron/carbon ratios, comparable to ratios found in most extant organisms. These iron-rich domains represent the oldest evidence for organic iron complexes in the geologic record and are consistent with Fe-isotopic evidence for metabolic iron fractionation in > 3,700 Ma Isua banded iron formation (Czaja et al., 2013; Whitehouse & Fedo, 2007).

Capabilities of an optical direct push probe for 2D-subsurface imaging.

Characterization of hydrological conditions at polluted sites is critical for understanding of contaminant distribution and transport. Standard techniques for site characterization, such as soil coring together with well installation for piezometric measurements and water sampling, allow only some insights into subsurface properties and processes. To obtain additional data, direct-push techniques are often used in soils and unconsolidated formations. The various available techniques provide high resolution information on cm to mm scale. Recently, the Optical Imaging Profiler (OIP) was developed for detection of fluorescent contaminants. Here, we have investigated the applicability of the OIP for groundwater tracing using fluorophores. Our laboratory experiments show that it is possible to qualitatively trace various fluorophores meaning that light emitted by the fluorophores can be detected by a standard digital camera sensor. The measured fluorescence depends on the number of fluorophore molecules present in the pore space adjacent to the OIP and decreases with smaller pore size as well as fluorophore concentration. In a field trial, an injected eosin Y solution could be very clearly detected after the injection within a radius of 0.5 m around the injection point. When the OIP is equipped with a second light source emitting visible light, images of the soil texture and color can be captured. Sediment color can act as a proxy for various soil properties. Tests at a second field site, indicate that detected variation in soil color depend on water saturation and redox processes. Hence, the OIP is a flexible, cost effective and multifunctional tool for characterization of contaminated sites.

Canalicular Junctions in the Osteocyte Lacuno-Canalicular Network of Cortical Bone.

The osteocyte lacuno-canalicular network (LCN) is essential for bone remodeling because osteocytes regulate cell recruitment. This has been proposed to occur through liquid-flow-induced shear forces in the canaliculi. Models of the LCN have thus far assumed that it contains canaliculi connecting the osteocyte lacunae. However, here, we reveal that enlarged spaces occur at places where several canaliculi cross; we name these spaces canalicular junctions. We characterize them in detail within mice cortical bone using synchrotron nanotomography at two length scales, with 50 and 130 nm voxel size, and show that canalicular junctions occur at a density similar to that of osteocyte lacunae and that canalicular junctions tend to cluster. Through confocal laser scanning microscopy, we show that canalicular junctions are widespread as we have observed them in cortical bone from several species, even though the number density of the canalicular junctions was not universal. Fluid flow simulations of a simple model system with and without a canalicular junction clearly show that liquid mass transport and flow velocities are altered by the presence of canalicular junctions. We suggest that these canalicular junctions may play an important role in osteocyte communication and possibly also in canalicular fluid flow. Therefore, we believe that they constitute an important component in the bone osteocyte network.

Hierarchically porous, ultra-strong reduced graphene oxide-cellulose nanocrystal sponges for exceptional adsorption of water contaminants.

Self-assembly of graphene oxide (GO) nanosheets into porous 3D sponges is a promising approach to exploit their capacity to adsorb contaminants while facilitating the recovery of the nanosheets from treated water. Yet, forming mechanically robust sponges with suitable adsorption properties presents a significant challenge. Ultra-strong and highly porous 3D sponges are formed using GO, vitamin C (VC), and cellulose nanocrystals (CNCs) - natural nanorods isolated from wood pulp. CNCs provide a robust scaffold for the partially reduced GO (rGO) nanosheets resulting in an exceptionally stiff nanohybrid. The concentration of VC as a reducing agent plays a critical role in tailoring the pore architecture of the sponges. By using excess amounts of VC, a unique hierarchical pore structure is achieved, where VC grains act as soft templates for forming millimeter-sized pores, the walls of which are also porous and comprised of micron-sized pores. The unique hierarchical pore structure ensures the interconnectivity of pores even at the core of large sponges as evidenced by micro and nano X-ray computed tomography. The unique pore architecture translates into an exceptional specific surface area for adsorption of a wide range of contaminants, such as dyes, heavy metals, pharmaceuticals and cyanotoxin from water.

Properties of reactive sputtered alumina-silica mixtures.

Standard antireflective coatings applied to hard substrates like sapphire suffer from poor abrasion resistance. Silica is used as low refractive index layer in many multilayer systems although it has a lower hardness than the substrate. In this work an attempt was made to enhance the hardness by the addition of alumina. Magnetron sputtering was used in two different ways because it delivers dense coatings with high durability. Nanoindention hardness measurements of mixed alumina-silica films are presented in comparison to haze measurements after a sand trickling test. The hardness of silica is unexpectedly lowered by the addition of small amounts of alumina. Two different stacks were coated in which the low refractive index layers were sputtered as pure material and material mixtures. The thickness loss results after an oscillation abrasion test are presented.

Slurry concentration effects on the bed morphology and separation efficiency of capillaries packed with sub-2 µm particles.

Transcolumn dispersion limitations on the separation efficiency of chromatographic columns suggest the need for packing methods that increase bed homogeneity and minimize potential wall effects. Here we address the influence of the slurry concentration in the slurry packing process on the resulting morphology and separation efficiency of ultrahigh-pressure liquid chromatography capillary columns.30­75 µm i.d. capillaries were packed with fully porous 0.9, 1.7, and 1.9 µm bridged-ethyl hybrid particles and 1.9 µm Kinetex core­shell particles. Capillaries prepared with higher slurry concentrations(20­100 mg/mL) showed higher separation efficiencies than those prepared using a low slurry con-centration (2­3 mg/mL). The effect is explained by an analysis of transcolumn bed heterogeneities in three-dimensional reconstructions acquired from the packed capillaries using confocal laser scanning microscopy. The three-dimensional analysis of porosity distributions and local particle size illustrates that beds packed with higher slurry concentrations suppress particle size segregation, however, at the expense of a larger amount of packing voids. In core­shell packings, where only few packing voids were found, the higher slurry concentration allowed for an additional densification of the bed's wall region, as revealed by a radial analysis of the mean particle distances. Overall, wall effects are attenuated in packed columns prepared with both wide and narrow particle size distributions, which will allow for improved chromatographic performance.

Influence of particle properties on the wall region in packed capillaries.

Analytical columns (4.6 mm i.d.) packed with core-shell particles have shown a significantly reduced eddy dispersion contribution to band broadening compared to conventional fully porous particles. It has been speculated if this is caused by the narrow particle size distribution (PSD) of the core-shell particles, as an intrinsic advantage, or by an improved packing structure that specifically reduces the transcolumn velocity biases caused by wall effects. A recent simulation study has pointed against the former proposition [A. Daneyko et al., Anal. Chem. 83 (2011) 3903]. It is more likely that the slurry packing process for core-shell particles results in bed morphologies with reduced wall effects compared to the fully porous particles with a wide PSD. To access the latter proposition experimentally we slurry packed capillary columns (100 µm i.d.) with different fully porous (wide PSDs) and core-shell (narrow PSDs) particles and imaged their bed structures three-dimensionally using confocal laser scanning microscopy. This allowed us to resolve and analyze the bed morphology in these columns locally on all length scales contributing to eddy dispersion. On the transcolumn scale we observed a systematic difference between core-shell and fully porous particles: In the vicinity of the column wall the core-shell particles packed denser (closer to the bulk packing densities) and with a higher regularity than the fully porous particles. The bulk regions of all packings were effectively indistinguishable. This provides experimental evidence that the reduced eddy dispersion contribution with core-shell packings should be attributed to a higher transcolumn homogeneity rather than to an improved bed morphology on smaller length scales, e.g., to a reduced short-range disorder.

Morphology and separation efficiency of low-aspect-ratio capillary ultrahigh pressure liquid chromatography columns.

We derive a quantitative relationship between the bed morphology and the chromatographic separation efficiency of capillary columns packed with sub-2 µm particles, covering capillary inner diameters from 10 to 75 µm. Our study focuses on wall effects and their impact on band broadening at increasing column-to-particle diameter (aspect) ratios. We approach these complex effects by a morphological analysis of reconstructed column segments composed of several thousand particles that were imaged by confocal laser scanning microscopy. Radial interparticle porosity profiles including wall effects are quantified through an integral porosity deviation, a scalar measure that proves to be a general descriptor of transcolumn porosity heterogeneity. It correlates with the associated transcolumn eddy dispersion, which dominates band broadening in the capillaries and is visualized in the plate height curves by a simple velocity-proportional term. Our comprehensive approach identifies the packing structure features that contribute to decreased efficiency as reflected, e.g., in subtle variations of the wall effect at different aspect ratios, or a particle size-segregation effect in larger-diameter columns as a result of an increased number of packing voids near the wall-bed interface.

Morphology and separation efficiency of a new generation of analytical silica monoliths.

The heterogeneous morphology of current silica monoliths hinders this column type to reach its envisioned performance goals. We present a new generation of analytical silica monoliths that deliver a substantially improved separation efficiency achieved through several advances in monolith morphology. Analytical silica monoliths from the 1st and 2nd Chromolith generation are characterized and compared by chromatographic methods, mercury intrusion porosimetry, scanning electron microscopy, and confocal laser scanning microscopy. The latter method is instrumental to quantify morphological differences between the monolith generations and to probe the radial variation of morphological properties. Compared with the 1st generation, the new monoliths possess not only smaller macropores, a more homogeneous macropore space, and a thinner silica skeleton, but also radial homogeneity of these structural parameters as well as of the local external or macroporosity. The 66.5% reduction in minimum plate height observed between silica monoliths of the 1st and 2nd Chromolith generation can thus be attributed to two key improvements: a smaller domain size at simultaneously increased macropore homogeneity and the absence of radial morphology gradients, which are behind the considerable peak asymmetry of the 1st generation.

Morphological analysis of physically reconstructed capillary hybrid silica monoliths and correlation with separation efficiency.

We report an experimental study on the structural (especially radial) heterogeneity of eleven 100 µm i.d. capillary tetramethoxysilane-methyltrimethoxysilane hybrid silica monoliths with different pore and skeleton sizes, which were imaged by an optimized confocal laser scanning microscopy method. This method allows the optical sectioning of the monoliths, which is a prerequisite for quantitative morphological image analysis. Both radial porosity profiles and chord length distributions were calculated in the macropore domain for each column from at least 100 complete cross-sectional views along the column axis. The statistical approach visualized radial heterogeneities on different length scales in the monolithic structures. Chord length distributions followed a simplified k-gamma function, and a structural parameter obtained from this function is introduced to provide a scalar measure of column heterogeneity. It enables the comparison of monoliths with different pore sizes and helps to establish correlations between the microscopic properties of a column, eddy dispersion, and its separation efficiency.