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Carbon and hydrogen stable isotope effects associated with methane formation by the corrosive archaeon Methanobacterium strain IM1 were determined during growth with hydrogen and iron. Isotope analyses were complemented by structural, elemental and molecular composition analyses of corrosion crusts. During growth with H2 , strain IM1 formed methane with average δ13 C of -43.5‰ and δ2 H of -370‰. Corrosive growth led to methane more depleted in 13 C, with average δ13 C ranging from -56‰ to -64‰ during the early and the late growth phase respectively. The corresponding δ2 H were less impacted by the growth phase, with average values ranging from -316 to -329‰. The stable isotope fractionation factors, α13CCO2/CH4 , were 1.026 and 1.042 for hydrogenotrophic and corrosive growth respectively. Corrosion crusts formed by strain IM1 have a domed structure, appeared electrically conductive and were composed of siderite, calcite and iron sulfide, the latter formed by precipitation of sulfide (from culture medium) with ferrous iron generated during corrosion. Strain IM1 cells were found attached to crust surfaces and encrusted deep inside crust domes. Our results may assist to diagnose methanogens-induced corrosion in the field and suggest that intrusion of sulfide in anoxic settings may stimulate corrosion by methanogenic archaea via formation of semiconductive crusts.

Photosymbiosis is widespread and ecologically important in the oceanic plankton but remains poorly studied. Here, we used multimodal subcellular imaging to investigate the photosymbiosis between colonial Collodaria and their microalga dinoflagellate (Brandtodinium) collected in surface seawaters. We showed that this symbiosis is a very dynamic system whereby symbionts interact with different host cells via extracellular vesicles within the “greenhouse-like” colony. 3D electron microscopy revealed that the volume of the photosynthetic apparatus (plastid and pyrenoid) of the microalgae increased in symbiosis compared to free-living while the mitochondria volume was similar. Stable isotope probing coupled with NanoSIMS showed that carbon and nitrogen were assimilated and stored in the symbiotic microalga in starch granules and purine crystals, respectively. Nitrogen was also allocated to the algal nucleus (nucleolus). After 3 hours, low13C and15N transfer was detected in the host Golgi. Metal mapping revealed that intracellular iron concentration was similar in free-living and symbiotic microalgae (ca 40 ppm) and two-fold higher in the host, whereas copper concentration increased in symbiotic microalgae (up to 6900 ppm) and was detected in the host cell and extracellular vesicles. Sulfur mapping also pinpointed the importance of this nutrient for the algal metabolism. This study, which revealed subcellular changes of the morphology and nutrient homeostasis in symbiotic microalgae, improves our understanding on the metabolism of this widespread and abundant oceanic symbiosis and paves the way for more studies to investigate the metabolites exchanged.

Ethane is the second most abundant component of natural gas in addition to methane, and—similar to methane—is chemically unreactive. The biological consumption of ethane under anoxic conditions was suggested by geochemical profiles at marine hydrocarbon seeps1–3, and through ethane-dependent sulfate reduction in slurries4–7. Nevertheless, the microorganisms and reactions that catalyse this process have to date remained unknown8. Here we describe ethane-oxidizing archaea that were obtained by specific enrichment over ten years, and analyse these archaea using phylogeny-based fluorescence analyses, proteogenomics and metabolite studies. The co-culture, which oxidized ethane completely while reducing sulfate to sulfide, was dominated by an archaeon that we name ‘Candidatus Argoarchaeum ethanivorans’; other members were sulfate-reducing Deltaproteobacteria. The genome of Ca. Argoarchaeum contains all of the genes that are necessary for a functional methyl-coenzyme M reductase, and all subunits were detected in protein extracts. Accordingly, ethyl-coenzyme M (ethyl-CoM) was identified as an intermediate by liquid chromatography–tandem mass spectrometry. This indicated that Ca. Argoarchaeum initiates ethane oxidation by ethyl-CoM formation, analogous to the recently described butane activation by ‘Candidatus Syntrophoarchaeum’9. Proteogenomics further suggests that oxidation of intermediary acetyl-CoA to CO2 occurs through the oxidative Wood–Ljungdahl pathway. The identification of an archaeon that uses ethane (C2H6) fills a gap in our knowledge of microorganisms that specifically oxidize members of the homologous alkane series (CnH2n+2) without oxygen. Detection of phylogenetic and functional gene markers related to those of Ca. Argoarchaeum at deep-sea gas seeps10–12 suggests that archaea that are able to oxidize ethane through ethyl-CoM are widespread members of the local communities fostered by venting gaseous alkanes around these seeps. An archaeon, ‘Candidatus Argoarchaeum ethanivorans’, which is involved in the oxidation of ethane observed in anoxic marine habitats, is identified and metabolically characterized.

Materials 14(15), 4243 (1-10) (2021). doi:10.3390/ma14154243, The results of the calculation of the energy band structure and luminescent research of CeF$_3$ crystals are presented. The existence of two 5d1 and 5d2 subbands of the conduction band genetically derived from 5d states of Ce$^{3+}$ ions with different effective electron masses of 4.9 me and 0.9 me, respectively, is revealed. The large electron effective mass in the 5d1 subband facilitates the localization of electronic excitations forming the 4f-5d cerium Frenkel self-trapped excitons responsible for the CeF$_3$ luminescence. The structure of the excitation spectra of the exciton luminescence peaked at 290 nm, and the defect luminescence at 340 nm confirms the aforementioned calculated features of the conduction band of CeF$_3$ crystals. The peculiarities of the excitation spectra of the luminescence of CaF$_2$:Ce crystals dependent on the cerium concentration are considered with respect to the phase formation possibility of CeF$_3$., Published by MDPI, Basel

During the past decades, several stand-alone and combinatorial methods have been developed to investigate the chemistry (i.e., mapping of elemental, isotopic, and molecular composition) and the role of microbes in soil and rhizosphere. However, none of these approaches are currently applicable to characterize soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows for simultaneous microbial identification and chemical analysis of the rhizosphere at micro− to nano-meter spatial resolution. Our approach includes (i) a resin embedding and sectioning method suitable for simultaneous correlative characterization of Zea mays rhizosphere, (ii) an analytical work flow that allows up to six instruments/techniques to be used correlatively, and (iii) data and image correlation. Hydrophilic, immunohistochemistry compatible, low viscosity LR white resin was used to embed the rhizosphere sample. We employed waterjet cutting and avoided polishing the surface to prevent smearing of the sample surface at nanoscale. The quality of embedding was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil were identified by Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) to avoid interferences from high levels of autofluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was done by Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray analysis (SEM-EDX), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), nano-focused Secondary Ion mass Spectrometry (nanoSIMS), and confocal Raman spectroscopy (μ-Raman). High-resolution correlative characterization by six different techniques followed by image registration shows that this method can meet the demanding requirements of multiple characterization techniques to identify spatial organization of bacteria and chemically map the rhizosphere. Finally, we presented individual and correlative workflows for imaging and image registration to analyze data. We hope this method will be a platform to combine various 2D analytics for an improved understanding of the rhizosphere processes and their ecological significance.

Most microorganisms in the biosphere remain uncultured and poorly characterized. Although the surge in genome sequences has enabled insights into the genetic and metabolic properties of uncultured microorganisms, their physiology and ecological roles cannot be determined without direct probing of their activities in natural habitats. Here we employed an experimental framework coupling genome reconstruction and activity assays to characterize the largely uncultured microorganisms responsible for aerobic biodegradation of biphenyl as a proxy for a large class of environmental pollutants, polychlorinated biphenyls. We used 13C-labeled biphenyl in contaminated soils and traced the flow of pollutant-derived carbon into active cells using single-cell analyses and protein–stable isotope probing. The detection of 13C-enriched proteins linked biphenyl biodegradation to the uncultured Alphaproteobacteria clade UBA11222, which we found to host a distinctive biphenyl dioxygenase gene widely retrieved from contaminated environments. The same approach indicated the capacity of Azoarcus species to oxidize biphenyl and suggested similar metabolic abilities for species of Rugosibacter. Biphenyl oxidation would thus represent formerly unrecognized ecological functions of both genera. The quantitative role of these microorganisms in pollutant degradation was resolved using single-cell-based uptake measurements. Our strategy advances our understanding of microbially mediated biodegradation processes and has general application potential for elucidating the ecological roles of uncultured microorganisms in their natural habitats.

International audience; Endosymbioses have shaped the evolutionary trajectory of life and remain ecologically important. Investigating oceanic photosymbioses can illuminate how algal endosymbionts are energetically exploited by their heterotrophic hosts and inform on putative initial steps of plastid acquisition in eukaryotes. By combining three-dimensional subcellular imaging with photophysiology, carbon flux imaging, and transcriptomics, we show that cell division of endosymbionts ( Phaeocystis ) is blocked within hosts (Acantharia) and that their cellular architecture and bioenergetic machinery are radically altered. Transcriptional evidence indicates that a nutrient-independent mechanism prevents symbiont cell division and decouples nuclear and plastid division. As endosymbiont plastids proliferate, the volume of the photosynthetic machinery volume increases 100-fold in correlation with the expansion of a reticular mitochondrial network in close proximity to plastids. Photosynthetic efficiency tends to increase with cell size, and photon propagation modeling indicates that the networked mitochondrial architecture enhances light capture. This is accompanied by 150-fold higher carbon uptake and up-regulation of genes involved in photosynthesis and carbon fixation, which, in conjunction with a ca.15-fold size increase of pyrenoids demonstrates enhanced primary production in symbiosis. Mass spectrometry imaging revealed major carbon allocation to plastids and transfer to the host cell. As in most photosymbioses, microalgae are contained within a host phagosome (symbiosome), but here, the phagosome invaginates into enlarged microalgal cells, perhaps to optimize metabolic exchange. This observation adds evidence that the algal metamorphosis is irreversible. Hosts, therefore, trigger and benefit from major bioenergetic remodeling of symbiotic microalgae with potential consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step toward plastid acquisition.

International audience; To better understand the physiology and acclimation capability of the cell, one of the great challenges of the future is to access the interior of a cell and unveil its chemical landscape (composition and distribution of elements and molecules). Chemical imaging has greatly improved in sensitivity and spatial resolution to visualize and quantify nutrients, metabolites, toxic elements, and drugs in single cells at the subcellular level. This review aims to present the current potential of these emerging imaging technologies and to guide biologists towards a strategy for interrogating biological processes at the nanoscale. We also describe various solutions to combine multiple imaging techniques in a correlative way and provide perspectives and future directions for integrative subcellular imaging across different disciplines

Fungal–bacterial interactions are highly diverse and contribute to many ecosystem processes. Their emergence under common environmental stress scenarios however, remains elusive. Here we use a synthetic microbial ecosystem based on the germination of Bacillus subtilis spores to examine whether fungal and fungal-like (oomycete) mycelia reduce bacterial water and nutrient stress in an otherwise dry and nutrient-poor microhabitat. We find that the presence of mycelia enables the germination and subsequent growth of bacterial spores near the hyphae. Using a combination of time of flight- and nanoscale secondary ion mass spectrometry (ToF- and nanoSIMS) coupled with stable isotope labelling, we link spore germination to hyphal transfer of water, carbon and nitrogen. Our study provides direct experimental evidence for the stimulation of bacterial activity by mycelial supply of scarce resources in dry and nutrient-free environments. We propose that mycelia may stimulate bacterial activity and thus contribute to sustaining ecosystem functioning in stressed habitats., The maintenance of bacterial and fungal activity is essential for ecosystem functioning, particularly in dry soils where the two phyla co-exist. Here, Worrich and colleagues show experimentally that mycelia traffic water and nutrients and thereby stimulate bacterial activity in stressful conditions.

Nowadays, high-resolution imaging techniques are extensively applied in a complementary way to gain insights into complex phenomena. For a truly complementary analytical approach, a common sample carrier is required that is suitable for the different preparation methods necessary for each analytical technique. This sample carrier should be capable of accommodating diverse analytes and maintaining their pristine composition and arrangement during deposition and preparation. In this work, a new type of sample carrier consisting of a silicon wafer with a hydrophilic polymer coating was developed. The robustness of the polymer coating toward solvents was strengthened by cross-linking and stoving. Furthermore, a new method of UV-ozone cleaning was developed that enhances the adhesion of the polymer coating to the wafer and ensures reproducible surface-properties of the resulting sample carrier. The hydrophilicity of the sample carrier was recovered applying the new method of UV-ozone cleaning, while avoiding UV-induced damages to the polymer. Noncontact 3D optical profilometry and contact angle measurements were used to monitor the hydrophilicity of the coating. The hydrophilicity of the polymer coating ensures its spongelike behavior so that upon the deposition of an analyte suspension, the solvent and solutes are separated from the analyte by absorption into the polymer. This feature is essential to limit the coffee-ring effect and preserve the native identity of an analyte upon deposition. The suitability of the sample carrier for various sample types was tested using nanoparticles from suspension, bacterial cells, and tissue sections. To assess the homogeneity of the analyte distribution and preservation of sample integrity, optical and scanning electron microscopy, helium ion microscopy, laser ablation inductively coupled plasma mass spectrometry, and time-of-flight secondary ion mass spectrometry were used. This demonstrates the broad applicability of the newly developed sample carrier and its value for complementary imaging.

Heterotrophic bacteria associated with microphytoplankton, particularly those colonizing the phycosphere, are major players in the remineralization of algal-derived carbon. Ocean warming might impact dissolved organic carbon (DOC) uptake by microphytoplankton-associated bacteria with unknown biogeochemical implications. Here, by incubating natural seawater samples at three different temperatures, we analysed the effect of experimental warming on the abundance and C and N uptake activity of Rhodobacteraceae and Flavobacteria, two bacterial groups typically associated with microphytoplankton. Using a nano-scale secondary ion mass spectrometry (nanoSIMS) single-cell analysis, we quantified the temperature sensitivity of these two taxonomic groups to the uptake of algal-derived DOC in the microphytoplankton associated fraction with 13 C-bicarbonate and 15 N-leucine as tracers. We found that cell-specific 13 C uptake was similar for both groups (~0.42 fg C h-1 μm-3 ), but Rhodobacteraceae were more active in 15 N-leucine uptake. Due to the higher abundance of Flavobacteria associated with microphytoplankton, this group incorporated fourfold more carbon than Rhodobacteraceae. Cell-specific 13 C uptake was influenced by temperature, but no significant differences were found for 15 N-leucine uptake. Our results show that the contribution of Flavobacteria and Rhodobacteraceae to C assimilation increased up to sixfold and twofold, respectively, with an increase of 3°C above ambient temperature, suggesting that warming may differently affect the contribution of distinct copiotrophic bacterial taxa to carbon cycling.

Microalgae biofilms may play an important role in the mitigation and prevention of eutrophication caused by domestic, agricultural and industrial wastewater effluents. Despite their potential, the biofilm development and role in nutrient removal are not well understood. Its clarification requires comprehensive studies of the complex three-dimensional architecture of the biofilm. In this study, we established a multimodal imaging approach to provide key information regarding architecture development and nutrient distribution in the biofilm of two green algae organisms: Chlorella pyrenoidosa and Chlorella vulgaris. Helium ion microscopy (HIM), scanning electron microscopy coupled with energy dispersive X-ray analysis (SEM-EDX) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were employed for i) elucidation of spatial arrangement, ii) elemental mapping and iii) 3D chemical imaging of the biofilm. The fine structure of the algal biofilm was resolved by HIM, the evidence of the accumulation of phosphate in hot spots was provided by SEM-EDX and the localization of phosphate oxides granules throughout the whole sample was clarified by ToF-SIMS. The reported results shed light on the phosphorus distribution during Chlorella's biofilm formation and highlight the potential of such correlative approach to solve fundamental question in algal biotechnology research.

Increasing production of rare earth elements (REE) might lead to future contamination of the environment. REE have been shown to accumulate in high concentrations in roots of plants. Plant experiments with Zea mays exposed to a nutrient solution containing gadolinium (Gd) or yttrium (Y) with 10 mg L −1 Gd or Y were carried out to investigate this accumulation behaviour. Total concentrations of 3.17 g kg −1 and 8.43 g kg −1 of Gd and Y were measured in treated plant roots. Using a novel combination of laser ablation mass spectrometry and time-of-flight secondary ion mass spectrometry, imaging of location and concentration of Gd and Y was carried out in root thin sections of treated roots. Single spots of elevated REE concentration were found at the epidermis, while inside the cortex, weak signals of Gd + and Y + were aligning with the root cell structures. The composition of Gd-containing secondary ions proves an REE-oxide phase accumulated at the epidermis, limiting REE availability for further uptake.

Ethylbenzene and toluene degradation under nitrate-, Mn(IV)-, or Fe(III)-reducing conditions was investigated by compound specific stable isotope analysis (CSIA) using three model cultures (Aromatoleum aromaticum EbN1, Georgfuchsia toluolica G5G6, and a Azoarcus-dominated mixed culture). Systematically lower isotope enrichment factors for carbon and hydrogen were observed for particulate Mn(IV). The increasing diffusion distances of toluene or ethylbenzene to the solid Mn(IV) most likely caused limited bioavailability and hence resulted in the observed masking effect. The data suggests further ethylbenzene hydroxylation by ethylbenzene dehydrogenase (EBDH) and toluene activation by benzylsuccinate synthase (BSS) as initial activation steps. Notably, significantly different values in dual isotope analysis were detected for toluene degradation by G. toluolica under the three studied redox conditions, suggesting variations in the enzymatic transition state depending on the available TEA. The results indicate that two-dimensional CSIA has significant potential to assess anaerobic biodegradation of ethylbenzene and toluene at contaminated sites.

International audience; Photosymbiosis between single-celled hosts and microalgae is common in oceanic plankton, especially in oligotrophic surface waters. However, the functioning of this ecologically important cell-cell interaction and the subcellular mechanisms allowing the host to accommodate and benefit from its microalgae remain enigmatic. Here, using a combination of quantitative single-cell structural and chemical imaging techniques (FIB-SEM, nanoSIMS, Synchrotron X-ray fluorescence), we show that the structural organization, physiology, and trophic status of the algal symbionts (the haptophyte Phaeocystis) significantly change within their acantharian hosts compared to their free-living phase in culture. In symbiosis, algal cell division is blocked, photosynthesis is enhanced, and cell volume is increased by up to 10-fold with a higher number of plastids (from 2 to up to 30) and thylakoid membranes. The multiplication of plastids can lead to a 38-fold increase of the total plastid volume in a cell. Subcellular mapping of nutrients (nitrogen and phosphorous) and their stoichiometric ratios shows that symbiotic algae are impoverished in phosphorous and suggests a higher investment in energy-acquisition machinery rather than in growth. Nanoscale imaging also showed that the host supplies a substantial amount of trace metals (e.g., iron and cobalt), which are stored in algal vacuoles at high concentrations (up to 660 ppm). Sulfur mapping reveals a high concentration in algal vacuoles that may be a source of antioxidant molecules. Overall, this study unveils an unprecedented morphological and metabolic transformation of microalgae following their integration into a host, and it suggests that this widespread symbiosis is a farming strategy wherein the host engulfs and exploits microalgae.

Coastal sediments are rich in conductive particles, possibly affecting microbial processes for which acetate is a central intermediate. In the methanogenic zone, acetate is consumed by methanogens and/or syntrophic acetate-oxidizing (SAO) consortia. SAO consortia live under extreme thermodynamic pressure, and their survival depends on successful partnership. Here, we demonstrate that conductive particles enable the partnership between SAO bacteria (i.e., Geobacter spp.) and methanogens (Methanosarcina spp.) from the coastal sediments of the Bothnian Bay of the Baltic Sea. Baltic methanogenic sediments were rich in conductive minerals, had an apparent isotopic fractionation characteristic of CO2-reductive methanogenesis, and were inhabited by Geobacter and Methanosarcina. As long as conductive particles were delivered, Geobacter and Methanosarcina persisted, whereas exclusion of conductive particles led to the extinction of Geobacter. Baltic Geobacter did not establish a direct electric contact with Methanosarcina, necessitating conductive particles as electrical conduits. Within SAO consortia, Geobacter was an efficient [13C]acetate utilizer, accounting for 82% of the assimilation and 27% of the breakdown of acetate. Geobacter benefits from the association with the methanogen, because in the absence of an electron acceptor it can use Methanosarcina as a terminal electron sink. Consequently, inhibition of methanogenesis constrained the SAO activity of Geobacter as well. A potential benefit for Methanosarcina partnering with Geobacter is that together they competitively exclude acetoclastic methanogens like Methanothrix from an environment rich in conductive particles. Conductive particle-mediated SAO could explain the abundance of acetate oxidizers like Geobacter in the methanogenic zone of sediments where no electron acceptors other than CO2 are available., IMPORTANCE Acetate-oxidizing bacteria are known to thrive in mutualistic consortia in which H2 or formate is shuttled to a methane-producing Archaea partner. Here, we discovered that such bacteria could instead transfer electrons via conductive minerals. Mineral SAO (syntrophic acetate oxidation) could be a vital pathway for CO2-reductive methanogenesis in the environment, especially in sediments rich in conductive minerals. Mineral-facilitated SAO is therefore of potential importance for both iron and methane cycles in sediments and soils. Additionally, our observations imply that agricultural runoff or amendments with conductive chars could trigger a significant increase in methane emissions.

Branched polymeric surfactants composed of grafted non-ionic polyethylene glycol (PEG) and anionic polyelectrolyte chains were synthesized via radical polymerization initiated by the comb-like PEG-containing polyperoxide. Above definite concentration in solution, these surfactants form micelle-like structures (MLS). The MLS formed by branched polymeric molecules are of larger size in comparison with size of MLS formed by the initial PEG-containing polyperoxide that is caused mainly by different mechanism of their self-organization and morphology of formed MLS. The availability of grafted polyelectrolyte chains in the MLS provides a possibility of their use as the containers for immobilization of bio-active substances and nucleation of the inorganic nanoparticles, as well as formation of their stable colloidal systems in water in a wide pH range. Noticeable compaction and narrowed size distribution of the MLS were revealed after immobilization of doxorubicin (Dox) molecules or Fe 2 O 3 nanocrystals. MLS-based systems were used for delivery of Dox and maghemite particles at treatment of tumor cells. Both MLS-based formulations of Dox and Fe 2 O 3 were efficiently engulfed by rat glioma C6 cells. A significant (10 times) decrease in the effective therapeutic dose of Dox was found when this drug was delivered by a MLS-based formulation of Dox. That effect might be explained by a specific structure and functionality of the novel carrier used for immobilization of drug that should be delivered to target cells.

Characterization of nanoparticle (NP) interaction with an organic host requires identification and localization of nano-sized phases via high-resolution 3D imaging. Simultaneous molecular and spatial resolution causes many analytical approaches to face inherent performance limits, but this challenge can be overcome with time-of-flight secondary ion mass spectrometry (ToF-SIMS). To implement ToF-SIMS analysis of complex organic systems, a careful sample preparation is required to ensure ultra-high vacuum compatibility and to preserve the native chemical composition and spatial arrangement. In this present study, we developed a new method for the deposition of NPs from suspensions employing a polymer-coated substrate to avoid NP aggregation and to eliminate dissolved contaminants. We achieved a lateral resolution of 108 nm simultaneously with a mass resolving power (MRP = M/ΔM) above 5000 in delayed extraction mode. Optimized ToF-SIMS performance allowed for (i) 3D localization of TiO2 NPs in Chlorella vulgaris biofilm, (ii) precise isotope-pattern-based identification of TiO2-related secondary ions and (iii) elucidation of their composition dependence on the nearest molecular surrounding of the NPs. The developed sample preparation method in combination with the high resolution 3D visualization of NPs in algal biofilm paves the way to study NP fate and localization in complex environments simultaneously.