The ability to simultaneously monitor the flux of iron atoms within the solution and solid phases can provide considerable insight into mechanisms of iron oxide mineral transformations. The autocatalytic interaction between hematite and Fe(ii)-oxalate has long been of interest for its environmental and industrial relevance. In this study we take advantage of iron isotopic labelling and mass-sensitive imaging at the single particle scale to determine how changes in solution composition correlate with the morphologic evolution of faceted, micrometer-sized hematite platelets. Net dissolution is confirmed through analyses of aqueous iron chemistry, as well as by quantitative atomic force microscopy. Isotopic mapping techniques show that Fe(ii) readily adsorbs to (001) and (012) surfaces in the absence of oxalate, but when oxalate is present selective dissolution of the (001) surface prevails and Fe deposition via recrystallization is not observed. Comparison between particle microtopographies following reaction with Fe(ii), oxalate, and Fe(ii)-oxalate show substantially different behaviors, consistent with distinct mechanisms of interaction with hematite surfaces. The extensive characterization conducted on the coupled solution/solid dynamics in this system provides new insight for distinguishing crystal growth, dissolution, and recrystallization processes.

Soil organic matter (SOM) is a key reservoir for global carbon (C), supporting soil fertility and influencing greenhouse gas emissions. Microbial residues, composed of dead cells and cellular fragments, are major contributors to SOM formation. Yet, mechanisms by which minerals enhance the accumulation of microbial residues remain poorly understood. Here, we used 13C-labeled glucose in a year-long incubation to trace microbial residue in sandy and silty soils. Across both soils, approximately 89% of retained microbial 13C was recovered in the fine (<53 μm) mineral-associated organic matter (MAOM) pool. Within this pool, the light MAOM fraction, enriched in poorly crystalline Fe minerals, held 4.3 times more 13C than the heavy, phyllosilicate-dominated MAOM fraction, despite accounting for only 17.2% of the total MAOM mass and 12.3% of the total soil mass. Along with 13C enrichment, the light MAOM fraction showed greater abundance of N-containing groups, e.g., (amides and amino groups), indicative of microbial-derived compounds like proteins and amino sugars. Fe oxides in light MAOM from both soils were spatially dispersed. Microbial residue accumulation was greater in finer-textured silty soil. These findings demonstrate that mineral composition and texture jointly regulate microbial necromass accrual, highlighting light MAOM as a key pool for enhancing soil C storage.

Stable isotope probing (SIP) experiments in conjunction with Raman microspectroscopy (Raman) or nano-scale secondary ion mass spectrometry (NanoSIMS) are frequently used to explore single cell metabolic activity in pure cultures as well as complex microbiomes. Despite the increasing popularity of these techniques, the comparability of isotope incorporation measurements using both Raman and NanoSIMS directly on the same cell remains largely unexplored. This knowledge gap creates uncertainty about the consistency of single-cell SIP data obtained independently from each method. Here, we conducted a comparative analysis of 543 Escherichia coli cells grown in M9 minimal medium in the absence or presence of heavy water (2H2O) using correlative Raman and NanoSIMS measurements to quantify the results between the two approaches. We demonstrate that Raman and NanoSIMS yield highly comparable measurements of 2H incorporation, with varying degrees of similarity based on the mass ratios analyzed using NanoSIMS. The 12C2H/12C1H and 12C22H/12C21H mass ratios provide targeted measurements of C-H bonds but may suffer from biases and background interference, while the 2H/1H ratio captures all hydrogen with lower detection limits, making it suitable for applications requiring comprehensive 2H quantification. Importantly, despite its higher mass resolution requirements, the use of C22H/C21H may be a viable alternative to the use of C2H/C1H due to lower background and higher overall count rates. Furthermore, using an empirical approach in determining Raman wavenumber ranges via the second derivative improved the data equivalency of 2H quantification between Raman and NanoSIMS, highlighting its potential for enhancing cross-technique comparability. These findings provide a robust framework for leveraging both techniques, enabling informed experimental design and data interpretation. By enhancing cross-technique comparability, this work advances SIP methodologies for investigating microbial metabolism and interactions in diverse systems.IMPORTANCEAccurate and reliable measurements of cellular properties are fundamental to understand the function and activity of microbes. This study addresses to what extent Raman microspectroscopy and nano-scale secondary ion mass spectrometry (NanoSIMS) measurements of single cell anabolic activity can be compared. Here, we study the relationship of the incorporation of a stable isotope (2H through incorporation of 2H2O) as determined by the two techniques and calculate a correlation coefficient to support the use of either technique when analyzing cells incubated with 2H2O. The ability to discern between the comparative strengths and limitations of these techniques is invaluable in refining experimental protocols, enhancing data comparability between studies, data interpretation, and ultimately advancing the quality and reliability of outcomes in microbiome research.

Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte-poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only similar to 11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through N-15(2) enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant-endophyte interactions.

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single-cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing 8 new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nanoscale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal noncanonical amino acid tagging (BONCAT), we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle.

Abstract Understanding biomineralization relies on imaging chemically heterogeneous organic–inorganic interfaces across a hierarchy of spatial scales. Further, organic minority phases are often responsible for emergent inorganic structures from the atomic arrangement of different polymorphs, to nano- and micrometer crystal dimensions, up to meter size mollusk shells. The desired simultaneous chemical and elemental imaging to identify sparse organic moieties across a large field-of-view with nanometer spatial resolution has not yet been achieved. Here, we combine nanoscale secondary ion mass spectroscopy (NanoSIMS) with spectroscopic IR s-SNOM imaging for simultaneous chemical, molecular, and elemental nanoimaging. At the example of Pinctada margaritifera mollusk shells we identify and resolve ~ 50 nm interlamellar protein sheets periodically arranged in regular ~ 600 nm intervals. The striations typically appear ~ 15 µm from the nacre-prism boundary at the interface between disordered neonacre to mature nacre. Using the polymorph distinctive IR-vibrational carbonate resonance, the nacre and prismatic regions are consistently identified as aragonite ( $${\overline{\nu }}_{a}=860$$ ν ¯ a = 860 cm−1) and calcite ( $${\overline{\nu }}_{c}=880$$ ν ¯ c = 880 cm−1), respectively. We observe previously unreported morphological features including aragonite subdomains encapsulated in extensions of the prism-covering organic membrane and regions of irregular nacre tablet formation coincident with dispersed organics. We also identify a ~ 200 nm region in the incipient nacre region with less well-defined crystal structure and integrated organics. These results show with the identification of the interlamellar protein layer how correlative nano-IR chemical and NanoSIMS elemental imaging can help distinguish different models proposed for shell growth in particular, and how organic function may relate to inorganic structure in other biomineralized systems in general.

Organicnitrate ester compounds (RONO2) are an importantsink for nitrogen oxides (NO x & EQUIV;NO + NO2) that impact ozone and organic aerosol formationin the lower atmosphere. While RONO2 formation from gasphase oxidation chemistry and heterogeneous reactions involving reactiveuptake of NO3 into organic aerosols is well documented, in situ bulk-aqueous-phase production of RONO2 within organic aerosols has not been explored as extensively. Here,we provide evidence that RONO2 species are produced duringaqueous phase reactions of nitronium (NO2 (+))with humic and fulvic acids, which are used as surrogates for aerosolorganic matter. X-ray photoelectron spectroscopy (XPS) and high-resolutionmass spectrometry were used to characterize nitrogen functional groupsand changes in bulk chemical composition during nitration reactions.Correlations between the organic N abundance in reacted humic andfulvic acids and functional group abundance measured by solid-state C-13 NMR indicate that NO2 (+) targets thelipid fraction of the organic matter. Fourier transform infrared spectroscopy(FTIR) analysis of model lipid compounds shows that the reaction ofNO(2) (+) with terminal alcohols (ROH) and alkenesis a source of stable RONO2 compounds. Further, the low-pHenvironment of most nitrate aerosols is expected to enhance the totalterminal ROH pool through acid-catalyzed ester hydrolysis within thelipid-like fraction of natural organic matter. Our findings demonstratethat multiphase oxidation of organic matter by NO2 (+), along with air-particle partitioning of products from gas-phasechemistry, may be an important source of particle-phase organic nitrates.

Pyrite trace element chemistry is important for understanding evolution of ore fluids in mineralizing systems. Much of the understanding of how metals are held in pyrite is based on micro-scale techniques such as LA-ICPMS and electron microprobe. This requires assumptions to be made when interpreting the results regarding whether elements are held as micro-inclusions versus as substitutions into the pyrite lattice. In this contribution, we use micro- to nano-scale analyses including LA-ICPMS, NanoSIMS, and atom probe tomography (APT) to investigate trace element deportment in pyrite from sites characterized by high trace element concentrations (Black Butte SEDEX deposit) and one characterized by medium to low trace element concentrations (Leicester pyrite member, New York). We further use synchrotron based XANES to investigate the redox state of As and how that relates to trace element enrichment. We find that trace elements are highly heterogeneous at all scales investigated and that interpretation of how trace elements are incorporated into pyrite using even micro-scale techniques must be pursued with caution.

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing eight new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nano-scale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal non-canonical amino acid tagging (BONCAT) we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle. Significance statement The emergence of multicellular lifeforms represents a pivotal milestone in Earth’s history, ushering in a new era of biological complexity. Because of the relative scarcity of multicellularity in the domains Bacteria and Archaea , research on the evolution of multicellularity has predominantly focused on eukaryotic model organisms. In this study, we explored the complexity of the only known bacteria without a unicellular stage in their life cycle, consortia of multicellular magnetotactic bacteria (MMB). Genomic and physiological analyses revealed that cells within individual MMB consortia are not clonal and exhibit metabolic differentiation. This implies a higher level of complexity than previously assumed for MMB consortia, prompting a reevaluation of the evolutionary factors that have led to the emergence of multicellularity. Because of their unique biology MMB consortia are ideally suited to become a model system to explore the underpinnings of bacterial multicellularity.

Oxygen isotopes are a versatile tool to address a wide range of questions in the Earth sciences. Applications include geothermometry, paleoclimatology, tracing of geochemical reservoirs, fluid-rock interaction, magmatic petrogenesis, and identification of extra-terrestrial materials. Zircon arguably provides one of the most robust records of primary magmatic O isotope ratio due to low diffusion rates in crystalline grains. The ability to correlate zircon O isotopes with temporal and petrogenetic information (e.g. U-Pb geochronology, Lu-Hf isotopes, and trace elements) makes this mineral a key archive for understanding Earth's crustal evolution. Consequently, zircon O isotope geochemistry has found widespread usage to address fundamental questions across the earth and planetary sciences. The general apparent ease of O isotopic acquisition through the advancement of rapid in situ techniques (i.e. secondary ion mass spectrometry; SIMS) and associated dedicated national laboratories has led to the generation of large O isotopic data sets of variable quality, highlighting the importance of a coherent workflow for data collection, reduction, and presentation. This paper presents a set of approaches for measurement, assessment, and reporting of zircon O isotope data. The focus in this contribution is on in situ analysis via secondary ion mass spectrometry using large geometry instruments, but other commonly used techniques are briefly reviewed for context. This work aims to provide an analytical framework necessary for geologically meaningful interpretation of O isotope data. In addition, we describe inherent geological (e.g. radiation-induced disturbance of the zircon O isotopic system) and analytical (e.g. fractionation due to sample topography effects) challenges and outline means to identify and avoid such issues as a prerequisite to the generation of robust primary O isotopic signatures for geological interpretation.

Fe-rich mobile colloids play vital yet poorly understood roles in the biogeochemical cycling of Fe in groundwater by influencing organic matter (OM) preservation and fluxes of Fe, OM, and other essential (micro-)nutrients. Yet, few studies have provided molecular detail on the structures and compositions of Fe-rich mobile colloids and factors controlling their persistence in natural groundwater. Here, we provide comprehensive new information on the sizes, molecular structures, and compositions of Fe-rich mobile colloids that accounted for up to 72% of aqueous Fe in anoxic groundwater from a redox-active floodplain. The mobile colloids are multi-phase assemblages consisting of Si-coated ferrihydrite nanoparticles and Fe(II)-OM complexes. Ferrihydrite nanoparticles persisted under both oxic and anoxic conditions, which we attribute to passivation by Si and OM. These findings suggest that mobile Fe-rich colloids generated in floodplains can persist during transport through redox-variable soils and could be discharged to surface waters. These results shed new light on their potential to transport Fe, OM, and nutrients across terrestrial-aquatic interfaces.

Microscopic and spectroscopic techniques are commonly applied to study microbial cells but are typically used on separate samples, resulting in population-level datasets that are integrated across different cells with little spatial resolution. To address this shortcoming, we developed a workflow that correlates several microscopic and spectroscopic techniques to generate an in-depth analysis of individual cells. By combining stable isotope probing (SIP), fluorescence in situ hybridization (FISH), scanning electron microscopy (SEM), confocal Raman microspectroscopy (Raman), and nano-scale secondary ion mass spectrometry (NanoSIMS), we illustrate how individual cells can be thoroughly interrogated to obtain information about their taxonomic identity, structure, physiology, and metabolic activity. Analysis of an artificial microbial community demonstrated that our correlative approach was able to resolve the activity of single cells using heavy water SIP in conjunction with Raman and/or NanoSIMS and establish their taxonomy and morphology using FISH and SEM. This workflow was then applied to a sample of yet uncultured multicellular magnetotactic bacteria (MMB). In addition to establishing their identity and activity, backscatter electron microscopy (BSE), NanoSIMS, and energy-dispersive X-ray spectroscopy (EDS) were employed to characterize the magnetosomes within the cells. By integrating these techniques, we demonstrate a cohesive approach to thoroughly study environmental microbes on a single-cell level.