Rates of microbial growth are fundamental to understanding environmental geochemistry and ecology. However, measuring the heterogeneity of microbial activity at the single-cell level, especially within complex populations and environmental matrices, remains a forefront challenge. Stable Isotope Probing (SIP) is a method for assessing microbial growth and involves measuring the incorporation of an isotopic label into microbial biomass. Here, we assess Raman microspectroscopy as a SIP technique, specifically focusing on the measurement of deuterium (2H), a tracer of microbial biomass production. We correlatively measured cells grown in varying concentrations of deuterated water with both Raman spectroscopy and nanoscale secondary ion mass spectrometry (nanoSIMS), generating isotopic calibrations of microbial 2H. Relative to Raman, we find that nanoSIMS measurements of 2H are subject to substantial dilution due to rapid exchange of H during sample washing. We apply our Raman-derived calibration to a numerical model of microbial growth, explicitly parameterizing the factors controlling growth rate quantification and demonstrating that Raman-SIP can sensitively measure the growth of microorganisms with doubling times ranging from hours to years. The measurement of single-cell growth with Raman spectroscopy, a rapid, non-destructive technique, represents an important step towards application of single-cell analysis into complex sample matrices or cellular assemblages.

Microbial degradation of organic carbon in sediments is impacted by the availability of oxygen and substrates for growth. To better understand how particle size and redox zonation impact microbial organic carbon incorporation, techniques that maintain spatial information are necessary to quantify elemental cycling at the microscale. In this study, we produced hydrogel microspheres of various diameters (100, 250, and 500 μm) and inoculated them with an aerobic heterotrophic bacterium isolated from a freshwater wetland (Flavobacterium sp.), and in a second experiment with a microbial community from an urban lacustrine wetland. The hydrogel-embedded microbial populations were incubated with 13C-labeled substrates to quantify organic carbon incorporation into biomass via nanoSIMS. Additionally, luminescent nanosensors enabled spatially explicit measurements of oxygen concentrations inside the microspheres. The experimental data were then incorporated into a reactive-transport model to project long-term steady-state conditions. Smaller (100 μm) particles exhibited the highest microbial cell-specific growth per volume, but also showed higher absolute activity near the surface compared to the larger particles (250 and 500 μm). The experimental results and computational models demonstrate that organic carbon availability was not high enough to allow steep oxygen gradients and as a result, all particle sizes remained well-oxygenated. Our study provides a foundational framework for future studies investigating spatially dependent microbial activity in aggregates using isotopically labeled substrates to quantify growth.

Guaymas Basin, located in the Gulf of California, is a hydrothermally active marginal basin. Due to steep geothermal gradients and localized heating by sill intrusions, microbial substrates like short-chain fatty acids and hydrocarbons are abiotically produced from sedimentary organic matter at comparatively shallow depths. We analyzed the effect of hydrocarbons on uptake of hydrocarbons by microorganisms via nano-scale secondary ion mass spectrometry (NanoSIMS) and microbial sulfate reduction rates (SRR), using samples from two drill sites sampled by IODP Expedition 385 (U1545C and U1546D). These sites are in close proximity of each other (ca. 1 km) and have very similar sedimentology. Site U1546D experienced the intrusion of a sill that has since then thermally equilibrated with the surrounding sediment. Both sites currently have an identical geothermal gradient, despite their different thermal history. The localized heating by the sill led to thermal cracking of sedimentary organic matter and formation of potentially bioavailable organic substrates. There were low levels of hydrocarbon and nitrogen uptake in some samples from both sites, mostly in surficial samples. Hydrocarbon and methane additions stimulated SRR in near-seafloor samples from Site U1545C, while samples from Site U1546D reacted positively only on methane. Our data indicate the potential of microorganisms to metabolize hydrocarbons even in the deep subsurface of Guaymas Basin.

OBJECTIVE: The objective of this work was to understand how triglyceride plant oils can deliver strength and softness benefits to hair by their penetration. These plant oils are complex mixtures of TAGs, so the initial studies performed were with pure TAGs and then these data compared to plant oils and their measured TAG compositions.
METHODS: LC-MS was used to identify the di and triglycerides in coconut oil, Camellia oleifera oil and safflower seed oil. Penetration of these plant oils and pure individual triglycerides was measured by a differential extraction method. Cross-sections of oils treated with 13C-labelled triolein were studied by NanoSIMS to visualize location of triglyceride inside hair. Fatigue strength was measured using constant stress to generate a survival distribution. Models of the lipid-rich cell membrane complex (CMC) were created with the equimolar ratio of 18-methyl-eicosanoic acid (MEAS), palmitic acid (C16:0) and oleic acid (C18:1).
RESULTS: Penetration of the individual pure TAGs was confirmed for all chain lengths and degree of unsaturation tested with higher penetration for shorter chain lengths and unsaturated fatty acids. Detailed compositional analysis of selected plant oils showed a wide variety of TAGs and penetration was also demonstrated for these oils. NanoSIMS and modelling confirmed these TAGs are penetrating the lipid-rich CMC of hair and are interacting with the fatty acids that make up the CMC. All plant oils delivered a fatigue strength improvement by penetration into the CMC and it is proposed that these oils prevent formation and/or propagation of flaws in the CMC network that leads to breakage.
CONCLUSIONS: Many plant oils with a wide range of triglyceride compositions can penetrate into hair and NanoSIMS data confirmed these oils partition into the lipid-rich cell membrane complex. Penetration studies of individual TAGs shown to be present in these oils confirmed TAGs of varying chain length can penetrate and there is a correlation between increased penetration efficacy and shorter chain lengths and presence of unsaturation in the fatty acid chains. All the oils studied delivered single fibre fatigue strength benefits.
OBJECTIVE: L\'objectif de ce travail était de comprendre comment les huiles végétales à base de triglycérides peuvent apporter aux cheveux des bienfaits en termes de résistance et de douceur grâce à leur pénétration. Ces huiles végétales sont des mélanges complexes de TAG, donc les études réalisées initiales ont porté sur des TAG purs et ces données ont été comparées à des huiles végétales et leurs compositions en TAG mesurées. MÉTHODES: La LC–MS a été utilisée pour identifier les di‐ et triglycérides dans l\'huile de noix de coco, l\'huile de Camellia oleifera et l\'huile de graines de carthame. La pénétration de ces huiles végétales et des triglycérides individuels purs a été mesurée par une méthode d\'extraction différentielle. Des coupes transversales d\'huiles traitées avec de la trioléine marquée au C13 ont été étudiées par NanoSIMS pour visualiser l\'emplacement des triglycérides à l\'intérieur des cheveux. La résistance à la fatigue a été mesurée à l\'aide d\'une sollicitation constante pour générer une distribution de la survie. Des modèles du complexe de membrane cellulaire riche en lipides (CMC) ont été créés avec le rapport équimolaire en acide 18‐méthyleicosanoïque (MEAS), acide palmitique (C16:0) et acide oléique (C18:1). RÉSULTATS: La pénétration des TAG purs individuels a été confirmée pour toutes les longueurs de chaîne et le degré d\'insaturation a été testé avec une pénétration plus élevée pour les chaînes plus courtes et les acides gras insaturés. Une analyse détaillée de la composition de certaines huiles végétales a montré une grande variété de TAG et la pénétration a également été démontrée pour ces huiles. Le NanoSIMS et la modélisation ont confirmé que ces TAG pénètrent dans la CMC riche en lipides des cheveux et interagissent avec les acides gras qui composent le CMC. Toutes les huiles végétales ont produit une amélioration de la résistance à la fatigue par pénétration dans le CMC et il est proposé que ces huiles préviennent la formation et/ou la propagation de défauts dans le réseau CMC qui entraînent une rupture.
CONCLUSIONS: De nombreuses huiles végétales avec un large éventail de compositions de triglycérides peuvent pénétrer dans les cheveux et les données du NanoSIMS ont confirmé que ces huiles se divisent en complexe de membrane cellulaire riche en lipides. Les études de pénétration des TAG individuels qui se sont avérés présents dans ces huiles ont confirmé que les TAG de longueur de chaîne variable peuvent pénétrer et il existe une corrélation entre l\'augmentation de l\'efficacité de pénétration et les longueurs de chaîne plus courtes et la présence d\'une insaturation dans les chaînes d\'acides gras. Toutes les huiles étudiées ont montré des bienfaits en matière de résistance à la fatigue pour une seule fibre.

Nutrient-induced blooms of the globally abundant freshwater toxic cyanobacterium Microcystis cause worldwide public and ecosystem health concerns. The response of Microcystis growth and toxin production to new and recycled nitrogen (N) inputs and the impact of heterotrophic bacteria in the Microcystis phycosphere on these processes are not well understood. Here, using microbiome transplant experiments, cyanotoxin analysis, and nanometer-scale stable isotope probing to measure N incorporation and exchange at single cell resolution, we monitored the growth, cyanotoxin production, and microbiome community structure of several Microcystis strains grown on amino acids or proteins as the sole N source. We demonstrate that the type of organic N available shaped the microbial community associated with Microcystis, and external organic N input led to decreased bacterial colonization of Microcystis colonies. Our data also suggest that certain Microcystis strains could directly uptake amino acids, but with lower rates than heterotrophic bacteria. Toxin analysis showed that biomass-specific microcystin production was not impacted by N source (i.e. nitrate, amino acids, or protein) but rather by total N availability. Single-cell isotope incorporation revealed that some bacterial communities competed with Microcystis for organic N, but other communities promoted increased N uptake by Microcystis, likely through ammonification or organic N modification. Our laboratory culture data suggest that organic N input could support Microcystis blooms and toxin production in nature, and Microcystis-associated microbial communities likely play critical roles in this process by influencing cyanobacterial succession through either decreasing (via competition) or increasing (via biotransformation) N availability, especially under inorganic N scarcity.

The marine subsurface is a long-term sink of atmospheric carbon dioxide with significant implications for climate on geologic timescales. Subsurface microbial cells can either enhance or reduce carbon sequestration in the subsurface, depending on their metabolic lifestyle. However, the activity of subsurface microbes is rarely measured. Here, we used nanoscale secondary ion mass spectrometry (nanoSIMS) to quantify anabolic activity in 3,203 individual cells from the thermally altered deep subsurface in the Guaymas Basin, Mexico (3-75 m below the seafloor, 0-14°C). We observed that a large majority of cells were active (83%-100%), although the rates of biomass generation were low, suggesting cellular maintenance rather than doubling. Mean single-cell activity decreased with increasing sediment depth and temperature and was most strongly correlated with porewater sulfate concentrations. Intracommunity heterogeneity in microbial activity decreased with increasing sediment depth and age. Using a dual-isotope labeling approach, we determined that all active cells analyzed were heterotrophic, deriving the majority of their cellular carbon from organic sources. However, we also detected inorganic carbon assimilation in these heterotrophic cells, likely via processes such as anaplerosis, and determined that inorganic carbon contributes at least 5% of the total biomass carbon in heterotrophs in this community. Our results demonstrate that the deep marine biosphere at Guaymas Basin is largely active and contributes to subsurface carbon cycling primarily by not only assimilating organic carbon but also fixing inorganic carbon. Heterotrophic assimilation of inorganic carbon may be a small yet significant and widespread underappreciated source of labile carbon in the global subsurface.
OBJECTIVE: The global subsurface is the largest reservoir of microbial life on the planet yet remains poorly characterized. The activity of life in this realm has implications for long-term elemental cycling, particularly of carbon, as well as how life survives in extreme environments. Here, we recovered cells from the deep subsurface of the Guaymas Basin and investigated the level and distribution of microbial activity, the physicochemical drivers of activity, and the relative significance of organic versus inorganic carbon to subsurface biomass. Using a sensitive single-cell assay, we found that the majority of cells are active, that activity is likely driven by the availability of energy, and that although heterotrophy is the dominant metabolism, both organic and inorganic carbon are used to generate biomass. Using a new approach, we quantified inorganic carbon assimilation by heterotrophs and highlighted the importance of this often-overlooked mode of carbon assimilation in the subsurface and beyond.

Plastic pollution in the marine realm is a severe environmental problem. Nevertheless, plastic may also serve as a potential carbon and energy source for microbes, yet the contribution of marine microbes, especially marine fungi to plastic degradation is not well constrained. We isolated the fungus Parengyodontium album from floating plastic debris in the North Pacific Subtropical Gyre and measured fungal-mediated mineralization rates (conversion to CO2) of polyethylene (PE) by applying stable isotope probing assays with 13C-PE over 9 days of incubation. When the PE was pretreated with UV light, the biodegradation rate of the initially added PE was 0.044 %/day. Furthermore, we traced the incorporation of PE-derived 13C carbon into P. album biomass using nanoSIMS and fatty acid analysis. Despite the high mineralization rate of the UV-treated 13C-PE, incorporation of PE-derived 13C into fungal cells was minor, and 13C incorporation was not detectable for the non-treated PE. Together, our results reveal the potential of P. album to degrade PE in the marine environment and to mineralize it to CO2. However, the initial photodegradation of PE is crucial for P. album to metabolize the PE-derived carbon.

The study of the whole of the genetic material contained within the microbial populations found in a certain environment is made possible by metagenomics. This technique enables a thorough knowledge of the variety, function, and interactions of microbial communities that are notoriously difficult to research. Due to the limitations of conventional techniques such as culturing and PCR-based methodologies, soil microbiology is a particularly challenging field. Metagenomics has emerged as an effective technique for overcoming these obstacles and shedding light on the dynamic nature of the microbial communities in soil. This review focuses on the principle of metagenomics techniques, their potential applications and limitations in soil microbial diversity analysis. The effectiveness of target-based metagenomics in determining the function of individual genes and microorganisms in soil ecosystems is also highlighted. Targeted metagenomics, including high-throughput sequencing and stable-isotope probing, is essential for studying microbial taxa and genes in complex ecosystems. Shotgun metagenomics may reveal the diversity of soil bacteria, composition, and function impacted by land use and soil management. Sanger, Next Generation Sequencing, Illumina, and Ion Torrent sequencing revolutionise soil microbiome research. Oxford Nanopore Technology (ONT) and Pacific Biosciences (PacBio)\'s third and fourth generation sequencing systems revolutionise long-read technology. GeoChip, clone libraries, metagenomics, and metabarcoding help comprehend soil microbial communities. The article indicates that metagenomics may improve environmental management and agriculture despite existing limitations.Metagenomics has revolutionised soil microbiology research by revealing the complete diversity, function, and interactions of microorganisms in soil. Metagenomics is anticipated to continue defining the future of soil microbiology research despite some limitations, such as the difficulty of locating the appropriate sequencing method for specific genes.

BACKGROUND: Global warming is causing large-scale disruption of cnidarian-Symbiodiniaceae symbioses fundamental to major marine ecosystems, such as coral reefs. However, the mechanisms by which heat stress perturbs these symbiotic partnerships remain poorly understood. In this context, the upside-down jellyfish Cassiopea has emerged as a powerful experimental model system.
RESULTS: We combined a controlled heat stress experiment with isotope labeling and correlative SEM-NanoSIMS imaging to show that host starvation is a central component in the chain of events that ultimately leads to the collapse of the Cassiopea holobiont. Heat stress caused an increase in catabolic activity and a depletion of carbon reserves in the unfed host, concurrent with a reduction in the supply of photosynthates from its algal symbionts. This state of host starvation was accompanied by pronounced in hospite degradation of algal symbionts, which may be a distinct feature of the heat stress response of Cassiopea. Interestingly, this loss of symbionts by degradation was concealed by body shrinkage of the starving animals, resulting in what could be referred to as \"invisible\" bleaching.
CONCLUSIONS: Overall, our study highlights the importance of the nutritional status in the heat stress response of the Cassiopea holobiont. Compared with other symbiotic cnidarians, the large mesoglea of Cassiopea, with its structural sugar and protein content, may constitute an energy reservoir capable of delaying starvation. It seems plausible that this anatomical feature at least partly contributes to the relatively high stress tolerance of these animals in rapidly warming oceans. Video Abstract.

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 ~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 15N2 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.