ISME Communications

Heterotrophic flagellates play a major role in nutrient recycling and shaping aquatic microbial communities. Many motile species have evolved diverse predatory strategies to exploit microbial prey, from bacteria to small eukaryotes. Excavates, deep-branching eukaryotic lineages with distinctive biflagellate morphology and conserved cytoskeletal features, exhibit varied feeding behaviours, including grazing, raptorial feeding, and filter-feeding. However, the temporal dynamics of their motility, feeding, and spatial distribution in relation to bacterial population density remain under-explored. Here, we quantify the behavioural ecology of excavates using high-resolution, single-cell tracking and quantitative analysis. Focussing on two Discoba species - the marine Jakoba libera and freshwater Reclinomonas americana - we identify different behavioural patterns: J. libera swims, forages and feeds, while R. americana swims and feeds. Prey availability differentially affects motility: J. libera increases the number of swimming cells when food is scarce, while R. americana shows the opposite trend. Spatial statistics reveal fundamental differences in their feeding and redistribution strategies. J. libera acts as an active forager, whereas R. americana behaves as a strict filter feeder. A third species, the anaerobic Kipferlia bialata (from a different clade), adopts a foraging strategy similar to J. libera. Together, these results elucidate the adaptive relationship between prey availability, feeding modes, and motility in bacteriovorous excavate flagellates, offering new insights into the ecological and evolutionary dynamics of microbial predators.

Anoxygenic phototrophic green sulfur bacteria (GSB; Chlorobiia) are important primary producers in anoxic and sulfidic environments, whose significance in aquatic ecosystems will expand with coastal deoxygenation. Here, we employed a uniquely comprehensive analytical approach to investigate the formation, maintenance, and collapse of a GSB bloom. We combined multi-omics -- V4V5 and synthetic long read 16S rRNA amplicon sequencing, metagenomics, and metaproteomics -- with total and GSB-specific cell counts, biogeochemical measurements, and isotopic analysis. The GSB bloom exceeded 109 cells ml-1, among the highest environmental cell densities reported to date, with up to 96 % of the bloom consisting of a single strain-level Prosthecochloris lineage (GSB-TRL01). Extreme sulfide concentrations (> 17 mM) coincided with peaking cell density and a shift in isotopic composition. Bloom persistence is supported by tightly coupled sulfur cycling, high rates of nitrogen fixation, mechanisms to tolerate oxidative stress and maintain redox balance, and nutrient acquisition through outer membrane transport systems. A shift in sulfur oxidizing enzymes towards enzymes with higher sulfide affinity proceeded bloom demise. Prosthecochloris GSB-TRL01 differs from closely related lineages in several outer membrane transport systems, including porins to transport phosphate and tonB-dependent transporters. Collectively, these findings identify physiological and metabolic strategies that may enable near-clonal Prosthecochloris populations to attain extraordinary biomass while coupling the sulfur, carbon, and nitrogen cycles in coastal euxinic environments.

Lipids have the potential to trace trophic interactions through an ecosystem, but their use has been largely limited to predator/prey-scale studies. Carnivore fecal lipids offer an unexplored way to strategically monitor the health and spatial distribution of ecosystems that are difficult to access. This hypothesis is explored here in seagrass meadows, which are a globally significant long-term carbon storage reservoir. This study tested the hypothesis that lipids can be used to passively monitor seagrass meadow health by tracing fatty acids transferred up the food chain and onto nearby beaches via coastal bird stool. By comparing the lipids produced by seagrasses and those made by diverse microbial eukaryotes, such as diatoms and dinoflagellates, our analysis could distinguish between healthy and collapsed meadows in a pilot dataset. A metabolite that is produced by the seagrass wasting disease pathogen was also recovered in our data, providing a potential diagnostic for seagrass health. The advantages to this non-invasive monitoring approach using lipids are that beaches are far more accessible than seagrass meadows, and birds spatially sample a larger area than researchers could do efficiently. Although this pilot study was limited in samples and field sites, our results provide compelling reasons to pursue carnivorous fecal dietary lipids as a source of ecosystem data.

Cyanobacteria are key prokaryotic primary producers in diverse ecosystems, yet the role of cyanobacterial siderophores in shaping their associated microbiomes remains unexplored. Our study demonstrates the benefits provided to the heterotrophic co-habitants of filamentous cyanobacteria in terrestrial microbial biofilms, focusing on the recently discovered widespread siderophores cyanochelins. To address the acceptance of cyanochelin B (CychB) across multiple bacterial classes, we first investigated its role in providing iron to a model siderophore producer P. aeruginosa PAO1 and selected Pseudomonas natural isolates, which were found to utilize CychB under iron limiting conditions while downregulating endogenous siderophore production. In response to CychB, PAO1 expresses a siderophore internalization cluster, which is localized in multiple Pseudomonas natural isolates. Using metagenome analysis, we characterized the bacterial community recruited along with CychB producing Phormidesmis cyanobacteria under long-term iron starvation. Potential CychB acceptor bacteria associated with the CychB producer were predominantly lacking endogenous siderophore machineries. Using siderophore selective pressure, we isolated a genuine CychB acceptor, gram-negative bacterium Methyloversatilis sp. S146 and demonstrated that its genome hosts an iron processing cluster overexpressed after CychB feeding, recognizing Methyloversatilis as a candidate for further mechanistic investigation of iron acquisition-driven microbial interactions. Our results indicate that CychB supports a specific subset of co-habiting heterotrophic bacteria during iron starvation, further emphasizing the role of cyanobacteria as key drivers of nutrient flows within globally important microbial soil crust ecosystems, supporting microbial life in nutrient-limited environments. These findings provide a mechanistic foundation to elucidate the role of cyanochelins as a public good in these communities.

Harmful algal bloom (HAB) succession in eutrophic lakes arises from seasonal rebalancing of selection, dispersal, and drift across interacting microbial domains, yet process attribution remains limited due to the scarcity of cross-domain analyses. Using multi-marker eDNA metabarcoding (16S rRNA, 18S rRNA, 23S rRNA), we show that bacterioplankton, cyanobacteria, phytoplankton, and zooplankton in Lake Taihu undergo coordinated spring-summer transitions, with inter-seasonal turnover far exceeding spatial heterogeneity. Assembly regimes shifted coherently across domains: spring communities were dominated by strong homogeneous selection--especially among cyanobacteria and microeukaryotes--whereas summer communities moved toward drift- or neutrality-like dynamics under bloom-stage mixing that enhanced dispersal. Null- and neutral-model diagnostics supported these trends. Cross-domain co-occurrence networks restructured seasonally, with summer networks becoming more connected and substantially more robust, indicating that bloom conditions foster cohesive, robust interaction structures rather than destabilization. These findings provide evidence that bloom progression restructures aquatic microbiomes through a seasonal rebalance of selection, drift, and dispersal coupled with adaptive strengthening of cross-domain connectivity, providing a process-explicit framework for understanding community stability and ecosystem resilience in eutrophic lakes.

The Deep Chlorophyll Maximum (DCM) is likely the most important feature organizing the marine epipelagic environment. Within this layer, opposing gradients of light and nutrients create a stratified habitat that supports high phytoplankton biomass and a substantial fraction of oceanic primary production. Despite its ecological importance, most studies treat the DCM as a single depth, overlooking its fine-scale heterogeneity. Here we investigated prokaryotic community organization across the DCM in the northwestern Mediterranean Sea through high-resolution sampling of four profiles collected over two days. Free-living (0.2-3 {micro}m) and particle-associated (3-20 {micro}m) communities were characterized using 16S rRNA gene amplicon sequencing. Prokaryotic communities changed progressively along the vertical gradient, revealing the DCM as a microbial coenocline with continuous community turnover. Fuzzy clustering identified distinct assemblages associated with environmental transitions from warm surface waters to the chlorophyll maximum, the nitrite peak below the DCM, and deeper nitrate-rich layers. In both the free-living and particle-associated fractions, most ASVs remained consistently associated with the same depth-defined clusters across all samplings, indicating stable niche partitioning over short timescales. However, these temporally stable ASVs accounted for a substantially smaller fraction of community sequences in particle-associated communities, suggesting higher dynamism, likely driven by particle-mediated transport. Nevertheless, phylogenetic analyses revealed that closely related ASVs tended to occupy similar depth niches, indicating that habitat preferences are phylogenetically conserved in both size fractions. Our results demonstrate prokaryotic niche partitioning over scales of only a few meters within the DCM, highlighting the importance of fine-scale sampling for understanding microbial community structure and responses to ocean change.

Sialic acids (Sias) and related nonulosonic acids are critical components of glycoconjugates involved in host-pathogen interactions, immune regulation, and cell signalling. Despite their biotechnological relevance, the diversity of enzymes involved in Sia biosynthesis remains largely underexplored due to limitations in culture-dependent methods and the lack of (ultra)high-throughput screening strategies. Here, we report the development of a highly sensitive droplet-based microfluidic screening platform enabling the functional discovery of sialic acid aldolases in environmental metagenomes. The method integrates a fluorescence-coupled enzymatic cascade compatible with fluorescence-activated droplet sorting (FADS), allowing the screening of >10 droplets per experiment, as well as a downstream validation strategy for the selected hits. Although some limitations were identified, the system demonstrated high sensitivity and was utilised for the screening of a metagenomic library from garden soil. During this campaign, a potential new sialic acid aldolase enzyme was identified. This work establishes a generalizable framework for measuring complex, multi-step enzymatic functions at ultrahigh throughput using coupled cascades in droplets

As circular agricultural inputs are increasingly sought, feather hydrolysate (FH), derived from keratin-rich poultry waste, has emerged as a promising fertilizer and biostimulant. However, how FH shapes soil microbiome succession over time, and what functional potential this restructuring carries, remains poorly understood. Here, we used multi-marker high-throughput sequencing of 16S, 18S, and ITS rDNA to track soil microbial communities over 30 days following FH amendment, with sampling at days 0, 1, 4, 14, and 30. FH induced a rapid and reproducible microbial succession characterized by an early decline in alpha diversity and a pronounced day-1 bloom of copiotrophic taxa, as inferred from predicted rrn operon copy-number profiles. This initial response was accompanied by enrichment of bacterial genera previously associated with plant growth-promoting traits, including pathogen suppression, nutrient solubilization, siderophore production, phytohormone synthesis, and ACC deaminase activity. Although these groups gradually declined after the initial peak, several remained at significantly elevated abundances throughout the 30-day experimental period. In parallel, FH-treated soils showed a sustained reduction in the cumulative relative abundance of genera annotated as putative phytopathogens. By integrating temporal community profiling, rrn-based life-strategy inference, and literature-derived functional annotation, we show that FH acts as a strong ecological filter, shifting the soil microbiome toward copiotrophic and putatively plant-beneficial taxa. These findings provide a temporal framework for understanding FH-mediated microbiome restructuring and highlight the need for future studies linking inferred microbial functional potential to measured disease incidence, nutrient fluxes, and plant performance across diverse soil contexts.

The Arctic Ocean microbiomes experience extreme seasonal fluctuations in light, nutrient availability, and organic carbon supply. In this environment, neutral lipid storage may provide a key survival strategy. Here, we investigated the diversity, distribution, and ecological role of neutral lipid metabolism in Arctic microbiomes using metagenome-resolved analyses and global ocean comparisons. Arctic photic-zone microbiomes were strongly enriched in triacylglycerol (TAG) biosynthesis genes relative to other oceans, primarily due to picoeukaryotic phytoplankton, including the ecologically dominant Micromonas and Bathycoccus. In contrast, prokaryotic communities exhibited diverse TAG-degrading taxa and fatty acid transport systems, supporting a previously unrecognized lipotrophic bacterial guild exploiting phytoplankton-derived lipids as carbon and energy sources. Genome-resolved analyses further revealed distinct bacterial lipid-storage strategies: TAG-producing taxa preferentially encoded fatty acid uptake and carbohydrate utilization pathways, whereas polyhydroxyalkanoate-producing taxa were associated with aromatic compound degradation, linking terrestrial organic matter to lipid storage. Our results expand the known diversity of marine microbes capable of neutral lipid metabolism and identify microbial lipid cycling as a previously overlooked component of the Arctic carbon cycle. We propose that neutral lipid storage and turnover support microbial survival through the polar night while enhancing carbon transfer within Arctic microbial food webs under ongoing climate change.

Aerobiological studies have largely focused on near-surface sampling and horizontal biogeographic patterns, while vertical structuring of airborne microbial communities within the atmospheric boundary layer (ABL) remains poorly understood. Here, we investigated microbial communities across the lower and upper ABL in a low-orography coastal site on the Antarctic Peninsula, representative of the Southern Ocean marine ABL and with low direct human influence. Airborne microorganisms were sampled simultaneously using ground-based and aerial platforms on five occasions. Community composition, abundance, and cell morphometry were assessed using metabarcoding and epifluorescence microscopy and interpreted alongside atmospheric observations. Airborne bacterial and eukaryotic communities showed consistent vertical stratification, although partial taxonomic overlap indicates vertical connectivity between atmospheric layers. Lower ABL communities were more diverse than upper ABL counterpart, compositionally homogeneous, and dominated by marine-associated taxa, reflecting strong influence from local sources and turbulent mixing. In contrast, upper ABL communities were less diverse but more heterogeneous among sampling events, enriched in stress-tolerant, terrestrial and plant-associated taxa, consistent with atmospheric filtering, selective upward transport, and long-range atmospheric inputs. Upper-layer samples also exhibited higher microbial abundance and greater prevalence of elongated cell morphologies, suggesting particle accumulation aloft and aerodynamic selection permanence. Together, these findings identify the Southern Ocean ABL as a vertically structured microbial habitat organized into two partially decoupled sublayers, in which atmospheric dynamics regulate microbial dispersal, ecosystem connectivity, and biogeographic patterns.

Gram-negative bacteria belonging to the phylum Bacteroidota are prominent and ecologically important members of host-associated microbiomes. A feature of Bacteroidota species is the presence of diverse outer membrane TonB-dependent transporter and cognate surface exposed lipoprotein (TBDT-SLP) complexes. Many 'SusCD-like' TBDT-SLPs coordinate complex carbohydrate capture from the environment. Here, we show the model soil Bacteroidota species Flavobacterium johnsoniae synthesises two TBDT-SLP complexes (named PusCD1 and PusCD2) that are essential for growth on phosphatidylinositol, a member of an abundant lipid class within plant plasma membranes, as a phosphorus source. Structural characterisation of ligand-bound PusCD1 complex from F. johnsoniae membranes reveals a new binding mechanism for organophosphorus and broadens the known functional repertoire of this class of transporters. Diverse PusCD-like complexes are widespread among plant associated Bacteroidota, pointing to a major, previously unrecognised route for the recycling of organophosphorus compounds in the rhizosphere, with potential implications for engineering microbial-mediated phosphorus recovery from organic waste streams.

Phage tail-like elements (PTEs) -- tailocins, bacterial type VI secretion systems (T6SS), and extracellular contractile injection systems (eCIS) -- are contractile nanomachines that bacteria use to kill their neighbors and compete within their micro-ecosystems. PTEs help shape microbial community composition. Most PTE detection tools only detect a single PTE class. Moreover, most tailocin detection methods are largely restricted to Pseudomonas, leaving a key part of tailocin diversity uncharacterized. In this work, we present PhageTAILor (https://github.com/hjcho-bio/PhageTAILor), an integrative and fully automated pipeline that detects and classifies prophages and 3 PTE classes from bacterial genomes. PhageTAILor combines a 6-detector homology-based candidate search (geNomad, tail-gene, PHROGs-tail, SecReT6, eCIStem, and a divergence-tolerant tail-HMM detector) with a LightGBM classifier comprising 1 multiclass and 3 binary heads, trained on 6,501 bacterial genomes carrying 13,082 prophages and PTEs. A phylogeny-free feature matrix used in our model keeps predictions reproducible between model construction and user inference. PhageTAILor performs strongly at the genome level and generalizes beyond its Pseudomonas-rich training set. On a 76-strain cross-clade benchmark, PhageTAILor detected tailocins at F1 = 0.955. Furthermore, it identified 12 of 13 experimentally validated tailocins spanning five genera versus 2 of 13 for a Pseudomonas-restricted tool TattleTail. PhageTAILor also demonstrated sensitivity equivalent to viral detection tool geNomad while avoiding its higher false-positive rate. Applied to 7,925 plant- and soil-associated bacterial isolates, PhageTAILor showed that prophages in the phyllosphere and tailocins in plant-associated bacteria, whereas eCIS are enriched in soil. PhageTAILor is distributed as an open-source, modular pipeline with a command-line interface.

Many wet tropical soils alternate frequently between fully oxygenated and anaerobic conditions, constraining the terminal electron acceptors available for microbial metabolism, and the mineral-organic matter interactions that regulate many aspects of soil carbon (C) cycling. However, it is still unclear how fluctuating soil redox conditions influence the microbial community composition and if microbially mediated C flux is sensitive to extended oxic or anoxic periods like those observed during drought and flooding respectively. Using a 44-day redox manipulation of tropical soils that experience daily-to-weekly oxygen (O2) fluctuations in the field, we measured how different redox regimes shape soil biogeochemistry and microbial and metabolite composition. Replicate microcosms were exposed to four treatments (static oxic, static anoxic, high frequency fluctuation (4 day oxic/4 day anoxic), or low frequency fluctuation (8 day oxic/4 day anoxic)) regimes, and harvested for microbial, metabolite, carbon dioxide (CO2) flux, and biogeochemical assays at multiple timepoints. Oxic and fluctuating redox conditions caused the microbial community to shift in a manner correlated with soil iron content and directly orthogonal to communities from anoxic soils. The identity of both iron oxidizers and iron reducers was distinct in static anoxic soils but was resilient to redox fluctuation and prolonged O2 exposure. The total amount of CO2 respired was similar across all four redox regimes. Water-extractable organic matter composition was distinct across redox treatments, with anoxic soils accumulating higher levels of carbohydrate-, proteins-, amino sugar-, and lignin-like compounds consistent with reduced enzymatic decomposition and release of mineral-associated organic matter via iron reduction, while oxic soils showed elevated lipid- and unsaturated hydrocarbon-like compounds indicative of greater microbial biomass turnover. The microbial community adapted to dynamic redox conditions and the results substantiate cycling of distinct C compounds under varying redox conditions resulting from varying bioavailability (driven by mineral-OM dynamics) and/or shifted microbial metabolism.

The Eboliang Hu saline lakes in the hyper-arid Qaidam Basin is a high-altitude, weakly acidic hypersaline system with strong environmental gradients and limited nitrogen availability. To resolve its microbial ecology and evolutionary context, we performed genome-resolved metagenomic sequencing across four distinct habitats, reconstructing 46 medium- to high-quality metagenome-assembled genomes (MAGs) and a comprehensive gene catalog. The community shows pronounced spatial heterogeneity and is dominated by Thermodesulfobacteriota, Pseudomonadota, Bacteroidota, and archaeal lineages. Phylogenomic placement and large-scale sequence comparisons indicate that multiple dominant taxa exhibit affinity to marine- and subsurface-associated reference lineages, consistent with long-term isolation of a marine-derived ecosystem about 10-11 million years ago. Functional reconstruction reveals a distributed metabolic system in which carbon, nitrogen, and sulfur cycling are partitioned across taxa. Notably, hydrogen oxidation and arsenite oxidation are recurrent energy-producing strategies across dominant lineages, indicating redox flexibility under oligotrophic conditions. Comparative genomics further suggests lineage-specific adaptations to osmotic stress, UV exposure, and nutrient limitation. Horizontal gene transfer and phylogenetic incongruence among key metabolic genes indicate that co-evolutionary processes and gene exchange have contributed to functional innovation. These findings provide a framework for understanding microbial persistence and evolution in isolated extreme environments and offer potential analogs for extraterrestrial habitability.

Protists play important roles in food chains and symbioses in soil and aquatic environments, displaying an enormous morphological and functional diversity. While most commonly found protist species are well known to science, our global-scale environmental DNA survey across soil, water, and sediments reveals dozens of novel, phylum-level phylogenetic lineages that remain to be characterized for basic morphology and function. A vast majority of these undescribed taxa occur in marine water and sediments, but some are common in soil. Most of these novel taxa have distinct substrate and habitat preferences and biogeographic patterns. To accord these lineages scientific agency and enable unambiguous scientific communication, we propose formal names for 150 species to phylum-level taxa from 25 deep lineages based on eDNA and rRNA gene long-read sequence information.

Phytoplankton are primarily confined to the sunlit epipelagic zone, yet intact algal cells persist in the oceans mesopelagic twilight zone where light is minimal. However, their survival strategies in the deeper ocean remain unknown. Here, we isolate Chaetoceros sp. DS1 from 1000 m depth and reveal its survival mechanisms under prolonged darkness and high pressure, including membrane lipid remodeling and enhanced antioxidant defenses. Importantly, DS1 maintains a latent photosynthetic reserve that activates upon exposure to dim blue light and higher pressure typical of the twilight zone, enabling photosynthesis and nutrient uptake. It exhibits metabolic flexibility by utilizing stored carbon in darkness and assimilating organic carbon under dim light. Global meta-omics data show widespread, transcriptionally active Chaetoceros-like diatoms in the twilight zone, expressing blue-light sensors and protein synthesis genes. These findings identify the ocean twilight zone as a previously overlooked niche for metabolically active phytoplankton, expanding understanding of deep-ocean microbial ecology and carbon cycling.

Microbial symbioses drive the evolutionary and functional diversification of eukaryotic clades, from single-celled protists to large invertebrates. However, our knowledge of host-associated assemblages (the "holobiont") is limited in microscopic animal phyla with a body size <1mm, due to practical challenges such as low biomass and difficult taxonomy of host species. Marine nematodes represent an ideal case study for rapidly advancing our knowledge of bacterial-animal symbioses, representing a globally abundant invertebrate group with strong links to terrestrial and model organism species within the same phylum. Here, we sequenced the holobionts of 220 marine nematodes and generated 815 metagenome-assembled genomes (MAGs) of host-associated bacteria/archaea. Our data indicates that 20-34% of marine nematodes harbor an obligate intracellular symbiont, often with multiple endosymbionts co-occurring within the same host. Three bacterial phyla (Pseudomonadota Bacteroidota, and Verrucomicrobiota) account for three-quarters of all nematode-associated MAGs, and the majority of these holobiont MAGs represent deeply divergent lineages in the prokaryotic tree of life. The Flavobacteriaceae (a core microbiome taxon in C. elegans and other terrestrial nematodes), were consistently recovered across phylogenetically diverse marine nematode lineages, suggesting evolutionary conservation of holobiont taxa across marine and terrestrial environments. We also report a novel chemoautotroph family (Ca. Thionematobacter) recovered from nematode hosts in both deep-sea and shallow-water habitats, and report the first confirmed instance of Cardinium endosymbionts from marine invertebrates. Finally, [~]65% of nematode-associated MAGs are able to degrade chitin, via hexosaminidase, implying that benthic invertebrate holobionts make significant contributions to global carbon cycling. These results underline the importance of evaluating symbiosis in microscopic marine invertebrates, and accelerating our understanding of animal evolution and ecosystem dynamics in vast benthic habitats.

Rivers transport most land-derived plastic waste to the ocean, yet how this reshapes microbial metabolic potential remains poorly resolved. We characterized plastisphere and water-column communities across 14 stations along the River Rhine, integrating ATR-FTIR polymer characterization, 16S/18S rRNA amplicon profiles, and 120 metagenome-assembled genomes. Plastisphere communities were taxonomically distinct from water communities (PERMANOVA R = 0.258, p = 0.0001) and, despite no overall shift in functional centroid (R = 0.245, p = 0.125), were markedly more heterogeneous in functional composition across sites (permutest p = 0.0047). Aerobic corrin ring synthesis, the core B12 biosynthetic pathway, was among the most differentially dispersed functions and enriched on plastic at all seven paired stations (Wilcoxon exact test, W = 3, p = 0.004079). This signal coincided with a diatom-dominated eukaryotic plastisphere community; diatoms cannot synthesize B12 and depend on bacterial provisioning, linking functional and taxonomic restructuring via a plausible cross-domain mechanism. Plastisphere communities were also less tightly coupled to the river's dissolved nutrient gradient than water communities (envfit R = 0.64 vs. 0.82). Together, these results indicate that riverine plastic litter does not merely accumulate biomass passively but actively restructures specific metabolic capacities of its colonizers, exemplified by vitamin B12 metabolism.

Oligotrophic subtropical waters are some of the ocean's largest continuous ecosystems, yet their protist diversity and community succession remain poorly understood. We examined the protist community in the surface waters of the Gulf of Aqaba at near-daily resolution from March 2024 to July 2025 using an Imaging FlowCytobot. The record revealed a clear seasonal succession. Total cell density increased nearly threefold during winter mixing, driven mainly by an increase in cells smaller than 5 microns. Cells were generally larger during summer, making total biovolume similar in summer and winter. Biovolume reached its highest sustained values during the spring bloom. Despite its low cell density, the summer community was dynamic, with sudden appearances of euglenophytes, flagellates and dinoflagellates at high densities. Coccolithophores and cryptophytes increased during winter, followed by diatoms and ciliates in spring. We also provide a dataset of 31,700 annotated IFCB images.

The murine gut microbiota provides robust colonization resistance against Clostridioides difficile infection (CDI), yet murine-associated microbes remain notoriously difficult to cultivate in vitro, limiting mechanistic investigation. To identify the ecological and nutritional basis of this cultivation barrier, we leveraged CDI susceptibility as a functional readout of microbial community metabolism to infer in vivo nutrient utilization. Germ-free C57BL/6 mice colonized with varying dilutions of ethanol-treated murine microbiota were challenged with C. difficile resulting in a spectrum of CDI outcomes. Comparative metabolomics of pre-challenge fecal samples revealed a consistent carbohydrate signature: glucose accumulated in communities that resisted C. difficile challenge, whereas complex carbohydrates, including raffinose, sucrose, trehalose, lactose, sorbitol, and mannitol, were significantly depleted. The broad depletion of these complex carbohydrates supports their functional importance within the collective microbial community. Conventional glucose-based media (CMA, BHI+I, RCMT) failed to support robust growth or subculture of murine gut microbiota. Guided by the metabolomics findings, we developed Peptone Yeast Extract with Six Salts and Sugars (PYE6S), a glucose-free medium supplemented with the complex carbohydrates identified as depleted. PYE6S enabled cultivation of 22 unique Firmicutes ASVs, 82% of which lacked named cultured representatives in reference databases. These findings suggest a plausible explanation for why conventional media fail and support a metabolomics-guided framework for rational cultivation of host-associated microbiota across diverse systems. This strategy may be extended to guide media design for other host-associated microbiotas.