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Viral communities from long-term anaerobic alkane-oxidizing enrichment cultures encode predicted cell surface adhesion functions.

The anaerobic oxidation of methane and C2+-alkanes is a dominant metabolism within hydrocarbon-rich deep-sea sediments and is largely mediated by alkane-oxidizing archaea in metabolic partnership with syntrophic sulfate-reducing bacteria. Although these processes fuel a diverse ecosystem, the viral component of alkane-rich sediments has historically been overlooked. We analyzed the viral assemblages in long-term sediment-free cultures of alkane-degrading organisms and found that abiotic factors such as incubation temperature had a greater correlation with community composition than with the phylogenetic patterns among individual viral species. No auxiliary metabolic genes (AMGs) directly involved in hydrocarbon oxidation or sulfate reduction were found, but the presence of candidate AMGs involved in heme synthesis pathways common in methane oxidizers hints at a possible viral impact on alkane degradation. We also examined potential host-virus pairs using CRISPR- and tRNA-based methods. Lastly, we identified the presence of nosD-like proteins in viruses from sediment-derived systems that are not present in water column datasets; their distribution, genomic context, and lack of canonical nosD characteristics suggest an alternate adhesion-related role in sediment communities. The number of new viruses obtained from these multi-year enrichment cultures and their potential roles in mediating host physiology illustrate the importance of studying the viral component in laboratory and environmental systems.

Geologic Sediments

Flavobacteria consume nitrous oxide produced by partial denitrifiers in coastal sediments.

Nearly one-fifth of global emissions of the potent greenhouse gas nitrous oxide (N2O) originate from the ocean, particularly from nutrient-polluted coastal regions. Permeable (sandy) sediments, which cover half of the continental shelf worldwide, are potential sources of N2O due to increasing nutrient inputs from urbanization and agriculture. Yet, the microbial processes determining N2O emissions in these dynamic and unique ecosystems remain understudied. Here, we combined environmental measurements, bacterial cultivation, and genomic analyses to understand the microbes and processes controlling N2O cycling in permeable sediments from Port Phillip Bay (Australia). We established a genomic resource comprising 249 metagenome-assembled genomes and 95 new isolate genomes. Genome-based metabolic reconstructions and culture-based gas measurements revealed that diverse bacteria in these sediments produce N2O through incomplete denitrification pathways. However, these bacteria co-occurred with highly abundant clade II N2O-reducing bacteria from the Flavobacteriaceae family. Kinetic profiling showed that both clade II nosZ flavobacterial isolates and whole sand communities exhibited a low apparent affinity for N2O under the tested experimental conditions, expanding the currently limited kinetic data available for N2O reducing microorganisms from coastal permeable sediments, including flavobacterial clade II N2O reducers. Collectively, these findings indicate that abundant N₂O reducing communities can substantially consume N2O within permeable sediments, thus limiting N2O accumulation despite active N2O production. Together with previous hydrodynamic models predicting low N2O release from permeable sediments, our results highlight the important role of specialized microbial communities in regulating N2O cycling under increasing nutrient pollution.

Nitrous Oxide

A prolonged hydrothermal past at Santorini Caldera revealed by sedimentary trace metal and microbial signatures.

Hydrothermal systems in volcanic calderas are critical in signalling volcanic unrest, forming ore deposits, and sustaining chemosynthetic microorganisms. Analysis of a ~3500-year sequence of sediments collected from the Santorini caldera, Greece, during International Ocean Discovery Program (IODP) Expedition 398 reveals the behaviour of a prolonged paleo-hydrothermal system. Sediment geochemical and metagenomic data record vigorous hydrothermal activity and metal fluxes for ~1100 years, within a 2270-year window between two major eruptions. Sediment hydrothermally-derived trace metals are significantly enriched over background (~200-fold for As and Hg, and 10-50-fold for Mn, Sb, Mo, and V), with long-term metal fluxes (9 t yr-1 As, 2.5 t yr-1 Cu, 7 kg yr-1 Ag) comparable to fluxes from present-day geothermal fields in the Taupo Volcanic Zone. Metagenomic analysis identifies elevated metal resistance genes-signals of microbial adaptation to heightened hydrothermal stressors. Here we integrate geological and genomic evidence to decipher the paleoenvironmental and biogeochemical history of the past hydrothermal system at Santorini caldera.

Geologic Sediments

sedimix: a workflow for the analysis of hominin nuclear DNA sequences from sediments.

SUMMARY: Sediment DNA-the recovery of genetic material from archaeological sediments-is an exciting new frontier in ancient DNA research, offering the potential to study individuals at a given archaeological site without destructive sampling. In recent years, several studies have demonstrated the promise of this approach by extracting hominin DNA from prehistoric sediments, including those dating back to the Middle or Late Pleistocene. However, a lack of open-source workflows for analysis of hominin sediment DNA samples poses a challenge for data processing and reproducibility of findings across studies. Here, we introduce a snakemake workflow, sedimix, for processing genomic sequences from archaeological sediment DNA samples to identify hominin sequences and generate relevant summary statistics to assess the reliability of the pipeline. By performing simulations and comparing our results to two published studies with human DNA from ∼25,000 years ago (including shotgun data from a sediment sample and capture data from touch DNA recovered from a deer tooth pendant) we demonstrate that sedimix yields accurate and reliable inferences. sedimix offers a reliable and adaptable framework to aid in the analysis of sediment DNA datasets and improve reproducibility across studies. AVAILABILITY AND IMPLEMENTATION: sedimix is available as an open-source software with the associated code, example data, and user manual with installation instructions available at https://github.com/jierui-cell/sedimix. A permanent archived version of this release is available via Zenodo: https://doi.org/10.5281/zenodo.17244854.

Animals

Tracking microplastic contamination across seasons in a freshwater reservoir: Evidence from surface water, sediments, and fishes.

Microplastics (MPs) are prevalent contaminants in aquatic environments, posing substantial ecological and health risks. These particles migrate within the different layers of aquatic bodies with time and affect the respective biota. Thus, to get an in-depth understanding of the particles, this current study investigated the seasonal distribution, morphological and chemical characteristics, along with potential ecological and human health impacts in the samples including surface water, sediment, and fish from a drinking water supplying reservoir in eastern India. Across three different seasons, pre-monsoon, monsoon, and post-monsoon samples were collected using optimized methods. Results revealed distinct seasonal trends: MP abundance in surface water peaked during the monsoon (mean: 1.15 MPs/L), while sediment showed the highest concentrations in the pre-monsoon (mean: 596 MPs/kg), indicating temporal accumulation dynamics influenced by runoff, hydrodynamics, and sedimentation. Fish gut analysis confirmed ingestion of MPs across five species, with concentrations ranging from 26 to 100 MPs/kg, depending on feeding habits. The most dominant MP type were fragments, followed by fibers, films, and beads. Polymer analysis via µFTIR identified polyethylene, polypropylene, and polyvinyl chloride as prevalent, with hazard assessments (i.e., Polymer Hazard Index (PHI)) indicating medium to very high ecological risks. Heavy metal association was more dominant in the MPs isolated from sediments than the waterborne MPs. Pollution Load Index (PLI) values were > 1 in most seasons, confirming contamination. Health risk analysis suggested potential exposure through both drinking water and fish consumption. This study emphasizes the need for seasonal monitoring, improved waste management, and mitigation strategies to address MP pollution in freshwater ecosystems.

Microplastics

Depth-dependent multi-kingdom microbial interactions and biogeochemical cycling genes in eutrophic shallow lake sediments.

Microorganisms are pivotal to lake ecosystem biogeochemical cycles, yet existing research often focuses on single microbial kingdoms or surface sediments, neglecting multi-kingdom interactions and depth-resolved dynamics. To address these gaps, we used metagenomic sequencing to characterize microbial communities and their functional associations across overlying water and 0-45 cm sediments in four shallow lakes of the middle Yangtze River basin, China. Despite increasing bacterial and fungal diversity with depth, the 0-9 cm surface sediments exhibited the strongest multi-kingdom network connectivity and the greatest microbial stability. Functional genes exhibited clear depth-dependent patterns: nitrogen cycling genes, including those involved in dissimilatory nitrate reduction to ammonium, were most enriched in the upper 0-9 cm of sediment; methane cycling genes were positively correlated with depth; phosphorus cycling genes and some sulfur cycling genes, such as assimilatory sulphate reduction, declined with depth. Sediment microbial assembly was dominated by deterministic processes, in which the vertical distribution of functional genes was primarily dictated by heavy metals and conventional environmental indicators. These findings highlight depth-specific multi-kingdom microbial interactions and their associations with biogeochemical cycling, advancing lacustrine microbial ecology understanding and providing references for lake conservation under environmental change.

Lakes

Comparative metagenomic analysis of microbial communities: unravelling microbial communities from the great Rann of Kachchh and coastal saltpans, Gujarat, India.

Hypersaline environments exhibit extreme physiochemical conditions yet support diverse microbial communities. These communities are not only ecologically important but also possess substantial potential for biotechnological exploitation. In this study, we employed a comparative metagenomic approach to assess microbial diversity using two distinct methodologies: (1) direct DNA extraction from raw sediment, and (2) DNA extraction following halophilic enrichment in selective media. Sediment samples were collected from multiple sites and pooled together within the Rann of Kachchh and close-by saltpans and were analysed using 16S rRNA sequencing coupled with bioinformatics pipelines. The results revealed pronounced differences in microbial community composition between the two approaches. Raw sediment samples exhibited significantly higher alpha diversity, with dominant taxa including Halobacterota, Cyanobacteria, and Desulfobacterota, with a substantial proportion of unclassified genera. In contrast, enriched samples were dominated by fast-growing, culturable genera such as Halobacterium, Alkalibacillus, and Candidatus haloredivivus. Principal Coordinate Analysis (PCoA) of beta diversity demonstrated distinct clustering between raw and enriched communities, even within samples from the same sites, underscoring the selective bias introduced by enrichment procedures. These findings emphasise that the methodological choice strongly influences the observed microbial diversity. The aim of this study was to compare microbial community composition in raw hypersaline sediments and enrichment cultures using metagenomic sequencing, to evaluate how enrichment selectively favours specific halophilic taxa. This comparative approach allows identification of the microbial groups that rapidly proliferate under controlled hypersaline conditions, thereby complementing direct environmental sequencing. By integrating both direct and enrichment-based metagenomic approaches, a more comprehensive understanding of microbial community structure in hypersaline environments can be achieved.

India

Getting to the Core of the Matter-Assessing the Role of Replication in Metabarcoding-Based sedaDNA.

Replication is central to most experimental and sampling designs, increasing inferential power and capturing fine-scale data heterogeneity. However, its importance remains poorly evaluated in some ecological and evolutionary settings. This is the case of metabarcoding studies using DNA recovered from sedimentary archives, in which biological signals integrate ecological information through depositional and burial processes, yet are commonly inferred from a single sediment core per site. Here, we evaluated the effect of different types of replication using sedimentary DNA metabarcoding data from two genetic markers (mitochondrial COI and nuclear 18S) using a nested sampling design. The design included three intertidal sites, three spatially separated sediment cores per site (biological replicates), two sediment horizons per core, and eight PCR (technical) replicates per sediment sample. Variance partitioning showed that site identity and sediment age group together explained > 70% of the variation in beta diversity, indicating that among-site spatial and stratigraphic differences were the dominant drivers of community composition. PERMANOVA likewise identified non-significant effects of biological replication. Among PCR replicates from the same sediment sample, richness varied substantially, whereas Shannon diversity was more consistent. Despite this variability, differences in community composition among technical replicates remained smaller than those associated with biological replication or site identity, indicating a limited influence on broader ecological patterns. Community composition was highly similar among replicate cores within sites, consistent with stratigraphic coherence. These results indicate limited within-site heterogeneity and suggest that, under stratigraphically coherent conditions, increasing biological replication may provide little additional information, whereas enhancing technical replication and stratigraphic resolution can improve ecological inference from sedimentary DNA metabarcoding datasets.

DNA Barcoding, Taxonomic

Bone Adhered Sediments as a Source of Target and Environmental DNA and Proteins.

In recent years, sediments from cave environments have provided invaluable insights into ancient hominids, as well as past fauna and flora. Unfortunately, however, sediments are not always collected during excavation. In this study, we analyzed an overlooked but abundant resource in archaeological collections - sediments adhered to bone. We performed metagenomics and metaproteomics analysis on sediment from several human skeletal elements, originating from Neolithic to Medieval sites in England. We were able to reconstruct a partial human genome, the genetic profile of which matches that recovered from the original skeletal element. Additionally, aDNA sequences matching the genomes of endogenous gut microbiome bacteria were identified. We also found the presence of genetic sequences corresponding to animals and plants. In particular, we managed to retrieve the partial genome and proteome of a Black Rat (Rattus rattus), sharing close genetic affinities to other medieval Rattus rattus. Our results demonstrate that material that is usually ignored or discarded, can be used to reveal information about the individual and the environmental conditions at the time of their death.

Animals

A deep metagenomic atlas of Qinghai-Xizang Plateau lakes reveals their microbial diversity and salinity adaptation mechanisms.

The Qinghai-Xizang Plateau (QXP), harboring the planet's highest density of plateau lakes, offers an exceptional biogeographic environment for studying extremophilic microbial communities and their adaptation to salinity. Through deep metagenomic sequencing, we construct the Qinghai-Xizang Lake Sediment Genome (QXLSG) catalog, a high-resolution genomic catalog comprising 5,866 metagenome-assembled genomes (MAGs), 58.16 million non-redundant protein encoding genes, and 19,008 biosynthetic gene clusters. Notably, 80.78% of the 2,742 species-level MAGs represent undescribed taxa, significantly expanding the known microbial diversity. Salinity emerges as the primary environmental factor influencing microbial community. Functional annotation highlights that the "salt-out" strategy, particularly the uptake of glycine betaine, is the main mechanism for salinity tolerance. This strategy is prevalent in both hypersaline lake communities and the dominant microbial phyla. Overall, this study provides a crucial genetic resource for future bioprospecting and deepens our understanding of the fundamental mechanisms of microbial adaptation to extreme saline environments.

Lakes

Description of Sulfitobacter gelatinilyticus sp. nov. and Sulfitobacter weihaiensis sp. nov., isolated from marine sediment.

Two Gram-stain-negative, yellowish-white, facultative anaerobic and rod-shaped bacterial strains, designated F26169LT and F26204T, were isolated from coastal sediment of Jingzi Port, Weihai. Based on phenotypic, physiological, biochemical, chemotaxonomic and phylogenomic analyses, the two strains were affiliated with the genus Sulfitobacter. They showed obvious differences in phenotypic, chemotaxonomic and genomic characteristics compared with closely related taxa in this genus, and the ANI, AAI and dDDH values between them and related species were all lower than the standard thresholds for bacterial species delineation. Genomic analysis revealed the presence of genes encoding a complete sulfur oxidation (SOX) pathway and dimethylsulfoniopropionate (DMSP) lyases in both strains. Accordingly, strains F26169LT and F26204T are proposed as two novel species of the genus Sulfitobacter, for which the names Sulfitobacter gelatinilyticus sp. nov. and Sulfitobacter weihaiensis sp. nov., are proposed, respectively. The type strains are F26169LT (= KCTC 92635 T = MCCC 1H01356T) and F26204T (= KCTC 92634 T = MCCC 1H01357T).

Geologic Sediments

Sedimentary Ancient DNA Tracks Multi-Kingdom Ecosystem Reorganizations Following Sequential Human Land Use at Crawford Lake.

Crawford Lake has an exceptional stratigraphic record that began recording biannual (varved) sedimentation in the lake basin ~750 years ago, preserving evidence of shifting cultural zones and agricultural practices, from Late Woodland Period Indigenous agriculturalists to the impacts of industrialization during the late 19th century. It was selected as the candidate site for the proposed 'Anthropocene' epoch in 2023-a proposal ultimately rejected in 2024-but the lake's significance extends beyond a formal stratigraphic boundary. Its sediments preserve a long record of human-ecosystem entanglement that captures the cumulative, reverberating nature of local human impacts and global change. While many proxies have been studied at the site, the lake's sedimentary ancient DNA (sedaDNA) record has yet to be investigated. Here, we report on sedaDNA preserved at Crawford Lake over the last ~1300 years. Sedentism and agriculture clearly impacted the entire lake ecosystem, with corresponding shifts observable in the sedaDNA of plants, animals, algae, fungi and bacteria. Canada goose (Branta canadensis) roosting on the lake-likely drawn by foraging opportunities in fields cleared for Three/Four Sisters agriculture and sedentism-contributed to repeated eutrophications and algal blooms that permanently shifted the lake's ecological structure. Subsequent impacts during the Euro-Canadian zone furthered anthropogenic succession, although local impacts have been minimal since closure of the sawmill in 1900 ce, allowing for sensitivity to global change. Beyond the molecular ecological history of the lake, we also evaluate the effectiveness of an Arctic/Subarctic bait-set for palaeoecological reconstructions of the Eastern Woodlands, and the preservation of lake sedaDNA.

Lakes

Heavy-metal stress shapes habitat-specific microbial survival strategies in estuarine environments.

Estuarine ecosystems face increasing heavy metal pollution from rapid urbanization and industrialization, yet the microbial adaptive strategies to multiple metal stressors across different habitats remain poorly understood. This study investigated the diversity and composition of bacterial and fungal communities across free-living (FL), particle-attached (PA), and sediment (SE) fractions from three estuaries with varying heavy metal contamination, and further investigated functional adaptations of bacterial communities. High-throughput amplicon sequencing revealed habitat-specific communities, with SE hosting the highest alpha diversity and enrichment of metal-resistant genera such as Woeseia and Sva1033. Environmental filtering, particularly by Zn, was the dominant driver shaping bacterial assemblages across all habitats, whereas fungal communities displayed greater stochastic assembly patterns. Analysis of 44 high-quality bacterial metagenome-assembled genomes (MAGs) revealed diverse metal resistance genes (cusA, znuB, and zntA), along with enriched metabolic pathways for carbon, nitrogen, and sulfur cycling. Notably, both active efflux/oxidative stress defense and indirect immobilization mechanisms were observed across all habitats, but their relative importance differed: FL and PA communities exhibited a greater reliance on active metal efflux (czcAB) and oxidative stress defense (trxAB) to maintain intracellular homeostasis, whereas SE communities displayed a stronger genomic potential for sulfate reduction (dsrAB) that may contribute to metal immobilization through sulfide precipitation. This metabolic partitioning highlights the complementary roles of different habitats in mediating metal toxicity and biogeochemical cycling, providing new insights into microbial resilience in polluted estuaries and underscoring the urgency of addressing heavy-metal contamination in these critical ecosystems.

Estuaries

Shifts of antibiotic resistance genes across an estuarine meandering bend and dissemination risks to offshore oceans.

Meandering is a fundamental geomorphic feature of rivers that plays a critical role in regulating pollutant attenuation. To elucidate its impact on antibiotic resistance genes (ARGs) distribution in estuarine intertidal sediments, samples were collected from both the landward side (freshwater-dominated) and the seaward side (tide-dominated) of a meander bend during ebb and flood tides. The total relative abundance of ARGs was approximately 2.7 times higher on the landward side, peaking during the ebb tide. Microbial composition analysis showed that genera Acinetobacter and Pseudomonas were dominant at the landward sites, while halophilic genera such as Marinobacter and Exiguobacterium were abundant at the seaward sites. Further analysis of metagenome-assembled genomes (MAGs) demonstrated that the dominant landward genus Acinetobacter acted as a key host of ARGs, with two of four MAGs encoding more than ten ARGs. Notably, the total relative abundance of mobile genetic elements was high but consistent between sides and tidal cycles (p > 0.05). Given this high dissemination risk, we further forecasted the ARGs transfer scenarios to oceanic settings based on a set of offshore MAGs (n = 3626). Three ARGs, i.e., acrA, vanSL, and AAC(2')-Ia, were inferred to have transfer potential, supported by neighboring MGEs detected in marine microorganisms. Analysis of the genomes of predicted recipients in the SRA database confirmed the predicted mobilizations. Together, this study highlights that the meandering planform may serve as a significant barrier, attenuating the discharge of ARGs from terrestrial sources into the marine environment.

Estuaries

Description and genomic characterization of Aquipuribacter aurantiacus sp. nov., isolated from saline lake sediment.

Strains MA13-6T and MA13-13, two Gram-stain-positive, aerobic, short rod-shaped actinobacteria, were isolated from a saline lake in Ngari Prefecture, Xizang Autonomous Region, China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these two strains belonged to the genus Aquipuribacter, with the closest relationship to Aquipuribacter hungaricus IV-75T (98.47% sequence similarity) and Aquipuribacter nitratireducens AMV4T (97.36% sequence similarity). Phylogenetic analysis based on genomes further confirmed their classification as a distinct cluster within the genus Aquipuribacter. The average nucleotide identity and digtal DNA-DNA hybridization values between these two strains and their closest relative Aquipuribacter hungaricus IV-75T, were 82.44-82.49% and 23.00%, respectively, clearly indicating that strains MA13-6T and MA13-13 represent a novel species. The 16S rRNA gene sequence similarity, average nucleotide identity and digital DNA-DNA hybridization values between these two strains were 99.79%, 99.97% and 99.40%, respectively, unequivocally confirming their classification within the same species. However, DNA fingerprinting analysis distinguished them as non-clonal variants. The polar lipids comprised phosphatidylglycerol, two unidentified phospholipids, two unidentified glycolipids, and two unidentified lipids. The predominant respiratory quinone was MK-10 (H4). The major fatty acids were anteiso-C15:0, C18:1ω9c, isoC16:0 and anteiso-C17:0. The cell wall diagnostic diamino acid was meso-diaminopimelic acid. Based on phylogenetic analyses combined with phenotypic and chemotaxonomic characterization, strains MA13-6T and MA13-13 represent a novel species of the genus Aquipuribacter, for which the name Aquipuribacter aurantiacus sp. nov. is proposed. The type strain is MA13-6T (=MCCC 1K10045T = KCTC 59572T).

Phylogeny

Comparative metagenomic assessment of Illumina-compatible library preparation methods, short-read lengths, and PacBio HiFi sequencing reveals differences in microbial and functional diversity recovery from a complex environmental sample.

UNLABELLED: Metagenomics enables comprehensive exploration of microbial communities but is influenced by library preparation and sequencing technologies, affecting recovery of microbial genomes and proteins. Here, we benchmarked six Illumina-compatible short-read library preparation conditions in triplicate at 2 × 150 bp and 2 × 250 bp read lengths alongside PacBio HiFi long-read sequencing using a composite environmental sample of marine mangrove sediment and terrestrial palm tree soil. Longer short reads (2 × 250 bp) combined with optimal library preparation approaches improved assembly quality, protein detection, and metagenome-assembled genome (MAG) recovery, achieving results approaching those of long-read sequencing. TruSeq libraries at 2 × 250 bp recovered more than sevenfold more unique proteins than the same kit at 2 × 150 bp (811,701 vs 110,108) using the same number of sequencing reads, while recovering a comparable number of high-quality MAGs to PacBio HiFi long-read sequencing (11 vs 18) and surpassing it in protein discovery by almost 10-fold (811,701 vs 87,745) at less than half of the sequencing cost. Furthermore, biosynthetic gene cluster analysis identified 46 biosynthetic gene clusters in TruSeq-250PE assemblies compared to 38 in PacBio HiFi, with several showing no close match in the MIBiG database. Although long reads yield more contiguity and complete genomes, longer short reads offer a cost-effective, scalable alternative for uncovering microbial and functional diversity. These findings provide critical guidance for metagenomic experimental design, demonstrating that strategic selection of library preparation chemistry and sequencing parameters can reveal more unknown microbial information in complex biomes without requiring additional sequencing depth. IMPORTANCE: Metagenomic outcomes are strongly influenced by library preparation and sequencing strategies, yet their combined effects in complex environmental samples remain poorly defined. Here, we provide the first direct comparison of Illumina NovaSeq short-read metagenomic sequencing at 2 × 150 bp and 2 × 250 bp across multiple library preparation kits, alongside PacBio HiFi long-read sequencing. We show that sequencing read length and library preparation critically shape assembly quality, protein recovery, and metagenome-assembled genome (MAG) reconstruction. These findings demonstrate that short-read sequencing at 2 × 250 bp, with appropriate library preparation, can match long-read technologies in MAG recovery while substantially surpassing them in protein discovery. With less than half of the sequencing price and a 3.5-fold reduction in cost per gigabase of usable data, this method facilitates more accessible large-scale metagenomic analysis within complex environmental systems.

Metagenomics

Deep subsurface organic-rich shale supports abundant, diverse, and novel fungi.

As Earth's principal reservoir of organic carbon and microbial biomass, the deep subsurface hosts microorganisms capable of mobilizing this once-sequestered carbon. Contrary to standard assumptions of eukaryotic scarcity, this study documents abundant fungal communities, ranging from 4.2 × 103 to 6.8 × 103 fungal cells ml-1, across a methane-producing organic-rich shale 247-556 meters below the surface. Although fungal:bacterial cell ratios ranged from 1:7028 to 1:713, application of biomass conversion factors developed for oceanic systems yielded a median fungal:bacterial biomass ratio of 1:4.7. 16S ribosomal ribonucleic acid (rRNA) gene amplicons revealed bacterial and archaeal communities mirroring those found in well-characterized extremophilic, carbon-degrading environments, while sequencing of 18S rRNA gene and internal transcribed spacer rRNA spacer amplicons collectively identified a eukaryotic hotspot with 689 fungal operational taxonomic units across six phyla. The dominant fungal classes, Agaricomycetes and Dothideomycetes, are well-established degraders of recalcitrant carbon compounds at the surface, suggesting they may similarly contribute to organic matter degradation and ecosystem maintenance in the subsurface. Cultivation and isolation efforts yielded 205 fungal strains, including 13 candidate novel taxa, underscoring the deep subsurface as an underexplored eukaryotic habitat. Stable carbon isotopes indicate methane is predominantly generated via microbial conversion of the fossil carbon, while water isotopes suggest in situ geochemical conditions have been relatively stable since the Late Pleistocene, with subglacial recharge as a plausible mechanism for microbial introduction. Collectively, these findings suggest that fungi are underrecognized contributors to organic matter transformation and functional diversity in the deep biosphere, revealing a critical gap in our understanding of deep subsurface ecosystem processes.

Fungi

Models of prebiological phosphorylation.

The hypothesis that contemporary metabolic pathways envolved from analogous chemical reaction sequences on the primitive Earth leads to a reexamination of models of prebiological phosphorylation. Present-day phosphate uptake by algae and bacteria seems to involve two transport systems: (a) A n active transport process occurring at low external phosphate concentrations (as in umpolluted natural waters), with a transport constant Ks of 10(-7) to 10(-6) M Pi. (b) Another (probably diffusive) process at higher phosphate concentrations (greater than 10-6 M)(as in the interstitial water of reducing sediments). Laboratory model experiments are described for the reation of reducing sugars with orthophosphate in the presence of cyanogen, producing glycosyl phosphates. These reactions proceed with appreciable yields only at high phosphate concentrations (greater than 10-3 M), and may thus possibly serve as simulations of prebiological phosphorylation with diffusive transport, as it may have occurred in the intestial water of reducing sediments.

Bacteria