Subsurface petroleum reservoirs host indigenous microorganisms that survive extreme conditions and long-term isolation. Microbial activity in these environments can contribute to adverse effects such as oil biodegradation and reservoir souring. Unlike the broader deep biosphere, oil reservoirs are frequently subjected to anthropogenic disturbances, particularly during production, when processes like water injection introduce external microbes and electron acceptors. In this study, we investigated microbial diversity, community structure, and the impact of water breakthrough using 16S rRNA gene and metagenomic sequencing of produced fluids, production water, and injection water samples from the Edvard Grieg oil reservoir offshore Norway. We found clear regional heterogeneity in community composition, characterized by overall low diversity, dominated by thermophilic, anaerobic, and halotolerant taxa. The southern region (wells A13, A17, A18, and A19) exhibited lower diversity, while the microbial community composition of well A07 showed a distinct signature. The prevailing genera included the strictly anaerobic bacterium Thermoanaerobacter and the hyperthermophilic archaeon Thermococcus. Water breakthrough triggered shifts in community structure, not because of widespread replacement by injected microbes, but due to the increase in sulfate-reducing bacteria. Comparison between sequence data from production fluids and water samples allowed the identification of microbial signatures that can act as cost-effective tools for monitoring oil reservoir processes and integrity.

Oil reservoirs are society's primary source of hydrocarbons. While microbial communities in industrially exploited oil reservoirs have been investigated in the past, pristine microbial communities in untapped oil reservoirs are little explored, as are distribution patterns of respective genetic signatures. Here, we show that a pristine oil sample contains a complex community consisting of bacteria and fungi for the degradation of hydrocarbons. We identified microorganisms and their pathways for the degradation of methane, n-alkanes, mono-aromatic, and polycyclic aromatic compounds in a metagenome retrieved from biodegraded petroleum encountered in a subsurface reservoir in the Barents Sea. Capitalizing on marker genes from metagenomes and public data mining, we compared the prokaryotes, putative viruses, and putative plasmids of the sampled site to those from 10 other hydrocarbon-associated sites, revealing a shared network of species and genetic elements across the globe. To test for the potential dispersal of the microbes and predicted elements via seawater, we compared our findings to the Tara Ocean dataset, resulting in a broad distribution of prokaryotic and viral signatures. Although frequently shared between hydrocarbon-associated sites, putative plasmids, however, showed little coverage in the Tara Oceans dataset, suggesting an undiscovered mode of transfer between hydrocarbon-affected ecosystems. Based on our analyses, genetic information is globally shared between oil reservoirs and hydrocarbon-associated sites, and we propose that currents and other physical occurrences within the ocean along with deep aquifers are major distributors of prokaryotes and viruses into these subsurface ecosystems.

Marine sediments harbor extremely diverse microbial communities that contribute to global biodiversity and play an essential role in the functioning of ecosystems. However, the metaproteome of marine sediments is still poorly understood. The extraction of proteins from environmental samples is still a challenge, especially from marine sediments, due to the complexity of the matrix. Therefore, methods for protein extraction from marine sediments need to be improved. To develop an effective workflow for protein extraction for clayey sediments, we compared, combined and enhanced different protein extraction methods. The workflow presented here includes blocking of protein binding sites on sediment particles with high concentrations of amino acids, effective cell lysis by ultrasonic capture, electro-elution, and simultaneous fractionation of proteins. To test the protocol's efficacy, we added Escherichia coli cells to sediment samples before protein extraction. By using our refined workflow, we were able to identify a comparable number of E. coli proteins from the supplemented sediment to those from pure E. coli cultures. This new protocol will enable future studies to identify active players in clay-rich marine sediments and accurately determine functional biodiversity based on their respective protein complements.

Neutral in situ leaching (ISL) is a prominent technique for extracting uranium (U) from sandstone-type U deposits. However, the intricate biogeochemical processes and associated microbial communities in environments subjected to prolonged neutral in situ U leaching remain insufficiently understood. Here, capitalizing on groundwater samples collected from an area subjected to two decades of ISL (Undergoing-ISL) and a background aquifer (Non-ISL) at the Qianjiadian U mining site (the first CO2 + O2 ISL site located in the Songliao Basin of northern China), we investigate the responses of aquifer microbiomes to CO2 + O2 ISL using genome-resolved metagenomics, resulting in a total of 315 medium- and high-quality bacterial metagenome-assembled genomes (MAGs) and 5 archeal genomes. Seven putative keystone MAGs were identified based on their high connection degree and low betweenness centrality within the co-occurrence network of reconstructed MAGs, notably highlighting the prevalence of microorganisms involved in sulfur, ammonium, and Fe(II) oxidation, which are integral to facilitating U(IV) oxidation during CO2 + O2 ISL. In particular, sulfur-oxidizing bacteria and Fe(II)-oxidizing bacteria (Rhodoferax spp. and SHZL01 spp.) played a key role in U mobilization during long-term neutral ISL. These findings enhance our understanding of subsurface microbial ecology and hold significant implications for bioremediation strategies and groundwater management in regions affected by mining.

The rapid evolution of microbiology as a field of research has led to the introduction of new terminology and the adaptation of existing terms. However, inconsistencies in the use of these terms, including variations across different scientific disciplines, can lead to confusion and miscommunication within the scientific community. This article discusses the importance of precise terminology in microbiome research, highlighting examples where terms have been misused or redefined without clear justification. We also present a list of frequently used terms in microbial ecology along with their specific definitions. We argue that the misuse of terminology can hinder scientific progress by creating ambiguity and misunderstanding. To address this, we propose a set of guidelines for the consistent use of key terms and provide clear definitions for some of the most commonly misused or newly introduced terms in the field. The definitions provided herein will also function as a guide for young researchers new to the field of microbial ecology. Accurate and consistent use of terminology is crucial for effective communication and collaboration in microbiology research. By adhering to standardised definitions, researchers can ensure that their work is clearly communicated and contributes meaningfully to the progress of science.

Candidatus Altiarchaea are widespread across aquatic subsurface ecosystems and possess a highly conserved core genome, yet adaptations of this core genome to different biotic and abiotic factors based on gene expression remain unknown. Here, we investigated the metatranscriptome of two Ca. Altiarchaeum populations that thrive in two substantially different subsurface ecosystems. In Crystal Geyser, a high-CO2 groundwater system in the USA, Ca. Altiarchaeum crystalense co-occurs with the symbiont Ca. Huberiarchaeum crystalense, while in the Muehlbacher sulfidic spring in Germany, an artesian spring high in sulfide concentration, Ca. A. hamiconexum is heavily infected with viruses. We here mapped metatranscriptome reads against their genomes to analyse the in situ expression profile of their core genomes. Out of 537 shared gene clusters, 331 were functionally annotated and 130 differed significantly in expression between the two sites. Main differences were related to genes involved in cell defence like CRISPR-Cas, virus defence, replication, transcription and energy and carbon metabolism. Our results demonstrate that altiarchaeal populations in the subsurface are likely adapted to their environment while influenced by other biological entities that tamper with their core metabolism. We consequently posit that viruses and symbiotic interactions can be major energy sinks for organisms in the deep biosphere.

Genomic analyses of microbial community composition are used to improve oil reservoir engineering and monitor reservoir dynamics. Given the challenges of extracting nucleic acids from oil, production water samples are often used as proxies from which to infer microbial community information from oil reservoirs. We employed 16S rRNA gene amplicon and metagenomic sequencing on samples of crude oil and production water from four North Sea oil fields. Taxonomic profiling revealed differences in microbial compositions and functions between production water and crude oil. Production water was more homogeneous, less diverse, harboured taxa associated with conditions non-native to the reservoir (e.g., seawater), and exhibited evidence of contact with atmospheric oxygen, most likely from passing through the water separators. Conversely, crude oil samples harboured microbial taxa typically associated with oil reservoirs. Despite long-term production and, in some cases, re-injection of production waters, the putative native microbial communities were still present in the oil. These findings demonstrate that crude oil samples are much more representative of oil reservoir microbiomes than their production water proxies. ### Competing Interest Statement The authors have declared no competing interest. Aker BP (Norway), https://ror.org/05g9srn20

The decomposition of organic matter is essential for sustaining the health of freshwater ecosystems by enabling nutrient recycling, sustaining food webs, and shaping habitat conditions, which collectively enhance ecosystem resilience and productivity. Bacteria and fungi play a crucial role in this process by breaking down coarse particulate organic matter (CPOM), such as leaf litter, into nutrients available for other organisms. However, the specific contribution of bacteria and their functional interactions with fungi in freshwater sediments have yet to be thoroughly explored. In the following study, we enriched organic matter through the addition of alder (Alnus glutinosa) leaves into artificial stream channels (AquaFlow mesocosms). We then investigated enzyme expression, metabolic pathways, and community composition of fungi and bacteria involved in the degradation of CPOM through metatranscriptomics and amplicon sequencing. Enzymes involved in the degradation of lignin, cellulose, and hemicellulose were selectively upregulated with increased organic matter. Analysis of ITS and 16S rRNA gene sequences revealed that during decomposition, fungal communities were predominantly composed of Basidiomycota and Ascomycota, while bacterial communities were largely dominated by Pseudomonadota and Bacteroidota. The similar gene expression patterns of CPOM degradation related enzymes observed between bacteria and fungi indicate potential functional interaction between these microbial groups. This correlation in enzyme expression may indicate that bacteria and fungi are jointly involved in the breakdown of coarse particulate organic matter, potentially through mutualistic interaction. This study uncovers the specific enzymatic activities of bacteria and fungi and the importance of microbial interactions in organic matter decomposition, revealing their central role in facilitating nutrient cycling and maintaining the ecological health and stability of freshwater ecosystems.

Aquatic ecosystems house a significant fraction of Earth’s biosphere, yet most prokaryotes inhabiting these environments remain uncultivated. While recently developed genome-resolved metagenomics and single-cell genomics techniques have underscored the immense genetic breadth and metabolic potential residing in uncultivated Bacteria and Archaea, cultivation of these microorganisms is required to study their physiology via genetic systems, confirm predicted biochemical pathways, exploit biotechnological potential, and accurately appraise nutrient turnover. Over the past two decades, the limitations of culture-independent investigations highlighted the importance of cultivation in bridging this vast knowledge gap. Here, we collected more than 80 highly sought-after uncultivated lineages of aquatic Bacteria and Archaea with global ecological impact. In addition to fulfilling critical roles in global carbon, nitrogen, and sulfur cycling, many of these organisms are thought to partake in key symbiotic relationships. This review highlights the vital contributions of uncultured microbes in aquatic ecosystems, from lakes and groundwater to the surfaces and depths of the oceans and will guide current and future initiatives tasked with cultivating our planet’s most elusive, yet highly consequential aquatic microflora.

Rivers face constant anthropogenic stress, resulting in significant changes in microbial community composition. What remains unclear is whether stream microbiomes exhibit distinct resilience patterns in composition and/or activity upon exposure to different stressors. By subjecting 64 river-connected mesocosms to multiple stressors, we show that sediment microbiomes of small lowland rivers are highly sensitive to low flow velocity. This stress results in altered community compositions incapable of mitigating the applied stressor within a two-week timeframe despite functional stability (inferred via metagenomics). Transcriptomics revealed a systematic heat shock response in the community and a highly active, metabolically versatile, uncharacterized anaerobic keystone species. Increases in temperature (+ 3.5°C) or salinity (+ 0.5 mS/cm) elicited minor responses at community and transcriptomic levels (e.g., upregulation of photosystems). Following a two-week recovery, transcriptomic-inferred stress responses vanished completely, underscoring the river microbiome resilience. Given the complex community responses observed at the activity and compositional levels, we conclude that maintaining natural river flow is vital to preventing energy loss and reduced microbiome activity in river sediments.

Science benefits from rapid open data sharing, but current guidelines for data reuse were established two decades ago, when databases were several million times smaller than they are today. These guidelines are largely unfamiliar to the scientific community, and, owing to the rapid increase in biological data generated in the past decade, they are also outdated. As a result, there is a lack of community standards suited to the current landscape and inconsistent implementation of data sharing policies across institutions. Here we discuss current sequence data sharing policies and their benefits and drawbacks, and present a roadmap to establish guidelines for equitable sequence data reuse, developed in consultation with a data consortium of 167 microbiome scientists. We propose the use of a Data Reuse Information (DRI) tag for public sequence data, which will be associated with at least one Open Researcher and Contributor ID (ORCID) account. The machine-readable DRI tag indicates that the data creators prefer to be contacted before data reuse, and simultaneously provides data consumers with a mechanism to get in touch with the data creators. The DRI aims to facilitate and foster collaborations, and serve as a guideline that can be expanded to other data types.

Stream hyporheic zones represent a unique ecosystem at the interface of stream water and surrounding sediments, characterized by high heterogeneity and accelerated biogeochemical activity. These zones are increasingly impacted by anthropogenic stressors and environmental changes at a global scale, directly altering their microbiomes. Despite their importance, the current body of literature lacks a systematic understanding of active nitrogen and sulfur cycling across stream sediment and surface water microbiomes, particularly across geographic locations and in response to environmental stressors. Based on previously published and unpublished datasets, 363 stream metagenomes were combined to build a comprehensive MAG and gene database from stream sediments and surface water including a full-factorial mesocosm experiment which had been deployed to unravel microbial stress response. Metatranscriptomic data from 23 hyporheic sediment samples collected across North America revealed that microbial activity in sediments was distinct from the activity in surface water, contrasting similarly encoded metabolic potential across the two compartments. The expressed energy metabolism of the hyporheic zone was characterized by increased cycling of sulfur and nitrogen compounds, governed by Nitrospirota and Desulfobacterota lineages. While core metabolic functions like energy conservation were conserved across sediments, temperature and stream order change resulted in differential expression of stress response genes previously observed in mesocosm studies. The hyporheic zone is a microbial hotspot in stream ecosystems, surpassing the activity of overlaying riverine surface waters. Metabolic activity in the form of sulfur and nitrogen cycling in hyporheic sediments is governed by multiple taxa interacting through metabolic handoffs. Despite the spatial heterogeneity of streams, the hyporheic sediment microbiome encodes and expresses conserved stress responses to anthropogenic stressors, e.g., temperature, in streams of separate continents. The high number of uncharacterized differentially expressed genes as a response to tested stressors is a call-to-action to deepen the study of stream systems. ### Competing Interest Statement The authors have declared no competing interest.

Most microorganisms cannot be cultured in isolation, necessitating sophisticated methods for studying their (eco)physiology. While numerous approaches can probe the activity of given microbes in enrichment cultures, no single technique can render simultaneous data on both metabolic capacities and mobile genetic elements. Here, we apply long-read sequencing to monitor the incorporation of non-canonical bases in genome-resolved metagenomic datasets and elucidate the replication patterns of both bacteria and phages. This technology enables the simultaneous reconstruction of both prokaryotic and viral genomes (alongside genomics downstream analyses like metabolic predictions), in addition to providing information regarding their replication in enrichment cultures. By spiking the base analog 5-bromo-2'-deoxyuridine (BrdU) into activated sludge microcosms, we determined that 114 of the 118 high-quality genomes recovered were actively replicating in enrichment cultures from activated sludge and identified both slow (low BrdU incorporation and change in abundance) and rapidly replicating organisms (high BrdU incorporation and change in abundance). Some of the genomes detected exhibited regions rich in BrdU that were predicted to represent prophages in their lytic cycle. Ultimately, this novel means of monitoring the replication responses of microbes, and deciphering their genomes and active mobile genetic elements will advance and empower strategies aimed at isolating previously uncultivated microbes in pure culture.

Significant progress in high-throughput analytical techniques has paved the way for novel approaches to integrating data sets from different compartments. This study leverages nontarget screening (NTS) via liquid chromatography-high-resolution mass spectrometry (LC-HRMS), a crucial technique for analyzing organic micropollutants and their transformation products, in combination with biological indicators. We propose a combined multivariate data processing framework that integrates LC-HRMS-based NTS data with other high-throughput data sets, exemplified here by 18S V9 rRNA and full-length 16S rRNA gene metabarcoding data sets. The power of data fusion is demonstrated by systematically evaluating the impact of treated wastewater (TWW) over time on an aquatic ecosystem through a controlled mesocosm experiment. Highly compressed NTS data were compiled through the implementation of the region of interest-multivariate curve resolution-alternating least-squares (MCR-ALS) method, known as ROIMCR. By integrating ANOVA-simultaneous component analysis with structural learning and integrative decomposition (SLIDE), the innovative SLIDE-ASCA approach enables the decomposition of global and partial common, as well as distinct variation sources arising from experimental factors and their possible interactions. SLIDE-ASCA results indicate that temporal variability explains a much larger portion of the variance (74.6%) than the treatment effect, with both contributing to global shared space variation (41%). Design structure benefits include enhanced interpretability, improved detection of key features, and a more accurate representation of complex interactions between chemical and biological data. This approach offers a greater understanding of the natural and wastewater-influenced temporal patterns for each data source, as well as reveals associations between chemical and biological markers in an exemplified perturbed aquatic ecosystem.

Understanding intricate microbial interactions in the environment is crucial. This is especially true for the relationships between nutrients and bacteria, as phosphorus, nitrogen and organic carbon availability are known to influence bacterial population dynamics. It has been suggested that low nutrient conditions prompt the evolutionary process of genome streamlining. This process helps conserve scarce nutrients and allows for proliferation. Genome streamlining is associated with genomic properties such as %GC content, genes encoding sigma factors, percent coding regions, gene redundancy, and functional shifts in processes like cell motility and ATP binding cassette transporters, among others. The current study aims to unveil the impact of nutrition on the genome size, %GC content, and functional properties of pelagic freshwater bacteria. We do this at finer taxonomic resolutions for many metagenomically characterized communities. Our study confirms the interplay of trophic level and genomic properties. It also highlights that different nutrient types, particularly phosphorus and nitrogen, impact these properties differently. We observed a covariation of functional traits with genome size. Larger genomes exhibit enriched pathways for motility, environmental interaction, and regulatory genes. ABC transporter genes reflect the availability of nutrients in the environment, with small genomes presumably relying more on metabolites from other organisms. We also discuss the distinct strategies different phyla adopt to adapt to oligotrophic environments. The findings contribute to our understanding of genomic adaptations within complex microbial communities.

The analysis of DNA sequences sheds light on microbial biology, but it is difficult to assess the function of genes that have little or no similarity to characterized genes. Here, scientists discuss this challenge from genomic and microbial perspectives. Ways to find and investigate previously unidentified microbial genes.

Ecosystems subject to mantle degassing are of particular interest for understanding global biogeochemistry, as their microbiomes are shaped by prolonged exposure to high CO2 and have recently been suggested to be highly active. While the genetic diversity of bacteria and archaea in these deep biosphere systems have been studied extensively, little is known about how viruses impact these microbial communities. Here, we show that the viral community in a high-CO2 cold-water geyser (Wallender Born, Germany) undergoes substantial fluctuations over a period of 12 days, although the corresponding prokaryotic community remains stable, indicating a newly observed "infect to keep in check" strategy that maintains prokaryotic community structure. We characterized the viral community using metagenomics and metaproteomics, revealing 8 654 viral operational taxonomic units (vOTUs). CRISPR spacer-to-protospacer matching linked 278 vOTUs to 32 hosts, with many vOTUs sharing hosts from different families. High levels of viral structural proteins present in the metaproteome (several structurally annotated based on AlphaFold models) indicate active virion production at the time of sampling. Viral genomes expressed many proteins involved in DNA metabolism and manipulation, and encoded for auxiliary metabolic genes, which likely bolster phosphate and sulfur metabolism of their hosts. The active viral community encodes genes to facilitate acquisition and transformation of host nutrients, and appears to consist of many nutrient-demanding members, based on abundant virion proteins. These findings indicate viruses are inextricably linked to the biogeochemical cycling in this high-CO2 environment and substantially contribute to prokaryotic community stability in the deep biosphere hotspots.

The human gastrointestinal tract contains diverse microbial communities, including archaea. Among them, Methanobrevibacter smithii represents a highly active and clinically relevant methanogenic archaeon, being involved in gastrointestinal disorders, such as IBD and obesity. Herein, we present an integrated approach using sequence and structure information to improve the annotation of M. smithii proteins using advanced protein structure prediction and annotation tools, such as AlphaFold2, trRosetta, ProFunc, and DeepFri. Of an initial set of 873 481 archaeal proteins, we found 707 754 proteins exclusively present in the human gut. Having analysed archaeal proteins together with 87 282 994 bacterial proteins, we identified unique archaeal proteins and archaeal-bacterial homologs. We then predicted and characterized functional domains and structures of 73 unique and homologous archaeal protein clusters linked the human gut and M. smithii . We refined annotations based on the predicted structures, extending existing sequence similarity-based annotations. We identified gut-specific archaeal proteins that may be involved in defense mechanisms, virulence, adhesion, and the degradation of toxic substances. Interestingly, we identified potential glycosyltransferases that could be associated with N-linked and O-glycosylation. Additionally, we found preliminary evidence for interdomain horizontal gene transfer between Clostridia species and M. smithii , which includes sporulation stage V proteins AE and AD. Our study broadens the understanding of archaeal biology, particularly M. smithii, and highlights the importance of considering both sequence and structure for the prediction of protein function.

Bacteria of the candidate phyla radiation (CPR), constituting about 25