How do the ocean’s most abundant microbes regulate Earth’s primary carbon sink? Most investigations of microbial carbon fixation remain regionally focused or limited to single pathways, obscuring global patterns in the biosphere’s largest habitat. Here, we compile and analyze global metagenomic, transcriptomic, and proteomic data to map the in situ activity of all major inorganic carbon assimilation pathways—from photo- and chemo-autotrophy to anaplerotism—across marine ecosystems. Our meta-analysis reveals a high diversity of active pathways across regions and depths, with microbial communities operating along a continuous metabolic carbon assimilation spectrum. Lineages occupy conserved positions on this spectrum, and the spectrum’s balance is predictable from environmental gradients. Together, our results identify the main drivers of ocean carbon fixation, the widespread nature of these pathways, their taxonomic carriers, niche partitioning, and environmental controls, directly linking microbial carbon assimilation strategies to ocean biogeochemistry and climate sensitivity

Abstract Extreme conditions, including darkness, elevated hydrostatic pressure, low temperature, and limited nutrient availability create unique deep-sea environments. Previous studies on deep-sea organisms/microorganisms have not only revealed the metabolic characteristics of various deep-sea life forms and their dynamic communities but also contributed to the development and utilization of deep-sea biological resources. Genomics studies of deep-sea organisms heavily rely on high-quality samples. However, environmental alterations during sample retrieval, from the deep sea to the research vessel, lead to microbial community changes and RNA degradation. Hence, various automatic devices are developed for in situ fixation of samples and extraction of nucleic acids in the deep-sea environment, with subsequent processing conducted onboard. In recent years, in situ metagenomics, metatranscriptomics and metaproteomics (omics) have become powerful tools for exploring the metabolic activities and periodicity of the deep-sea inhabitants, which can provide insights for the survival mechanisms, element cycles, and dynamic changes of the ‘microbial dark matter’ in the deep ocean. This paper reviews the necessity of deep-sea in situ omics, outlines the development of ecological monitoring equipment for omics research in the deep sea, and discusses its applications in revealing novel genes and active substances in the deep sea, as well as understanding the distribution and ecological functions of deep-sea microorganisms. These in situ omics approaches are poised to revolutionize deep-sea research, enabling more accurate models of ecosystem dynamics.

BACKGROUND AND AIMS:Warming and marine heat waves (MHWs) are responsible for global declines in kelp forest ecosystems. Research to date focuses on impacts of temperature increase based on thermal tolerances of habitat-forming kelp species. Winter MHWs do not tend to surpass the thermal maxima of macroalgae, providing the opportunity to examine impacts in the absence of large-scale biomass loss, and in conjunction with other abiotic stressors. The transformation of organic carbon by heterotrophic marine bacteria, a key component of carbon cycling and foodweb productivity in kelp forests is strongly modulated by temperature. In this study, the metabolic responses of Macrocystis pyrifera heterotrophic bacterial communities to winter MHW conditions were examined in conjunction with hydrogen peroxide (H2O2) stress, a known inhibitor of bacterial activity. METHODS:Carbon uptake rates of planktonic and surface-associated (biofilm) microbial communities from M. pyrifera forests were assessed with exposure to H2O2 treatments alone, and in tandem with short-term heat-spike experiments simulating ambient, moderate, and extreme heatwave conditions in winter. KEY RESULTS:Carbon uptake rates of planktonic bacteria increased under extreme MHW conditions, with no significant response to increasing H2O2 concentrations. Conversely, M. pyrifera biofilm communities showed a significant decrease in carbon uptake rates at high H2O2 exposure compared to the intermediate treatment regardless of temperature regime applied. CONCLUSIONS:In winter, ambient water temperatures are limiting planktonic bacterial carbon incorporation rates in kelp forests, and MHW events stimulate increased carbon turnover. Increasing bacterial metabolism during winter may affect kelp forest ecosystem services through altering turnover and maintenance of organic carbon within kelp forests and annual carbon cycling patterns. We suggest that temperature is not limiting bacterial carbon uptake at the algal surface, and other stressors are driving carbon uptake patterns in M. pyrifera biofilms.

Phosphorus is a critically limiting nutrient in marine ecosystems, with alkaline phosphatases (APases) playing a vital role in liberating phosphate from organic compounds. However, the dominant taxa and APase families driving the marine phosphorus cycle, particularly in the deep ocean, remain poorly understood. Equally enigmatic remains the (multi)functional diversity and mechanisms of action of different APases. To address these gaps, this study combines global multi-omic analyses, biochemical studies of purified recombinant proteins, and laboratory experiments with proteomics and enzymatic rate measurements. Here we show that multi-omics consistently identify Alteromonas as a primary contributor to APase expression and production, with PhoA as the dominant APase family, particularly in the deep ocean. Furthermore, all four major APase families (PhoA, PhoD, PhoX, PafA) exhibit multifunctionality, revealing distinct substrate preferences and regulatory mechanisms. Ultimately, this study expands the mechanistic understanding of the marine phosphorus cycle, while revealing the significance of enzyme multifunctionality in elemental cycles.

Understanding dissolved organic matter (DOM) relies on the development of methods capable of navigating its complexity. Although analytical techniques have continually advanced, the fate of individual compound classes remains nearly impossible to track with the current technology. Previously, we reported the synthesis of carboxylate-rich alicyclic molecule (CRAM) compounds that shared more similar analytical features with DOM than previously available standards. Here, we adopt an alternative approach to the conventional use of DOM as a bulk material by subjecting our synthesized CRAM compounds to simulated solar irradiation and microbial incubation experiments alongside molecules with chosen biological or chemical relevance. Irradiation experiments typically showed that compounds bearing only carboxylic acids and/or alcohols on a saturated carbon backbone were the most resistant to photochemical degradation but also that some of the investigated CRAM analogues were notably more stable in the presence of DOM. Within microbial incubations, all of our synthesized CRAMs were entirely stable after 8 months in various aquatic settings. These sets of experiments provide support for the proposed stability of CRAM within the environment as well as providing a platform from which a more diverse set of molecules can be used to assist in probing the stability of DOM.

Marine fungi play key roles in organic matter cycling, yet their distribution across particle size fractions remains understudied. We analyze 18S rDNA data from four size fractions (0.8–5, 5–20, 20-180, and 180–2000 μm) collected across the global sunlit ocean. Here, we show fungal diversity and relative abundance decline with increasing particle size. Fungal community structure is influenced by eukaryotic diversity and chlorophyll levels. Fungi co-occur with other eukaryotes, especially zooplankton, hinting at potential predator-prey interactions. Generalist fungi dominate smaller fractions, while specialists dominate larger fractions, likely due to stronger microenvironmental selection. Co-occurrence networks are dominated by positive interactions and driven by fungal specialists. Dispersal limitation emerges as the main ecological process shaping community assembly. Our findings reveal strong niche differentiation among marine fungi along the particle continuum and emphasize the role of particle size and biological interactions in structuring fungal diversity and biogeography. Size-fractionated analysis of marine fungi reveals strong ecological differentiation, with particle size and dispersal limitation shaping diversity, specialization, and interactions across the global ocean.

Learning about the metabolic activities and adaptations of deep-sea microbes is a challenging task, because the collection and retrieval of samples from the deep ocean induce RNA degradation and alteration of microbial communities. Here, we employed a in situ DNA/RNA co-extraction device to collect 18 time-course nucleotide acid samples for winter and summer seasons in the South China Sea to generate metatranscriptomes and metagenomes with the minimal possible sampling perturbation. Between the two seasons, the most active eukaryotic microbes were Ciliophora, whereas the most abundant but inactive eukaryotic microbes were Retaria. In the winter, autotrophic microorganisms contributed to organic matter production by CO2 fixation associated with nitrification. In the summer, the primary source of energy originated from heterotrophic microorganisms that can utilize alkanes, aromatic compounds and carbohydrates, partially relying on anaerobic respiration in the particles. This may relate with nutrient source variations as reflected by the different levels of microbial network complexity between two seasons. Altogether, we uncovered the metabolic activities and adaptations of active microbial groups in two seasons with in situ metatranscriptomes, paving the way to identification of the real microbial contributors to element cycles in the deep ocean.

Metagenomic analysis has recently unveiled the widespread presence of pelagic fungi in the global ocean, yet their quantitative contribution to carbon stocks remains elusive, hindering their incorporation into biogeochemical models. Here, we revealed the biomass of pelagic fungi in the open-ocean water column by combining ergosterol extraction, Calcofluor-White staining, catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), and microfluidic mass sensor techniques. We compared fungal biomass with the biomass of other more studied microbial groups in the ocean such as archaea and bacteria. Globally, fungi contributed 0.32 Gt C (CI: 0.19-0.46), refining previous uncertainty estimates from two orders of magnitude to less than one. While fungal biomass was lower than that of bacteria, it exceeded that of the archaea (archaea:fungi:bacteria biomass ratio of 1:9:44). Collectively, our findings reveal the important contribution of fungi to open-ocean biomass and, consequently, the marine carbon cycle, emphasizing the need for their inclusion in biogeochemical models.

The metabolic potential and activity of deep-sea microbes have not been fully explored by meta-transcriptomics using the samples obtained by different sampling methods. Here, we report active deep-sea microbes obtained by the methods of multiple in situ nucleic acid collection (MISNAC), in situ microbial filtration and fixation (ISMIFF), in situ microbial filtration without fixation (ISMIFU), and the Niskin bottle at a 1038 m depth in the South China Sea. Higher biodiversity and different dominant active microbial taxa in the metatranscriptomes were detected in the MISNAC and ISMIFF samples compared with the other two approaches. The transcriptional profiles of 40 conserved genes were similar between the MISNAC and ISMIFF samples, while the expression of a quarter of these genes was not detected in the ISMIFU sample. Genes related to the CO oxidation and nitrification processes were highly transcribed in the MISNAC and ISMIFF transcriptomes, whereas those for chemotaxis and low-oxygen adaptation were highly transcribed in the Niskin samples. Overall, our result highlights the importance of in situ sampling and preservation for more precise quantification of the ecological function of active deep-sea microbiomes.

Microbes are the engines driving the elemental cycles. In order to interact with their environment and the community, microbes secrete proteins into the environment (known collectively as the secretome), where they remain active for prolonged periods of time. Despite the environmental relevance of microbes, our knowledge of the marine secretome remains limited due to a lack of effective in silico methods for the study of secreted proteins. An alternative approach to characterise the secretome is to combine modern machine learning tools with the evolutionary adaptation changes of the proteome to the marine environment. In this study, we identify and describe adaptations of marine extracellular proteins, which vary between phyla, resulting in differences in ATP costs, amino acid composition and nitrogen and sulphur content. We develop 'Ayu', a machine prediction tool that does not employ homology-based predictors and achieves better and quicker performance than current state-of-the-art software. When applied to oceanic samples (Tara Oceans dataset), our method was able to recover more than double the proteins compared to the most widely used method to identify secreted proteins. The application of this tool to open ocean samples allows better characterisation of the composition of the marine secretome.

Learning about the metabolic activities and adaptations of deep-sea microbes is challenging, as sample collection and retrieval often cause RNA degradation and microbial community shifts. Here, we employed an in situ DNA/RNA co-extraction device to collect 18 time-series nucleic acid samples during winter and summer in the South China Sea, minimizing sampling perturbation for metatranscriptome and metagenome analyses. Between the two seasons, the prokaryotic microbiota showed seasonal variations in species composition. Burkholderiales dominated in summer, whereas Pseudomonadales, Bacillales, and Rhodobacterales were enriched in winter. However, the dominant transcriptionally active taxa affiliated with Nitrososphaerales, MGIII, SAR324, UBA11654, Marinisomatales, and Poseidoniales remained largely stable across seasons. Among eukaryotes, Ciliophora were the most active, whereas Retaria were abundant but inactive. Despite the stable active prokaryotic community, metabolic profiles differed significantly between seasons. In the winter, autotrophic microorganisms, particularly Nitrososphaerales, exhibited higher CO2 fixation activity via the 3HP/4HB cycle, accompanied by enhanced ammonia oxidation for energy generation. In addition, CO oxidation activity was also elevated. In the summer, the primary source of energy originated from heterotrophic microorganisms capable of utilizing fatty acids, benzoate, and H2, likely relying on anaerobic respiration within organic particles. This may relate to nutrient source variations as reflected by the different levels of microbial network complexity between the two seasons. Altogether, our in situ metatranscriptomes revealed the metabolic activities and adaptations of active microbial groups across seasons, providing a basis for identifying the microbial contributors to elemental cycles in the deep ocean.

Recent discoveries have uncovered pelagic fungi as significant contributors to the recycling of organic matter in the ocean. However, their drivers and whether the environmental filtering on the functional role of prokaryotes also applies to pelagic fungi remain unknown. In this study, we employed the metagenomic and metatranscriptomic approaches to explore the fungi mediated organic matter degradation in the sunlit ocean. Samples were collected from the subtropical Atlantic Ocean (non-polar) to the Southern Ocean (polar), and differentiated between small (0.2 - 3 µm, SF) and large ( >3 µm, LF) size fractions, to study niche partitioning in fungal communities and functions. Fungi accounted for 2-5% of eukaryotic genes and transcripts. Fungi contributed over 3% of eukaryotic carbohydrate-active enzymes (CAZymes) transcripts but less than 0.5% of protease transcripts, highlighting their specialized role in carbohydrate degradation. Non-polar and polar regions exhibited distinct fungal community composition and metabolic functions, potentially disrupting the balance of organic matter storage and cycling in these ecologically sensitive regions. Temperature emerged as a key driver of fungal CAZyme activity, revealing sensitivity to ocean warming. Our findings underscore the active role of pelagic fungi in organic matter degradation while revealing the environmental and ecological factors shaping their functional contributions across global oceanic regions.

Phosphorus is essential for life and critically influences marine productivity. Despite geochemical evidence of active phosphorus cycling in deep-sea cold seeps, the microbial processes involved remain poorly understood. Traditional sequence-based searches often fail to detect proteins with remote homology. To address this, we developed a deep learning model, LucaPCycle, integrating raw sequences and contextual embeddings based on the protein language model ESM2-3B. LucaPCycle identified 5241 phosphorus-cycling protein families from global cold seep gene and genome catalogs, substantially enhancing our understanding of their diversity, ecology, and function. Among previously unannotated sequences, we discovered three alkaline phosphatase families that feature unique domain organizations and preserved enzymatic capabilities. These results highlight previously overlooked ecological importance of phosphorus cycling within cold seeps, corroborated by data from porewater geochemistry, metatranscriptomics, and metabolomics. We revealed a previously unrecognized diversity of archaea, including Asgardarchaeota, anaerobic methanotrophic archaea and Thermoproteota, which contribute to organic phosphorus mineralization and inorganic phosphorus solubilization through various mechanisms. Additionally, auxiliary metabolic genes of cold seep viruses primarily encode the PhoR-PhoB regulatory system and PhnCDE transporter, potentially enhancing their hosts' phosphorus utilization. Overall, LucaPCycle are capable of accessing previously 'hidden' sequence spaces for microbial phosphorus cycling and can be applied to various ecosystems.

Anaplerotic carbon fixation is ubiquitous in heterotrophic organisms including those inhabiting the ocean1. Despite its prevalence, the drivers of this process and its significance in ocean carbon cycling remain poorly understood2,3. Here we combined global ocean metatranscriptomic analysis, laboratory experiments on a bacterial model strain, and microautoradiography combined with catalyzed reporter deposition fluorescence in situ hybridization (MICRO-CARD-FISH) on marine microbial communities, to uncover the global prevalence of anaplerotic processes in oceanic dark dissolved inorganic carbon (DIC) fixation. Metatranscriptomic analysis revealed high expression levels of key anaplerotic genes, especially in mesopelagic waters, comparable to those of photo- and chemolithoautotrophic DIC fixation genes. Alteromonas emerged as the main contributor to anaplerotic DIC fixation gene expression, highlighting its role in DIC assimilation in the global ocean. Laboratory incubations with a marine Alteromonas representative confirmed their capability to fix DIC, which varied with organic matter availability and temperature. MICRO-CARD-FISH on oceanic samples revealed that Alteromonas contributed 0–40% (14 ± 16%, mean ± s.d.) to the dark DIC fixation in the pelagic ocean. Considering that Alteromonas is an obligate heterotroph lacking chemoautotrophic DIC fixation genes, its contribution to DIC fixation should be attributed to anaplerotic processes. Based on these results, we estimated a contribution of anaplerotic processes to dark DIC fixation of 0–0.5 C Pg y-1 in the global dark ocean. Yet, since Alteromonas is not the only taxon performing anaplerotic DIC fixation, our results represent a baseline conservative estimate. Collectively, our findings place anaplerotic DIC fixation as a relevant processes in the oceanic carbon cycling.

Even though fungi are ubiquitous in the biosphere, the ecological knowledge of marine fungi remains rather rudimentary. Also, little is known about their tolerance to salinity and how it influences their activities. Extracellular enzymatic activities (EEAs) are widely used to determine heterotrophic microbes’ enzymatic capabilities and substrate preferences. Five marine fungal species belonging to the most abundant pelagic phyla (Ascomycota and Basidiomycota) were grown under non-saline and saline conditions (0 g/L and 35 g/L, respectively). Due to their sensitivity and specificity, fluorogenic substrate analogues were used to determine hydrolytic activity on carbohydrates (β-glucosidase, β-xylosidase, and N-acetyl-β-D-glucosaminidase); peptides (leucine aminopeptidase and trypsin); lipids (lipase); organic phosphorus (alkaline phosphatase), and sulfur compounds (sulfatase). Afterwards, kinetic parameters such as maximum velocity (Vmax) and half-saturation constant (Km) were calculated. All fungal species investigated cleaved these substrates, but some species were more efficient than others. Moreover, most enzymatic activities were reduced in the saline medium, with some exceptions like sulfatase. In non-saline conditions, the average Vmax ranged between 208.5 to 0.02 μmol/g biomass/h, and in saline conditions, 88.4 to 0.02 μmol/g biomass/h. The average Km ranged between 1553.2 and 0.02 μM with no clear influence of salinity. Taken together, our results highlight a potential tolerance of marine fungi to freshwater conditions and indicate that changes in salinity (due to freshwater input or evaporation) might impact their enzymatic activities spectrum and, therefore, their contribution to the oceanic elemental cycles.

Biodiversity, the source of origin, and ecological roles of fungi in groundwater are to this day a largely neglected field in fungal and freshwater ecology. We used DNA-based Illumina high-throughput sequence analysis of both fungal gene markers 5.8S and internal transcribed spacers region 2 (ITS2), improving taxonomic classification. This study focused on the groundwater and river mycobiome along an altitudinal and longitudinal transect of a pre-alpine valley in Austria in two seasons. Using Bayesian network modeling approaches, we identified patterns in fungal community assemblages that were mostly shaped by differences in landscape (climatic, topological, and geological) and environmental conditions. While river fungi were comparatively more diverse, unique fungal assemblages could be recovered from groundwater, including typical aquatic lineages such as Rozellomycota and Olpidiomycota. The most specious assemblages in groundwater were not linked to the input of organic material from the surface, and as such, seem to be sustained by characteristic groundwater conditions. Based on what is known from closely related fungi, our results suggest that the present fungal communities potentially contribute to mineral weathering, carbon cycling, and denitrification in groundwater. Furthermore, we were able to observe the effects of varying land cover due to agricultural practices on fungal biodiversity in groundwater ecosystems. This study contributes to improving our understanding of fungi in the subsurface aquatic biogeosphere.

Multifunctional enzymes can significantly impact biotechnological applications by performing activities beyond their primary functions. This review explores the role of the multifunctionality of alkaline phosphatase, a key enzyme in the phosphorus cycle, focusing on the molecular mechanisms influencing its activity and its biotechnological potential. We argue that understanding these aspects can enhance the utility of alkaline phosphatase in research and industry, fostering innovations in enzyme engineering, environmental biotechnology, and metabolic engineering. By exploring enzyme promiscuity, we highlight alkaline phosphatase's versatility, paving the way for advancements in sustainable agriculture, environmental remediation, and clinical diagnostics. Further research will unlock new applications and catalytic efficiencies, driving forward ecological and biotechnological progress.

Proteins in the open ocean represent a significant source of organic matter, and their profiles reflect the metabolic activities of marine microorganisms. Here, by analyzing metaproteomic samples collected from the Pacific, Atlantic and Southern Ocean, we reveal size-fractionated patterns of the structure and function of the marine microbiota protein pool in the water column, particularly in the dark ocean (>200 m). Zooplankton proteins contributed three times more than algal proteins to the deep-sea community metaproteome. Gammaproteobacteria exhibited high metabolic activity in the deep-sea, contributing up to 30% of bacterial proteins. Close virus-host interactions of this taxon might explain the dominance of gammaproteobacterial proteins in the dissolved fraction. A high urease expression in nitrifiers suggested links between their dark carbon fixation and zooplankton urea production. In summary, our results uncover the taxonomic contribution of the microbiota to the oceanic protein pool, revealing protein fluxes from particles to the dissolved organic matter pool. This study analysed metaproteomic samples collected from the Pacific, Atlantic and Southern Oceans to reveal size-fractionated patterns of the structure and function of the marine microbiota protein pool in the water column.

Abstract Background Phosphorus plays a crucial role in limiting marine ecosystems productivity. To overcome this limitation, organisms use alkaline phosphatases (APases) to obtain phosphate from organic compounds. Although traditionally viewed as isoenzymes with specific roles, recent research has uncovered the multifunctionality of PhoA, a specific APase family. However, the prevalence of enzyme multifunctionality across all APase families remains unexplored, as does the determination of the dominant APases and the microbial taxa expressing them in the oceanic water column. Results Our findings revealed that all major APase families are actively expressed in the global ocean and are dominated by Alteromonas, particularly in the deep ocean. Enzymatic assays with purified recombinant APases from Alteromonas mediterranea revealed multifunctional activities with substrate affinities indicating distinct ecological roles. Proteomic analysis under phosphorus limitation unveiled specific protein regulation in Alteromonas mediterranea. Investigation into the secretion signal-peptides of APase enzymes revealed that PhoA and PafA predominantly utilize the Sec pathway for secretion, whereas PhoD and PhoX are primarily transported through the Tat pathway. Conclusions Our findings underscore the relevance of multifunctional APases in the global ocean, illustrating how microbes utilize multifunctional alkaline phosphatase families to acquire phosphorus, while ultimately highlighting the role of multifunctional enzymes in marine biogeochemical cycles.