Anthropogenically induced changes in precipitation are projected to generate increased river runoff to semi-enclosed seas, increasing loads of terrestrial dissolved organic matter and decreasing salinity. To determine how bacterial community structure and functioning adjust to such changes, we designed microcosm transplant experiments with Baltic Proper (salinity 7.2) and Bothnian Sea (salinity 3.6) water. Baltic Proper bacteria generally reached higher abundances than Bothnian Sea bacteria in both Baltic Proper and Bothnian Sea water, indicating higher adaptability. Moreover, Baltic Proper bacteria growing in Bothnian Sea water consistently showed highest bacterial production and beta-glucosidase activity. These metabolic responses were accompanied by basin-specific changes in bacterial community structure. For example, Baltic Proper Pseudomonas and Limnobacter populations increased markedly in relative abundance in Bothnian Sea water, indicating a replacement effect. In contrast, Roseobacter and Rheinheimera populations were stable or increased in abundance when challenged by either of the waters, indicating an adjustment effect. Transplants to Bothnian Sea water triggered the initial emergence of particular Burkholderiaceae populations, and transplants to Baltic Proper water triggered Alteromonadaceae populations. Notably, in the subsequent re-transplant experiment, a priming effect resulted in further increases to dominance of these populations. Correlated changes in community composition and metabolic activity were observed only in the transplant experiment and only at relatively high phylogenetic resolution. This suggested an importance of successional progression for interpreting relationships between bacterial community composition and functioning. We infer that priming effects on bacterial community structure by natural episodic events or climate change induced forcing could translate into long-term changes in bacterial ecosystem process rates.

Throughout coastal Antarctica, ice shelves separate oceanic waters from sunlight by hundreds of meters of ice. Historical studies have detected activity of nitrifying microorganisms in oceanic cavities below permanent ice shelves. However, little is known about the microbial composition and pathways that mediate these activities. In this study, we profiled the microbial communities beneath the Ross Ice Shelf using a multi-omics approach. Overall, beneath-shelf microorganisms are of comparable abundance and diversity, though distinct composition, relative to those in the open meso- and bathypelagic ocean. Production of new organic carbon is likely driven by aerobic lithoautotrophic archaea and bacteria that can use ammonium, nitrite, and sulfur compounds as electron donors. Also enriched were aerobic organoheterotrophic bacteria capable of degrading complex organic carbon substrates, likely derived from in situ fixed carbon and potentially refractory organic matter laterally advected by the below-shelf waters. Altogether, these findings uncover a taxonomically distinct microbial community potentially adapted to a highly oligotrophic marine environment and suggest that ocean cavity waters are primarily chemosynthetically-driven systems.

Ergosterol has traditionally been used as a proxy to estimate fungal biomass as it is almost exclusively found in fungal lipid membranes. Ergosterol determination has been mostly used for fungal samples from terrestrial, freshwater, salt marsh- and mangrove-dominated environments or to describe fungal degradation of plant matter. In the open ocean, however, the expected concentrations of ergosterol are orders of magnitude lower than in terrestrial or macrophyte-dominated coastal systems. Consequently, the fungal biomass in the open ocean remains largely unknown. Recent evidence based on microscopy and -omics techniques suggests, however, that fungi contribute substantially to the microbial biomass in the oceanic water column, highlighting the need to accurately determine fungal biomass in the open ocean. We performed ergosterol extractions of an oceanic fungal isolate (Rhodotorula sphaerocarpa) with biomass concentrations varying over nine orders of magnitude. While after the initial chloroform-methanol extraction ~87% of the ergosterol was recovered, a second extraction recovered an additional ~10%. Testing this extraction method on samples collected from the open Atlantic Ocean, we successfully determined ergosterol concentrations as low as 0.12 pM. Thus, this highly sensitive method is well suited for measuring fungal biomass from open ocean waters, including deep-sea environments.

Although terrestrial and aquatic fungi are well-known decomposers of organic matter, the role of marine fungi remains largely unknown. Recent studies based on omics suggest that marine fungi potentially play a major role in elemental cycles. However, there is very limited information on the diversity of extracellular enzymatic activities performed by pelagic fungi in the ocean and how these might be affected by community composition and/or critical environmental parameters such as temperature. In order to obtain information on the potential metabolic activity of marine fungi, extracellular enzymatic activities (EEA) were investigated. Five marine fungal species belonging to the most abundant pelagic phyla (Ascomycota and Basidiomycota) were grown at 5 °C and 20 °C, and fluorogenic enzymatic assays were performed using six substrate analogues for the hydrolysis of carbohydrates (β-glucosidase, β-xylosidase, and N-acetyl-β-D-glucosaminidase), amino acids (leucine aminopeptidase), and of organic phosphorus (alkaline phosphatase) and sulfur compounds (sulfatase). Remarkably, all fungal strains were capable of hydrolyzing all the offered substrates. However, the hydrolysis rate (Vmax) and half-saturation constant (Km) varied among the fungal strains depending on the enzyme type. Temperature had a strong impact on the EEAs, resulting in Q10 values of up to 6.1 and was species and substrate dependent. The observed impact of temperature on fungal EEA suggests that warming of the global ocean might alter the contribution of pelagic fungi in marine biogeochemical cycles.

Molecular hydrogen (H2) is an abundant and readily accessible energy source in marine systems, but it remains unknown whether marine microbial communities consume this gas. Here we use a suite of approaches to show that marine bacteria consume H2 to support growth. Genes for H2-uptake hydrogenases are prevalent in global ocean metagenomes, highly expressed in metatranscriptomes and found across eight bacterial phyla. Capacity for H2 oxidation increases with depth and decreases with oxygen concentration, suggesting that H2 is important in environments with low primary production. Biogeochemical measurements of tropical, temperate and subantarctic waters, and axenic cultures show that marine microbes consume H2 supplied at environmentally relevant concentrations, yielding enough cell-specific power to support growth in bacteria with low energy requirements. Conversely, our results indicate that oxidation of carbon monoxide (CO) primarily supports survival. Altogether, H2 is a notable energy source for marine bacteria and may influence oceanic ecology and biogeochemistry.

Heterotrophic prokaryotes express extracellular hydrolytic enzymes to cleave large organic molecules before taking up the hydrolyzed products. According to foraging theory, extracellular enzymes should be cell associated in dilute systems such as deep sea habitats, but secreted into the surrounding medium in diffusion-limited systems. However, extracellular enzymes in the deep sea are found mainly dissolved in ambient water rather than cell associated. In order to resolve this paradox, we conducted a global survey of peptidases and carbohydrate-active enzymes (CAZymes), two key enzyme groups initiating organic matter assimilation, in an integrated metagenomics, metatranscriptomics, and metaproteomics approach. The abundance, percentage, and diversity of genes encoding secretory processes, i.e., dissolved enzymes, consistently increased from epipelagic to bathypelagic waters, indicating that organic matter cleavage, and hence prokaryotic metabolism, is mediated mainly by particle-associated prokaryotes releasing their extracellular enzymes into diffusion-limited particles in the bathypelagic realm.

Most of the research on the cycling of carbon in the open-ocean has focused on heterotrophic prokaryotes and eukaryotic phytoplankton, but the role of pelagic fungi remains largely enigmatic.

Increased atmospheric CO2 is driving ocean acidification (OA), and potential changes in marine ecosystems. Research shows that both planktonic and benthic communities are affected, but how these changes are linked remains unresolved. Here we show experimentally that decreasing seawater pH (from pH 8.1 to 7.8 and 7.4) leads to reduced biofilm formation and lower primary producer biomass within biofilms. These changes occurred concurrently with a re-arrangement of the biofilm microbial communities. Changes suggest a potential shift from autotrophic to heterotrophic dominated biofilms in response to reduced pH. In a complimentary experiment, biofilms reared under reduced pH resulted in altered larval settlement for a model species (Galeolaria hystrix). These findings show that there is a potential cascade of impacts arising from OA effects on biofilms that may drive important community shifts through altered settlement patterns of benthic species.

The Southern Ocean contributes substantially to the global biological carbon pump (BCP). Salps in the Southern Ocean, in particular Salpa thompsoni, are important grazers that produce large, fast-sinking fecal pellets. Here, we quantify the salp bloom impacts on microbial dynamics and the BCP, by contrasting locations differing in salp bloom presence/absence. Salp blooms coincide with phytoplankton dominated by diatoms or prymnesiophytes, depending on water mass characteristics. Their grazing is comparable to microzooplankton during their early bloom, resulting in a decrease of ~1/3 of primary production, and negative phytoplankton rates of change are associated with all salp locations. Particle export in salp waters is always higher, ranging 2- to 8- fold (average 5-fold), compared to non-salp locations, exporting up to 46% of primary production out of the euphotic zone. BCP efficiency increases from 5 to 28% in salp areas, which is among the highest recorded in the global ocean.

To decipher the response of mesopelagic prokaryotic communities to input of nutrients, we tracked changes in prokaryotic abundance, extracellular enzymatic activities, heterotrophic production, dark dissolved inorganic carbon (DIC) fixation, community composition (16S rRNA sequencing) and community gene expression (metatranscriptomics) in 3 microcosm experiments with water from the mesopelagic North Atlantic. Responses in 3 different treatments amended with thiosulfate, ammonium or organic matter (i.e., pyruvate plus acetate) were compared to unamended controls. The strongest stimulation was found in the organic matter enrichments, where all measured rates increased >10-fold. Strikingly, in the organic matter treatment, the dark DIC fixation rates-assumed to be related to autotrophic metabolisms-were equally stimulated as all the other heterotrophic-related parameters. This increase in DIC fixation rates was paralleled by an up-regulation of genes involved in DIC assimilation via anaplerotic pathways. Alkaline phosphatase was the metabolic rate most strongly stimulated and its activity seemed to be related to cross-activation by nonpartner histidine kinases, and/or the activation of genes involved in the regulation of elemental balance during catabolic processes. These findings suggest that episodic events such as strong sedimentation of organic matter into the mesopelagic might trigger rapid increases of originally rare members of the prokaryotic community, enhancing heterotrophic and autotrophic carbon uptake rates, ultimately affecting carbon cycling. Our experiments highlight a number of fairly unstudied microbial processes of potential importance in mesopelagic waters that require future attention.

Fjords are semi-enclosed marine systems with unique physical conditions that influence microbial community composition and structure. Pronounced organic matter and physical condition gradients within fjords provide a natural laboratory for the study of changes in microbial community structure and metabolic potential in response to environmental conditions. Photosynthetic production in euphotic zones sustains deeper aphotic microbial activity via organic matter sinking, augmented by large terrestrial inputs. Previous studies do not consider both prokaryotic and eukaryotic communities when linking metabolic potential and activity, community composition, and environmental gradients. To address this gap we profiled microbial functional potential (Biolog Ecoplates), bacterial abundance, heterotrophic production (3H-Leucine incorporation), and prokaryotic/eukaryotic community composition (16S and 18S rRNA amplicon gene sequencing). Similar factors shaped metabolic potential, activity and community (prokaryotic and eukaryotic) composition across surface/near surface sites. However, increased metabolic diversity at near bottom (aphotic) sites reflected an organic matter influence from sediments. Photosynthetically produced particulate organic matter shaped the upper water column community composition and metabolic potential. In contrast, microbial activity at deeper aphotic waters were strongly influenced by other organic matter input than sinking marine snow (e.g. sediment resuspension of benthic organic matter, remineralisation of terrestrially derived organic matter, etc.), severing the link between community structure and metabolic potential. Taken together, different organic matter sources shape microbial activity, but not community composition, in New Zealand fjords.

One of the central objectives of microbial ecology is to study the distribution of microbial communities and their association with their environments. Biogeographical studies have partitioned the oceans into provinces and regions, but the identification of their boundaries remains challenging, hindering our ability to study transition zones (i.e. ecotones) and microbial ecosystem heterogeneity. Fuzzy clustering is a promising method to do so, as it creates overlapping sets of clusters. The outputs of these analyses thus appear both structured (into clusters) and gradual (due to the overlaps), which aligns with the inherent continuity of the pelagic environment, and solves the issue of defining ecosystem boundaries.

Alkaline phosphatase (APase) is one of the marine enzymes used by oceanic microbes to obtain inorganic phosphorus (Pi) from dissolved organic phosphorus to overcome P-limitation. Marine APase is generally recognized to perform P-monoesterase activity. Here we integrated a biochemical characterization of a specific APase enzyme, examination of global ocean databases, and field measurements, to study the type and relevance of marine APase promiscuity. We performed an in silico mining of phoA homologs, followed by de novo synthesis and heterologous expression in E. coli of the full-length gene from Alteromonas mediterranea, resulting in a recombinant PhoA. A global analysis using the TARA Oceans, Malaspina and other metagenomic databases confirmed the predicted widespread distribution of the gene encoding the targeted PhoA in all oceanic basins throughout the water column. Kinetic assays with the purified PhoA enzyme revealed that this enzyme exhibits not only the predicted P-monoester activity, but also P-diesterase, P-triesterase and sulfatase activity as a result of a promiscuous behavior. Among all activities, P-monoester bond hydrolysis exhibited the highest catalytic activity of APase despite its lower affinity for phosphate monoesters. APase is highly efficient as a P-monoesterase at high substrate concentrations, whereas promiscuous activities of APase, like diesterase, triesterase, and sulfatase activities are more efficient at low substrate concentrations. Strong similarities were observed between the monoesterase:diesterase ratio of the purified PhoA protein in the laboratory and in natural seawater. Thus, our results reveal enzyme promiscuity of APase playing potentially an important role in the marine phosphorus cycle.

Marine microbes are an important control on the biogeochemical cycling of trace metals, but simultaneously, these metals can control the growth of microorganisms and the cycling of major nutrients like C and N. However, studies on the response/limitation of microorganisms to trace metals have traditionally focused on the response of autotrophic phytoplankton to Fe fertilization. Few reports are available on the response of heterotrophic prokaryotes to Fe, and even less to other biogeochemically relevant metals. We performed the first study coupling dark incubations with next generation sequencing to specifically target the functional and phylogenetic response of heterotrophic prokaryotes to Fe enrichment. Furthermore, we also studied their response to Co, Mn, Ni, Zn, Cu (individually and mixed), using surface and deep samples from either coastal or open-ocean waters. Heterotrophic prokaryotic activity was stimulated by Fe in surface open-ocean, as well as in coastal, and deep open-ocean waters (where Zn also stimulated). The most susceptible populations to trace metals additions were uncultured bacteria (e.g., SAR324, SAR406, NS9, and DEV007). Interestingly, hydrocarbon-degrading bacteria (e.g., Thalassolituus, Marinobacter, and Oleibacter) benefited the most from metal addition across all waters (regions/depths) revealing a predominant role in the cycling of metals and organic matter in the ocean.

Natural autofluorescence is a widespread phenomenon observed in different types of tissues and organisms. Depending on the origin of the autofluorescence, its intensity can provide insights on the physiological state of an organism. Fungal autofluorescence has been reported in terrestrial and human-derived fungal samples. Yet, despite the recently reported ubiquitous presence and importance of marine fungi in the ocean, the autofluorescence of pelagic fungi has never been examined. Here, we investigated the existence and intensity of autofluorescence in five different pelagic fungal isolates. Preliminary experiments of fungal autofluorescence at different growth stages and nutrient conditions were conducted, reflecting contrasting physiological states of the fungi. In addition, we analysed the effect of natural autofluorescence on co-staining with DAPI. We found that all the marine pelagic fungi that were studied exhibited autofluorescence. The intensity of fungal autofluorescence changed depending on the species and the excitation wavelength used. Furthermore, fungal autofluorescence varied depending on the growth stage and on the concentration of available nutrients. Collectively, our results indicate that marine fungi can be auto-fluorescent, although its intensity depends on the species and growth condition. Hence, oceanic fungal autofluorescence should be considered in future studies when fungal samples are stained with fluorescent probes (i.e., fluorescence in situ hybridization) since this could lead to misinterpretation of results.

Fungi have shaped the biosphere since the development of life on Earth. Despite fungi being present in all environments, most of the available fungal research has focused on soils. As a result, the role and composition of fungal communities in aquatic (marine and freshwater) environments remain largely unexplored. The use of different primers to characterise fungal communities has additionally complicated intercomparisons among studies. Consequently, we lack a basic global assessment of fungal diversity across major ecosystems. Here, we took advantage of a recently published 18S rRNA dataset comprising samples from major ecosystems (terrestrial, freshwater, and marine) to attempt a global assessment of fungal diversity and community composition. We found the highest fungal diversities for terrestrial > freshwater > marine environments, and pronounced gradients of fungal diversity along temperature, salinity, and latitude in all ecosystems. We also identified the most abundant taxa in each of these ecosystems, mostly dominated by Ascomycota and Basidiomycota, except in freshwater rivers where Chytridiomycota dominated. Collectively, our analysis provides a global analysis of fungal diversity across all major environmental ecosystems, highlighting the most distinct order and ASVs (amplicon sequencing variants) by ecosystem, and thus filling a critical gap in the study of the Earth's mycobiome.

Marine microbial communities rely on dissolved organic phosphorus (DOP) remineralisation to meet phosphorus (P) requirements. We extensively surveyed the genomic and metagenomic distribution of genes directing phosphonate biosynthesis, substrate-specific catabolism of 2-aminoethylphosphonate (2-AEP, the most abundant phosphonate in the marine environment), and broad-specificity catabolism of phosphonates by the C-P lyase (including methylphosphonate, a major source of methane). We developed comprehensive enzyme databases by curating publicly available sequences and then screened metagenomes from TARA Oceans and Munida Microbial Observatory Time Series (MOTS) to assess spatial and seasonal variation in phosphonate metabolism pathways. Phosphonate cycling genes were encoded in diverse gene clusters by 35 marine bacterial and archaeal classes. More than 65% of marine phosphonate cycling genes mapped to Proteobacteria with production demonstrating wider taxonomic diversity than catabolism. Hydrolysis of 2-AEP was the dominant phosphonate catabolism strategy, enabling microbes to assimilate carbon and nitrogen alongside P. Genes for broad-specificity catabolism by the C-P lyase were far less widespread, though enriched in the extremely P-deplete environment of the Mediterranean Sea. Phosphonate cycling genes were abundant in marine metagenomes, particularly from the mesopelagic zone and winter sampling dates. Disparity between prevalence of substrate-specific and broad-specificity catabolism may be due to higher resource expenditure from the cell to build and retain the C-P lyase. This study is the most comprehensive metagenomic survey of marine microbial phosphonate cycling to date and provides curated databases for 14 genes involved in phosphonate cycling.

Marine bacterioplankton are essential in global nutrient cycling and organic matter turnover. Time-series analyses, often at monthly sampling frequencies, have established the paramount role of abiotic and biotic variables in structuring bacterioplankton communities and productivities. However, fine-scale seasonal microbial activities, and underlying biological principles, are not fully understood. We report results from four consecutive years of high-frequency time-series sampling in the Baltic Proper. Pronounced temporal dynamics in most investigated microbial variables were observed, including bacterial heterotrophic production, plankton biomass, extracellular enzyme activities, substrate uptake rate constants of glucose, pyruvate, acetate, amino acids, and leucine, as well as nutrient limitation bioassays. Spring blooms consisting of diatoms and dinoflagellates were followed by elevated bacterial heterotrophic production and abundances. During summer, bacterial productivity estimates increased even further, coinciding with an initial cyanobacterial bloom in early July. However, bacterial abundances only increased following a second cyanobacterial bloom, peaking in August. Uptake rate constants for the different measured carbon compounds varied seasonally and inter-annually and were highly correlated to bacterial productivity estimates, temperature, and cyanobacterial abundances. Further, we detected nutrient limitation in response to environmental conditions in a multitude of microbial variables, such as elevated productivities in nutrient bioassays, changes in enzymatic activities, or substrate preferences. Variations among biotic variables often occurred on time scales of days to a few weeks, yet often spanning several sampling occasions. Such dynamics might not have been captured by sampling at monthly intervals, as compared to more predictable transitions in abiotic variables such as temperature or nutrient concentrations. Our study indicates that high resolution analyses of microbial biomass and productivity parameters can help out in the development of biogeochemical and food web models disentangling the microbial black box.

Compared to higher latitudes, tropical heterotrophic bacteria may be less responsive to warming because of strong bottom-up control. In order to separate both drivers, we determined the growth responses of bacterial physiological groups to temperature after adding dissolved organic matter (DOM) from mangroves, seagrasses and glucose to natural seawater from the Great Barrier Reef. Low (LNA) and high (HNA) nucleic acid content, membrane-intact (Live) and membrane-damaged (Dead) plus actively respiring (CTC+) cells were monitored for 4 days. Specific growth rates of the whole community were significantly higher (1.9 day-1 ) in the mangrove treatment relative to the rest (0.2-0.4 day-1 ) at in situ temperature and their temperature dependence, estimated as activation energy, was also consistently higher. Strong bottom-up control was suggested in the other treatments. Cell size depended more on DOM than temperature. Mangrove DOM resulted in significantly higher contributions of Live, HNA and CTC+ cells to total abundance, while the seagrass leachate reduced Live cells below 50%. Warming significantly decreased Live and CTC+ cells contributions in most treatments. Our results suggest that only in the presence of highly labile compounds, such as mangroves DOM, can we anticipate increases in heterotrophic bacteria biomass in response to warming in tropical regions.

Despite recent studies suggesting that marine fungi are ubiquitous in oceanic systems and involved in organic matter degradation, their role in the carbon cycle of the oceans is still not characterized and fungal respiration and production are understudied. This study focused on determining fungal growth efficiencies and its susceptibility to temperature differences and nutrient concentration. Hence, respiration and biomass production of three fungal isolates (Rhodotorula mucilaginosa, Rhodotorula sphaerocarpa, Sakaguchia dacryoidea) were measured in laboratory experiments at two temperatures and two nutrient concentrations. We found that fungal respiration and production rates differed among species, temperature, and nutrient concentration. Fungal respiration and production were higher at higher temperatures, but higher fungal growth efficiencies were observed at lower temperatures. Nutrient concentration affected fungal respiration, production, and growth efficiency, but its influence differed among species. Altogether, this study provides the first growth efficiency estimates of pelagic fungi, providing novel insights into the role of fungi as source/sink of carbon during organic matter remineralization. Further research is now needed to unravel the role of pelagic fungi in the marine carbon cycle, a topic that gains even more importance in times of increasing CO2 concentrations and global warming.