Climate change induces shifts in coastal Baltic Sea surface water microorganism stress and photosynthesis gene expression.

The world's oceans are challenged by climate change linked warming with typically highly populated coastal areas being particularly susceptible to these effects. Many studies of climate change on the marine environment use large, short-term temperature manipulations that neglect factors such as long-term adaptation and seasonal cycles. In this study, a Baltic Sea 'heated' bay influenced by thermal discharge since the 1970s from a nuclear reactor (in relation to an unaffected nearby 'control' bay) was used to investigate how elevated temperature impacts surface water microbial communities and activities. 16S rRNA gene amplicon based microbial diversity and population structure showed no difference in alpha diversity in surface water microbial communities, while the beta diversity showed a dissimilarity between the bays. Amplicon sequencing variant relative abundances between the bays showed statistically higher values for, e.g., Ilumatobacteraceae and Burkholderiaceae in the heated and control bays, respectively. RNA transcript-derived activities followed a similar pattern in alpha and beta diversity with no effect on Shannon's H diversity but a significant difference in the beta diversity between the bays. The RNA data further showed more elevated transcript counts assigned to stress related genes in the heated bay that included heat shock protein genes dnaKJ, the co-chaperonin groS, and the nucleotide exchange factor heat shock protein grpE. The RNA data also showed elevated oxidative phosphorylation transcripts in the heated (e.g., atpHG) compared to control (e.g., atpAEFB) bay. Furthermore, genes related to photosynthesis had generally higher transcript numbers in the control bay, such as photosystem I (psaAC) and II genes (psbABCEH). These increased stress gene responses in the heated bay will likely have additional cascading effects on marine carbon cycling and ecosystem services.

Ebullition dominates methane emissions in stratified coastal waters.

Coastal areas are an important source of methane (CH4). However, the exact origins of CH4 in the surface waters of coastal regions, which in turn drive sea-air emissions, remain uncertain. To gain a comprehensive understanding of the current and future climate change feedbacks, it is crucial to identify these CH4 sources and processes that regulate its formation and oxidation. This study investigated coastal CH4 dynamics by comparing water column data from six stations located in the brackish Tvärminne Archipelago, Baltic Sea. The sediment biogeochemistry and microbiology were further investigated at two stations (i.e., nearshore and offshore). These stations differed in terms of stratification, bottom water redox conditions, and organic matter loading. At the nearshore station, CH4 diffusion from the sediment into the water column was negligible, because nearly all CH4 was oxidized within the upper sediment column before reaching the sediment surface. On the other hand, at the offshore station, there was significant benthic diffusion of CH4, albeit the majority underwent oxidation before reaching the sediment-water interface, due to shoaling of the sulfate methane transition zone (SMTZ). The potential contribution of CH4 production in the water column was evaluated and was found to be negligible. After examining the isotopic signatures of δ13C-CH4 across the sediment and water column, it became apparent that the surface water δ13C-CH4 values observed in areas with thermal stratification could not be explained by diffusion, advective fluxes, nor production in the water column. In fact, these values bore a remarkable resemblance to those detected below the SMTZ. This supports the hypothesis that the source of CH4 in surface waters is more likely to originate from ebullition than diffusion in stratified brackish coastal systems.

Biotic interactions between benthic infauna and aerobic methanotrophs mediate methane fluxes from coastal sediments.

Coastal ecosystems dominate oceanic methane (CH4) emissions. However, there is limited knowledge about how biotic interactions between infauna and aerobic methanotrophs (i.e. CH4 oxidizing bacteria) drive the spatial-temporal dynamics of these emissions. Here, we investigated the role of meio- and macrofauna in mediating CH4 sediment-water fluxes and aerobic methanotrophic activity that can oxidize significant portions of CH4. We show that macrofauna increases CH4 fluxes by enhancing vertical solute transport through bioturbation, but this effect is somewhat offset by high meiofauna abundance. The increase in CH4 flux reduces CH4 pore-water availability, resulting in lower abundance and activity of aerobic methanotrophs, an effect that counterbalances the potential stimulation of these bacteria by higher oxygen flux to the sediment via bioturbation. These findings indicate that a larger than previously thought portion of CH4 emissions from coastal ecosystems is due to faunal activity and multiple complex interactions with methanotrophs.

Thiobacillus as a key player for biofilm formation in oligotrophic groundwaters of the Fennoscandian Shield.

Biofilm formation is a common adaptation for microbes in energy-limited conditions such as those prevalent in the vast deep terrestrial biosphere. However, due to the low biomass and the inaccessible nature of subsurface groundwaters, the microbial populations and genes involved in its formation are understudied. Here, a flow-cell system was designed to investigate biofilm formation under in situ conditions in two groundwaters of contrasting age and geochemistry at the Äspö Hard Rock Laboratory, Sweden. Metatranscriptomes showed Thiobacillus, Sideroxydans, and Desulforegula to be abundant and together accounted for 31% of the transcripts in the biofilm communities. Differential expression analysis highlighted Thiobacillus to have a principal role in biofilm formation in these oligotrophic groundwaters by being involved in relevant processes such as the formation of extracellular matrix, quorum sensing, and cell motility. The findings revealed an active biofilm community with sulfur cycling as a prominent mode of energy conservation in the deep biosphere.

Long-Term Pollution Does Not Inhibit Denitrification and DNRA by Adapted Benthic Microbial Communities.

Denitrification in sediments is a key microbial process that removes excess fixed nitrogen, while dissimilatory nitrate reduction to ammonium (DNRA) converts nitrate to ammonium. Although microorganisms are responsible for essential nitrogen (N) cycling, it is not yet fully understood how these microbially mediated processes respond to toxic hydrophobic organic compounds (HOCs) and metals. In this study, we sampled long-term polluted sediment from the outer harbor of Oskarshamn (Baltic Sea), measured denitrification and DNRA rates, and analyzed taxonomic structure and N-cycling genes of microbial communities using metagenomics. Results showed that denitrification and DNRA rates were within the range of a national reference site and other unpolluted sites in the Baltic Sea, indicating that long-term pollution did not significantly affect these processes. Furthermore, our results indicate an adaptation to metal pollution by the N-cycling microbial community. These findings suggest that denitrification and DNRA rates are affected more by eutrophication and organic enrichment than by historic pollution of metals and organic contaminants.

Climate change-related warming reduces thermal sensitivity and modifies metabolic activity of coastal benthic bacterial communities.

Besides long-term average temperature increases, climate change is projected to result in a higher frequency of marine heatwaves. Coastal zones are some of the most productive and vulnerable ecosystems, with many stretches already under anthropogenic pressure. Microorganisms in coastal areas are central to marine energy and nutrient cycling and therefore, it is important to understand how climate change will alter these ecosystems. Using a long-term heated bay (warmed for 50 years) in comparison with an unaffected adjacent control bay and an experimental short-term thermal (9 days at 6-35 °C) incubation experiment, this study provides new insights into how coastal benthic water and surface sediment bacterial communities respond to temperature change. Benthic bacterial communities in the two bays reacted differently to temperature increases with productivity in the heated bay having a broader thermal tolerance compared with that in the control bay. Furthermore, the transcriptional analysis showed that the heated bay benthic bacteria had higher transcript numbers related to energy metabolism and stress compared to the control bay, while short-term elevated temperatures in the control bay incubation experiment induced a transcript response resembling that observed in the heated bay field conditions. In contrast, a reciprocal response was not observed for the heated bay community RNA transcripts exposed to lower temperatures indicating a potential tipping point in community response may have been reached. In summary, long-term warming modulates the performance, productivity, and resilience of bacterial communities in response to warming.

Methane emissions offset atmospheric carbon dioxide uptake in coastal macroalgae, mixed vegetation and sediment ecosystems.

Coastal ecosystems can efficiently remove carbon dioxide (CO2) from the atmosphere and are thus promoted for nature-based climate change mitigation. Natural methane (CH4) emissions from these ecosystems may counterbalance atmospheric CO2 uptake. Still, knowledge of mechanisms sustaining such CH4 emissions and their contribution to net radiative forcing remains scarce for globally prevalent macroalgae, mixed vegetation, and surrounding depositional sediment habitats. Here we show that these habitats emit CH4 in the range of 0.1 - 2.9 mg CH4 m-2 d-1 to the atmosphere, revealing in situ CH4 emissions from macroalgae that were sustained by divergent methanogenic archaea in anoxic microsites. Over an annual cycle, CO2-equivalent CH4 emissions offset 28 and 35% of the carbon sink capacity attributed to atmospheric CO2 uptake in the macroalgae and mixed vegetation habitats, respectively, and augment net CO2 release of unvegetated sediments by 57%. Accounting for CH4 alongside CO2 sea-air fluxes and identifying the mechanisms controlling these emissions is crucial to constrain the potential of coastal ecosystems as net atmospheric carbon sinks and develop informed climate mitigation strategies.

Molecular diet analysis enables detection of diatom and cyanobacteria DNA in the gut of Macoma balthica.

Detritivores are essential to nutrient cycling, but are often neglected in trophic networks, due to difficulties with determining their diet. DNA analysis of gut contents shows promise of trophic link discrimination, but many unknown factors limit its usefulness. For example, DNA can be rapidly broken down, especially by digestion processes, and DNA provides only a snapshot of the gut contents at a specific time. Few studies have been performed on the length of time that prey DNA can be detected in consumer guts, and none so far using benthic detritivores. Eutrophication, along with climate change, is altering the phytoplankton communities in aquatic ecosystems, on which benthic detritivores in aphotic soft sediments depend. Nutrient-poor cyanobacteria blooms are increasing in frequency, duration, and magnitude in many water bodies, while nutrient-rich diatom spring blooms are shrinking in duration and magnitude, creating potential changes in diet of benthic detritivores. We performed an experiment to identify the taxonomy and quantify the abundance of phytoplankton DNA fragments on bivalve gut contents, and how long these fragments can be detected after consumption in the Baltic Sea clam Macoma balthica. Two common species of phytoplankton (the cyanobacteria Nodularia spumigena or the diatom Skeletonema marinoi) were fed to M. balthica from two regions (from the northern and southern Stockholm archipelago). After removing the food source, M. balthica gut contents were sampled every 24 hours for seven days to determine the number of 23S rRNA phytoplankton DNA copies and when the phytoplankton DNA could no longer be detected by quantitative PCR. We found no differences in diatom 18S rRNA gene fragments of the clams by region, but the southern clams showed significantly more cyanobacteria 16S rRNA gene fragments in their guts than the northern clams. Interestingly, the cyanobacteria and diatom DNA fragments were still detectable by qPCR in the guts of M. balthica one week after removal from its food source. However, DNA metabarcoding of the 23S rRNA phytoplankton gene found in the clam guts showed that added food (i.e. N. spumigena and S. marinoi) did not make up a majority of the detected diet. Our results suggest that these detritivorous clams therefore do not react as quickly as previously thought to fresh organic matter inputs, with other phytoplankton than large diatoms and cyanobacteria constituting the majority of their diet. This experiment demonstrates the viability of using molecular methods to determine feeding of detritivores, but further studies investigating how prey DNA signals can change over time in benthic detritivores will be needed before this method can be widely applicable to both models of ecological functions and conservation policy.

Microbial functional genes are driven by gradients in sediment stoichiometry, oxygen, and salinity across the Baltic benthic ecosystem.

BACKGROUND: Microorganisms in the seafloor use a wide range of metabolic processes, which are coupled to the presence of functional genes within their genomes. Aquatic environments are heterogenous and often characterized by natural physiochemical gradients that structure these microbial communities potentially changing the diversity of functional genes and its associated metabolic processes. In this study, we investigated spatial variability and how environmental variables structure the diversity and composition of benthic functional genes and metabolic pathways across various fundamental environmental gradients. We analyzed metagenomic data from sediment samples, measured related abiotic data (e.g., salinity, oxygen and carbon content), covering 59 stations spanning 1,145 km across the Baltic Sea. RESULTS: The composition of genes and microbial communities were mainly structured by salinity plus oxygen, and the carbon to nitrogen (C:N) ratio for specific metabolic pathways related to nutrient transport and carbon metabolism. Multivariate analyses indicated that the compositional change in functional genes was more prominent across environmental gradients compared to changes in microbial taxonomy even at genus level, and indicate functional diversity adaptation to local environments. Oxygen deficient areas (i.e., dead zones) were more different in gene composition when compared to oxic sediments. CONCLUSIONS: This study highlights how benthic functional genes are structured over spatial distances and by environmental gradients and resource availability, and suggests that changes in, e.g., oxygenation, salinity, and carbon plus nitrogen content will influence functional metabolic pathways in benthic habitats. Video Abstract.

Long-Term Warming of Baltic Sea Coastal Waters Affects Bacterial Communities in Bottom Water and Sediments Differently.

Coastal marine ecosystems are some of the most diverse natural habitats while being highly vulnerable in the face of climate change. The combination of anthropogenic influence from land and ongoing climate change will likely have severe effects on the environment, but the precise response remains uncertain. This study compared an unaffected "control" Baltic Sea bay to a "heated" bay that has undergone artificial warming from cooling water release from a nuclear power plant for ~50 years. This heated the water in a similar degree to IPCC SSP5-8.5 predictions by 2100 as natural systems to study temperature-related climate change effects. Bottom water and surface sediment bacterial communities and their biogeochemical processes were investigated to test how future coastal water warming alters microbial communities; shifts seasonal patterns, such as increased algae blooming; and influences nutrient and energy cycling, including elevated respiration rates. 16S rRNA gene amplicon sequencing and geochemical parameters demonstrated that heated bay bottom water bacterial communities were influenced by increased average temperatures across changing seasons, resulting in an overall Shannon's H diversity loss and shifts in relative abundances. In contrast, Shannon's diversity increased in the heated surface sediments. The results also suggested a trend toward smaller-sized microorganisms within the heated bay bottom waters, with a 30% increased relative abundance of small size picocyanobacteria in the summer (June). Furthermore, bacterial communities in the heated bay surface sediment displayed little seasonal variability but did show potential changes of long-term increased average temperature in the interplay with related effects on bottom waters. Finally, heated bay metabolic gene predictions from the 16S rRNA gene sequences suggested raised anaerobic processes closer to the sediment-water interface. In conclusion, climate change will likely alter microbial seasonality and diversity, leading to prolonged and increased algae blooming and elevated respiration rates within coastal waters.

High spatiotemporal variability of methane concentrations challenges estimates of emissions across vegetated coastal ecosystems.

Coastal methane (CH4 ) emissions dominate the global ocean CH4 budget and can offset the "blue carbon" storage capacity of vegetated coastal ecosystems. However, current estimates lack systematic, high-resolution, and long-term data from these intrinsically heterogeneous environments, making coastal budgets sensitive to statistical assumptions and uncertainties. Using continuous CH4 concentrations, δ13 C-CH4  values, and CH4  sea-air fluxes across four seasons in three globally pervasive coastal habitats, we show that the CH4 distribution is spatially patchy over meter-scales and highly variable in time. Areas with mixed vegetation, macroalgae, and their surrounding sediments exhibited a spatiotemporal variability of surface water CH4 concentrations ranging two orders of magnitude (i.e., 6-460 nM CH4 ) with habitat-specific seasonal and diurnal patterns. We observed (1) δ13 C-CH4  signatures that revealed habitat-specific CH4 production and consumption pathways, (2) daily peak concentration events that could change >100% within hours across all habitats, and (3) a high thermal sensitivity of the CH4 distribution signified by apparent activation energies of ~1 eV that drove seasonal changes. Bootstrapping simulations show that scaling the CH4 distribution from few samples involves large errors, and that ~50 concentration samples per day are needed to resolve the scale and drivers of the natural variability and improve the certainty of flux calculations by up to 70%. Finally, we identify northern temperate coastal habitats with mixed vegetation and macroalgae as understudied but seasonally relevant atmospheric CH4  sources (i.e., releasing ≥ 100 µmol CH4  m-2  day-1 in summer). Due to the large spatial and temporal heterogeneity of coastal environments, high-resolution measurements will improve the reliability of CH4 estimates and confine the habitat-specific contribution to regional and global CH4 budgets.

Weakened resilience of benthic microbial communities in the face of climate change.

Increased ocean temperature associated with climate change is especially intensified in coastal areas and its influence on microbial communities and biogeochemical cycling is poorly understood. In this study, we sampled a Baltic Sea bay that has undergone 50 years of warmer temperatures similar to RCP5-8.5 predictions due to cooling water release from a nuclear power plant. The system demonstrated reduced oxygen concentrations, decreased anaerobic electron acceptors, and higher rates of sulfate reduction. Chemical analyses, 16S rRNA gene amplicons, and RNA transcripts all supported sediment anaerobic reactions occurring closer to the sediment-water interface. This resulted in higher microbial diversities and raised sulfate reduction and methanogenesis transcripts, also supporting increased production of toxic sulfide and the greenhouse gas methane closer to the sediment surface, with possible release to oxygen deficient waters. RNA transcripts supported prolonged periods of cyanobacterial bloom that may result in increased climate change related coastal anoxia. Finally, while metatranscriptomics suggested increased energy production in the heated bay, a large number of stress transcripts indicated the communities had not adapted to the increased temperature and had weakened resilience. The results point to a potential feedback loop, whereby increased temperatures may amplify negative effects at the base of coastal biochemical cycling.

Interplay between eutrophication and climate warming on bacterial communities in coastal sediments differs depending on water depth and oxygen history.

Coastal aquatic systems suffer from nutrient enrichment, which results in accelerated eutrophication effects due to increased microbial metabolic rates. Climate change related prolonged warming will likely accelerate existing eutrophication effects, including low oxygen concentrations. However, how the interplay between these environmental changes will alter coastal ecosystems is poorly understood. In this study, we compared 16S rRNA gene amplicon based bacterial communities in coastal sediments of a Baltic Sea basin in November 2013 and 2017 at three sites along a water depth gradient with varying bottom water oxygen histories. The shallow site showed changes of only 1.1% in relative abundance of bacterial populations in 2017 compared to 2013, while the deep oxygen-deficient site showed up to 11% changes in relative abundance including an increase of sulfate-reducing bacteria along with a 36% increase in organic matter content. The data suggested that bacterial communities in shallow sediments were more resilient to seasonal oxygen decline, while bacterial communities in sediments subjected to long-term hypoxia seemed to be sensitive to oxygen changes and were likely to be under hypoxic/anoxic conditions in the future. Our data demonstrate that future climate changes will likely fuel eutrophication related spread of low oxygen zones.

Cyanophage Diversity and Community Structure in Dead Zone Sediments.

Up to 20% of prokaryotic organisms in the oceans are estimated to die every day due to viral infection and lysis. Viruses can therefore alter microbial diversity, community structure, and biogeochemical processes driven by these organisms. Cyanophages are viruses that infect and lyse cyanobacterial cells, adding bioavailable carbon and nutrients into the environment. Cyanobacteria are photosynthesizing bacteria, with some species capable of N2 fixation, which are known to form large blooms as well as resistant resting cells known as akinetes. Here, we investigated cyanophage diversity and community structure plus cyanobacteria in dead zone sediments. We sampled surface sediments and sequenced DNA and RNA, along an oxygen gradient-representing oxic, hypoxic, and anoxic conditions-in one of the world's largest dead zones located in the Baltic Sea. Cyanophages were detected at all stations and, based on partial genome contigs, had a higher alpha diversity and different beta diversity in the hypoxic-anoxic sediments, suggesting that cyanobacteria in dead zone sediments and/or environmental conditions select for specific cyanophages. Some of these cyanophages can infect cyanobacteria with potential consequences for gene expression related to their photosystem and phosphate regulation. Top cyanobacterial genera detected in the anoxic sediment included Dolichospermum/Anabaena, Synechococcus, and Cyanobium RNA transcripts classified to cyanobacteria were associated with numerous pathways, including anaerobic carbon metabolism and N2 fixation. Cyanobacterial blooms are known to fuel oxygen-depleted ecosystems with phosphorus (so-called internal loading), and our cyanophage data indicate the potential for viral lysis of cyanobacteria which might explain the high nutrient turnover in these environments.IMPORTANCE Cyanophages are viruses that target cyanobacteria and directly control their abundance via viral lysis. Cyanobacteria are known to cause large blooms in water bodies, substantially contributing to oxygen depletion in bottom waters resulting in areas called dead zones. Our knowledge of cyanophages in dead zones is very scarce, and so far, no studies have assembled partial cyanophage genomes and investigated their associated cyanobacteria in these dark and anoxic sediments. Here, we present the first study using DNA and RNA sequencing to investigate in situ diversity of cyanophages and cyanobacteria in dead zones. Our study shows that dead zone sediments contain different cyanophages compared to oxic sediments and suggest that these viruses are able to affect cyanobacterial photosystem and phosphate regulation. Furthermore, cyanophage-controlled lysis of cyanobacteria might also increase the turnover of carbon, phosphorus, and nitrogen in these oxygen-free environments at the bottom of the sea.

Active DNRA and denitrification in oxic hypereutrophic waters.

Since the start of synthetic fertilizer production more than a hundred years ago, the coastal ocean has been exposed to increasing nutrient loading, which has led to eutrophication and extensive algal blooms. Such hypereutrophic waters might harbor anaerobic nitrogen (N) cycling processes due to low-oxygen microniches associated with abundant organic particles, but studies on nitrate reduction in coastal pelagic environments are scarce. Here, we report on 15N isotope-labeling experiments, metagenome, and RT-qPCR data from a large hypereutrophic lagoon indicating that dissimilatory nitrate reduction to ammonium (DNRA) and denitrification were active processes, even though the bulk water was fully oxygenated (> 224 µM O2). DNRA in the bottom water corresponded to 83% of whole-ecosystem DNRA (water + sediment), while denitrification was predominant in the sediment. Microbial taxa important for DNRA according to the metagenomic data were dominated by Bacteroidetes (genus Parabacteroides) and Proteobacteria (genus Wolinella), while denitrification was mainly associated with proteobacterial genera Pseudomonas, Achromobacter, and Brucella. The metagenomic and microscopy data suggest that these anaerobic processes were likely occurring in low-oxygen microniches related to extensive growth of filamentous cyanobacteria, including diazotrophic Dolichospermum and non-diazotrophic Planktothrix. By summing the total nitrate fluxes through DNRA and denitrification, it results that DNRA retains approximately one fifth (19%) of the fixed N that goes through the nitrate pool. This is noteworthy as DNRA represents thus a very important recycling mechanism for fixed N, which sustains algal proliferation and leads to further enhancement of eutrophication in these endangered ecosystems.

Fish Skin Microbiomes Are Highly Variable Among Individuals and Populations but Not Within Individuals.

Fish skin-associated microbial communities are highly variable among populations and species and can impact host fitness. Still, the sources of variation in microbiome composition, and particularly how they vary among and within host individuals, have rarely been investigated. To tackle this issue, we explored patterns of variation in fish skin microbiomes across different spatial scales. We conducted replicate sampling of dorsal and ventral body sites of perch (Perca fluviatilis) from two populations and characterized the variation of fish skin-associated microbial communities with 16S rRNA gene metabarcoding. Results showed a high similarity of microbiome samples taken from the left and right side of the same fish individuals, suggesting that fish skin microbiomes can be reliably assessed and characterized even using a single sample from a specific body site. The microbiome composition of fish skin differed markedly from the bacterioplankton communities in the surrounding water and was highly variable among individuals. No ASV was present in all samples, and the most prevalent phyla, Actinobacteria, Bacteroidetes, and Proteobacteria, varied in relative abundance among fish hosts. Microbiome composition was both individual- and population specific, with most of the variation explained by individual host. At the individual level, we found no diversification in microbiome composition between dorsal and ventral body sites, but the degree of intra-individual heterogeneity varied among individuals. To identify how genetic and phenotypic characteristics of fish hosts impact the rate and nature of intra-individual temporal dynamics of the skin microbiome, and thereby contribute to the host-specific patterns documented here, remains an important task for future research.

High throughput shotgun sequencing of eRNA reveals taxonomic and derived functional shifts across a benthic productivity gradient.

Benthic macrofauna is regularly used in monitoring programmes, however the vast majority of benthic eukaryotic biodiversity lies mostly in microscopic organisms, such as meiofauna (invertebrates < 1 mm) and protists, that rapidly responds to environmental change. These communities have traditionally been hard to sample and handle in the laboratory, but DNA sequencing has made such work less time consuming. While DNA sequencing captures both alive and dead organisms, environmental RNA (eRNA) better targets living organisms or organisms of recent origin in the environment. Here, we assessed the biodiversity of three known bioindicator microeukaryote groups (nematodes, foraminifera, and ciliates) in sediment samples collected at seven coastal sites along an organic carbon (OC) gradient. We aimed to investigate if eRNA shotgun sequencing can be used to simultaneously detect differences in (i) biodiversity of multiple microeukaryotic communities; and (ii) functional feeding traits of nematodes. Results showed that biodiversity was lower for nematodes and foraminifera in high OC (6.2%-6.9%), when compared to low OC sediments (1.2%-2.8%). Dissimilarity in community composition increased for all three groups between Low OC and High OC, as well as the classified feeding type of nematode genera (with more nonselective deposit feeders in high OC sediment). High relative abundant genera included nematode Sabatieria and foraminifera Elphidium in high OC, and Cryptocaryon-like ciliates in low OC sediments. Considering that future sequencing technologies are likely to decrease in cost, the use of eRNA shotgun sequencing to assess biodiversity of benthic microeukaryotes could be a powerful tool in recurring monitoring programmes.

Depicting Temporal, Functional, and Phylogenetic Patterns in Estuarine Diazotrophic Communities from Environmental DNA and RNA.

Seasonally nitrogen-limited and phosphorus-replete temperate coastal waters generally host dense and diverse diazotrophic communities. Despite numerous studies in marine systems, little is known about diazotrophs and their functioning in oligohaline estuarine environments. Here we applied a combination of nifH transcript and metagenomic shotgun sequencing approaches to investigate temporal shifts in taxonomic composition and nifH activity of size-fractionated diazotrophic communities in a shallow and mostly freshwater coastal lagoon. Patterns in active nifH phylotypes exhibited a clear seasonal succession, which reflected their different tolerances to temperature change and nitrogen (N) availability. Thus, in spring, heterotrophic diazotrophs (Proteobacteria) dominated the nifH phylotypes, while increasing water temperature and depletion of inorganic N fostered heterocystous Cyanobacteria in summer. Metagenomic data demonstrated four main N-cycling pathways and three of them with a clear seasonal pattern: denitrification (spring) → N2 fixation (summer) → assimilative NO3- reduction (fall), with NH4+ uptake into cells occurring across all seasons. Although a substantial denitrification signal was observed in spring, it could have originated from the re-suspended benthic rather than planktonic community. Our results contribute to a better understanding of the realized genetic potential of pelagic N2 fixation and its seasonal dynamics in oligohaline estuarine ecosystems, which are natural coastal biogeochemical reactors.

Low Abundance of Methanotrophs in Sediments of Shallow Boreal Coastal Zones With High Water Methane Concentrations.

Coastal zones are transitional areas between land and sea where large amounts of organic and inorganic carbon compounds are recycled by microbes. Especially shallow zones near land have been shown to be the main source for oceanic methane (CH4) emissions. Water depth has been predicted as the best explanatory variable, which is related to CH4 ebullition, but exactly how sediment methanotrophs mediates these emissions along water depth is unknown. Here, we investigated the relative abundance and RNA transcripts attributed to methane oxidation proteins of aerobic methanotrophs in the sediment of shallow coastal zones with high CH4 concentrations within a depth gradient from 10-45 m. Field sampling consisted of collecting sediment (top 0-2 cm layer) from eight stations along this depth gradient in the coastal Baltic Sea. The relative abundance and RNA transcripts attributed to the CH4 oxidizing protein (pMMO; particulate methane monooxygenase) of the dominant methanotroph Methylococcales was significantly higher in deeper costal offshore areas (36-45 m water depth) compared to adjacent shallow zones (10-28 m). This was in accordance with the shallow zones having higher CH4 concentrations in the surface water, as well as more CH4 seeps from the sediment. Furthermore, our findings indicate that the low prevalence of Methylococcales and RNA transcripts attributed to pMMO was restrained to the euphotic zone (indicated by Photosynthetically active radiation (PAR) data, photosynthesis proteins, and 18S rRNA data of benthic diatoms). This was also indicated by a positive relationship between water depth and the relative abundance of Methylococcales and pMMO. How these processes are affected by light availability requires further studies. CH4 ebullition potentially bypasses aerobic methanotrophs in shallow coastal areas, reducing CH4 availability and limiting their growth. Such mechanism could help explain their reduced relative abundance and related RNA transcripts for pMMO. These findings can partly explain the difference in CH4 concentrations between shallow and deep coastal areas, and the relationship between CH4 concentrations and water depth.

N2 fixation dominates nitrogen cycling in a mangrove fiddler crab holobiont.

Mangrove forests are among the most productive and diverse ecosystems on the planet, despite limited nitrogen (N) availability. Under such conditions, animal-microbe associations (holobionts) are often key to ecosystem functioning. Here, we investigated the role of fiddler crabs and their carapace-associated microbial biofilm as hotspots of microbial N transformations and sources of N within the mangrove ecosystem. 16S rRNA gene and metagenomic sequencing provided evidence of a microbial biofilm dominated by Cyanobacteria, Alphaproteobacteria, Actinobacteria, and Bacteroidota with a community encoding both aerobic and anaerobic pathways of the N cycle. Dinitrogen (N2) fixation was among the most commonly predicted process. Net N fluxes between the biofilm-covered crabs and the water and microbial N transformation rates in suspended biofilm slurries portray these holobionts as a net N2 sink, with N2 fixation exceeding N losses, and as a significant source of ammonium and dissolved organic N to the surrounding environment. N stable isotope natural abundances of fiddler crab carapace-associated biofilms were within the range expected for fixed N, further suggesting active microbial N2 fixation. These results extend our knowledge on the diversity of invertebrate-microbe associations, and provide a clear example of how animal microbiota can mediate a plethora of essential biogeochemical processes in mangrove ecosystems.