Cluster 5 picocyanobacteria significantly contribute to primary productivity in aquatic ecosystems. Estuarine populations are highly diverse and consist of many co-occurring strains, but their physiology remains largely understudied. In this study, we characterized 17 novel estuarine picocyanobacterial strains. Phylogenetic analysis of the 16S rRNA and pigment genes (cpcBandcpeBA) uncovered multiple estuarine and freshwater-related clusters and pigment types. Assays with five representative strains (three phycocyanin rich and two phycoerythrin rich) under temperature (10–30°C), light(10–190 μmol  photons  m^-2s^-1), and salinity (2–14  PSU) gradients revealed distinct growth optima and tolerance, indicating that genetic variability was accompanied by physiological diversity. Adaptability to environmental conditions was associated with differential pigment content and photosynthetic performance. Amplicon sequence variants at a coastal and an offshore station linked population dynamics with phylogenetic clusters, supporting that strains isolated in this study represent key ecotypes within the Baltic Sea picocyanobacterial community. The functional diversity found within strains with the same pigment type suggests that understanding estuarine picocyanobacterial ecology requires analysis beyond the phycocyanin and phycoerythrin divide. This new knowledge of the environmental preferences in estuarine picocyanobacteria is important for understanding and evaluating productivity in current and future ecosystems.

Picophytoplankton in the Baltic Sea includes the simplest unicellular cyanoprokaryotes (Synechococcus/Cyanobium) and photosynthetic picoeukaryotes (PPE). Picophytoplankton are thought to be a key component of the phytoplankton community, but their seasonal dynamics and relationships with nutrients and temperature are largely unknown. We monitored pico- and larger phytoplankton at a coastal site in Kalmar Sound (K-Station) weekly during 2018. Among the cyanoprokaryotes, phycoerythrin-rich picocyanobacteria (PE-rich) dominated in spring and summer while phycocyanin-rich picocyanobacteria (PC-rich) dominated during autumn. PE-rich and PC-rich abundances peaked during summer (1.1 x 10(5) and 2.0 x 10(5) cells mL(-1)) while PPE reached highest abundances in spring (1.1 x 10(5) cells mL(-1)). PPE was the main contributor to the total phytoplankton biomass (up to 73%). To assess nutrient limitation, bioassays with combinations of nitrogen (NO3 or NH4) and phosphorus additions were performed. PE-rich and PC-rich growth was mainly limited by nitrogen, with a preference for NH4 at >15 degrees C. The three groups had distinct seasonal dynamics and different temperature ranges: 10 degrees C and 17-19 degrees C for PE-rich, 13-16 degrees C for PC-rich and 11-15 degrees C for PPE. We conclude that picophytoplankton contribute significantly to the carbon cycle in the coastal Baltic Sea and underscore the importance of investigating populations to assess the consequences of the combination of high temperature and NH4 in a future climate.

Picophytoplankton (<2 µm diameter), a diverse group of picocyanobacteria and photosynthetic picoeukaryotes, are significant contributors to primary production. Predatory mortality controls picophytoplankton biomass and thereby energy transfer in the marine food web. The 2 major pathways of picophytoplankton mortality are grazing and viral lysis. Grazing passes carbon directly to higher trophic levels, while lysis products are passed into the viral loop. Picophytoplankton are abundant in the Baltic Sea but little is known about their predatory mortality. Using a modification of the dilution approach, we calculated growth and mortality rates of picophytoplankton and studied the effect of predation on community structure during late August and September. The experiments were conducted coinciding with the peak in picophytoplankton abundance (similar to 10(5) cells ml(-1)) at the Linnaeus Microbial Observatory in the Baltic Sea Proper. The results showed that grazing is an important controller of picocyanobacteria and photosynthetic picoeukaryote populations, while no significant viral lysis effect was detected. Grazing on picocyanobacteria was proportional to growth rates, while grazing on photosynthetic picoeukaryotes exceeded growth. Selective grazing of phylogenetically distinct picocyanobacterial clades had a significant effect on community structure, suggesting that grazing has an impact on the seasonal dynamics of co-occurring clades. Picocyanobacteria had a higher carbon transfer contribution to higher trophic levels than photosynthetic picoeukaryotes at the time of the experiments. The study shows that picophytoplankton are important contributors to carbon cycling in the Baltic Sea microbial food web and should be considered for future ecological models.

Picocyanobacteria (< 2 mu m in diameter) are significant contributors to total phytoplankton biomass. Due to the high diversity within this group, their seasonal dynamics and relationship with environmental parameters, especially in brackish waters, are largely unknown. In this study, the abundance and community composition of phycoerythrin rich picocyanobacteria (PE-SYN) and phycocyanin rich picocyanobacteria (PC-SYN) were monitored at a coastal (K-station) and at an offshore station (LMO; similar to 10 km from land) in the Baltic Sea over three years (2018-2020). Cell abundances of picocyanobacteria correlated positively to temperature and negatively to nitrate (NO3) concentration. While PE-SYN abundance correlated to the presence of nitrogen fixers, PC-SYN abundance was linked to stratification/shallow waters. The picocyanobacterial targeted amplicon sequencing revealed an unprecedented diversity of 2169 picocyanobacterial amplicons sequence variants (ASVs). A unique assemblage of distinct picocyanobacterial clades across seasons was identified. Clade A/B dominated the picocyanobacterial community, except during summer when low NO3, high phosphate (PO4) concentrations and warm temperatures promoted S5.2 dominance. This study, providing multiyear data, links picocyanobacterial populations to environmental parameters. The difference in the response of the two functional groups and clades underscore the need for further high-resolution studies to understand their role in the ecosystem.

Despite the high abundance of Archaea in the global ocean, their metabolism and biogeochemical roles remain largely unresolved. We investigated the population dynamics and metabolic activity of Thaumarchaeota in polar environments, where these microorganisms are particularly abundant and exhibit seasonal growth. Thaumarchaeota were more abundant in deep Arctic and Antarctic waters and grew throughout the winter at surface and deeper Arctic halocline waters. However, in situ single-cell activity measurements revealed a low activity of this group in the uptake of both leucine and bicarbonate (<5% Thaumarchaeota cells active), which is inconsistent with known heterotrophic and autotrophic thaumarchaeal lifestyles. These results suggested the existence of alternative sources of carbon and energy. Our analysis of an environmental metagenome from the Arctic winter revealed that Thaumarchaeota had pathways for ammonia oxidation and, unexpectedly, an abundance of genes involved in urea transport and degradation. Quantitative PCR analysis confirmed that most polar Thaumarchaeota had the potential to oxidize ammonia, and a large fraction of them had urease genes, enabling the use of urea to fuel nitrification. Thaumarchaeota from Arctic deep waters had a higher abundance of urease genes than those near the surface suggesting genetic differences between closely related archaeal populations. In situ measurements of urea uptake and concentration in Arctic waters showed that small-sized prokaryotes incorporated the carbon from urea, and the availability of urea was often higher than that of ammonium. Therefore, the degradation of urea may be a relevant pathway for Thaumarchaeota and other microorganisms exposed to the low-energy conditions of dark polar waters.

Microbial communities in surface waters of temperate seas are exposed to recurring annual seasonal variation in temperature and nutrient concentrations. To what extent bacterioplankton populations in natural communities alter their functional repertoire as a result of seasonal succession has not been thoroughly investigated. Here we use metatranscriptomics and leverage a comprehensive catalogue of metagenome-assembled genomes (MAGs) to follow gene expression in individual populations over a two-year time period at an offshore station in the Baltic Sea. We show that the collective expression of the MAGs changed in a consistent manner across seasons in the two years, forming clusters representing the four seasons, and that more than 80% of these displayed a recurring seasonal pattern. Furthermore, we found that the changes in expression could partly be explained by modulation of expression within the prokaryotic populations, since intra-population expression patterns also changed with season. Taken together, our results demonstrate how natural microbial populations alter their expression on the gene level, and how these changes drive large scale changes on both population and community level. This work aims to broaden the understanding of how microbes respond and adapt to their environment by preferentially altering their expressed genetic repertoire, and how microbial community dynamics can be explained through the gene expression of various populations constituting the community. 

Aphanizomenon, together with Dolichospermum and Nodularia, constitute the major genera of bloom forming filamentous nitrogen fixing cyanobacteria in the Baltic Sea. Like the other genera, Aphanizomenon displays summer blooms that are highly variable in magnitude and duration but unlike the others it is considered a holoplanktonic species. Still, the molecular mechanisms enabling Aphanizomenon year-round presence in surface waters are currently unknown. Here we combine analysis of Aphanizomenon population dynamics at the Linnaeus Microbial Observatory (LMO) station in the Baltic Proper over nine years (2011-2019) with associated gene expression patterns during 2016-2017 to identify annual abundance, and metabolic and life cycle strategies. Aphanizomenon biomass showed large annual variability and a consistent biovolume peak in summer, with bloom intensity ranging from 78-1334 mm3 m-3. 16S rRNA gene amplicon sequence data showed that one Aphanizomenon amplicon sequence variant (ASV) dominated, and its relative abundance correlated with biovolume measurements. Metatranscriptomic reads that mapped to an Aphanizomenon metagenome- assembled genome (MAG) revealed annually repeating gene expression patterns, resulting in distinct gene expression profiles during different meteorological seasons. Genes encoding proteins involved in several important functional classes, e.g. carbon fixation, photosynthesis, and associated photopigments showed seasonal variation, but were detected year round. Other genes, particularly those involved in nitrogen fixation, were highly expressed in summer, while absent in winter. Vitamin metabolism and phosphorus scavenging genes were preferentially expressed during the colder periods of the annual cycle. Together, these data show that Aphanizomenon regulates the molecular machinery on the seasonal scale, providing context to the observed dynamics of Aphanizomenon in the Baltic Proper and a foundation for understanding the ecology of these cyanobacteria. 

Nitrogenase genes (nifH) from heterotrophic dinitrogen (N-2)-fixing bacteria appear ubiquitous in marine bacterioplankton, but the significance of these bacteria for N cycling is unknown. Quantitative data on the N-2-fixation potential of marine and estuarine heterotrophs are scarce, and the shortage of cultivated specimens currently precludes ecophysiological characterization of these bacteria. Through the cultivation of diazotrophs from suboxic (1.79molO(2)L(-1)) Baltic Sea water in an artificial seawater medium devoid of combined N, we report the cultivability of a considerable fraction of the diazotrophic community in the Gotland Deep. Two nifH clades were present both in situ and in enrichment cultures showing gene abundances of up to 4.6x10(5) and 5.8x10(5)nifH gene copies L-1 within two vertical profiles in the Baltic Sea. The distributions of the two clades suggested a relationship with the O-2 concentrations in the water column as abundances increased in the suboxic and anoxic waters. It was possible to cultivate and isolate representatives from one of these prevalent clades, and preliminary analysis of their ecophysiology demonstrated growth optima at 0.5-15molO(2)L(-1) and 186-194molO(2)L(-1) in the absence of combined N.

Future climate scenarios in the Baltic Sea project an increase of cyanobacterial bloom frequency and duration, attributed to eutrophication and climate change. Some cyanobacteria can be toxic and their impact on ecosystem services is relevant for a sustainable sea. Yet, there is limited understanding of the mechanisms regulating cyanobacterial diversity and biogeography. Here we unravel successional patterns and changes in cyanobacterial community structure using a 2-year monthly time series during the productive season in a 100 km coastal-offshore transect using microscopy and high-throughput sequencing of 16S rRNA gene fragments. A total of 565 cyanobacterial OTUs were found, of which 231 where filamentous/colonial and 334 picocyanobacterial. Spatial differences in community structure between coastal and offshore waters were minor. An "epidemic population structure" (dominance of a single cluster) was found for Aphanizomenon/Dolichospermum within the filamentous/colonial cyanobacterial community. In summer, this cluster simultaneously occurred with opportunistic clusters/OTUs, e.g., Nodulana spumigena and Pseudanabaena. Picocyanobacteria, Synechococcus/Cyanobium, formed a consistent but highly diverse group. Overall, the potential drivers structuring summer cyanobacterial communities were temperature and salinity. However, the different responses to environmental factors among and within genera suggest high niche specificity for individual OTUs. The recruitment and occurrence of potentially toxic filamentous/colonial clusters was likely related to disturbance such as mixing events and short-term shifts in salinity, and not solely dependent on increasing temperature and nitrogen-limiting conditions. Nutrients did not explain further the changes in cyanobacterial community composition. Novel occurrence patterns were identified as a strong seasonal succession revealing a tight coupling between the emergence of opportunistic picocyanobacteria and the bloom of filamentous/colonial clusters. These findings highlight that if environmental conditions can partially explain the presence of opportunistic picocyanobacteria, microbial and trophic interactions with filamentous/colonial cyanobacteria should also be considered as potential shaping factors for single-celled communities. Regional climate change scenarios in the Baltic Sea predict environmental shifts leading to higher temperature and lower salinity; conditions identified here as favorable for opportunistic filamentous/colonial cyanobacteria. Altogether, the diversity and complexity of cyanobacterial communities reported here is far greater than previously known, emphasizing the importance of microbial interactions between filamentous and picocyanobacteria in the context of environmental disturbances.

It is well known that the biological control of oceanic silica cycling is dominated by diatoms, with sponges and radiolarians playing additional roles. Recent studies have revealed that some smaller marine organisms (e.g. the picocyanobacterium Synechococcus) also take up silicic acid (dissolved silica, dSi) and accumulate silica, despite not exhibiting silicon dependent cellular structures. Here, we show biogenic silica (bSi) accumulation in five strains of picoeukaryotes (<2-3 mu m), including three novel isolates from the Baltic Sea, and two marine species (Ostreococcus tauri and Micromonas commoda), in cultures grown with added dSi (100 mu M). Average bSi accumulation in these novel biosilicifiers was between 30 and 92 amol Si cell(-1). Growth rate and cell size of the picoeukaryotes were not affected by dSi addition. Still, the purpose of bSi accumulation in these smaller eukaryotic organisms lacking silicon dependent structures remains unclear. In line with the increasing recognition of picoeukaryotes in biogeochemical cycling, our findings suggest that they can also play a significant role in silica cycling.

The symbiotic unicellular cyanobacterium Candidatus Atelocyanobacterium thalassa (UCYN-A) is one of the most abundant and widespread nitrogen (N-2)-fixing cyanobacteria in the ocean. Although it remains uncultivated, multiple sublineages have been detected based on partial nitrogenase (nifH) gene sequences, including the four most commonly detected sublineages UCYN-A1, UCYN-A2, UCYN-A3 and UCYN-A4. However, very little is known about UCYN-A3 beyond the nifH sequences from nifH gene diversity surveys. In this study, single cell sorting, DNA sequencing, qPCR and CARD-FISH assays revealed discrepancies involving the identification of sublineages, which led to new information on the diversity of the UCYN-A symbiosis. 16S rRNA and nifH gene sequencing on single sorted cells allowed us to identify the 16S rRNA gene of the uncharacterized UCYN-A3 sublineage. We designed new CARD-FISH probes that allowed us to distinguish and observe UCYN-A2 in a coastal location (SIO Pier; San Diego) and UCYN-A3 in an open ocean location (Station ALOHA; Hawaii). Moreover, we reconstructed about 13% of the UCYN-A3 genome from Tara Oceans metagenomic data. Finally, our findings unveil the UCYN-A3 symbiosis in open ocean waters suggesting that the different UCYN-A sublineages are distributed along different size fractions of the plankton defined by the cell-size ranges of their prymnesiophyte hosts.

Symbiosis between a marine alga and a N-2-fixing cyanobacterium (Cyanobacterium UCYN-A) is geographically widespread in the oceans and is important in the marine N cycle. UCYN-A is uncultivated and is an unusual unicellular cyanobacterium because it lacks many metabolic functions, including oxygenic photosynthesis and carbon fixation, which are typical in cyanobacteria. It is now presumed to be an obligate symbiont of haptophytes closely related to Braarudosphaera bigelowii. N-2-fixing cyanobacteria use different strategies to avoid inhibition of N-2 fixation by the oxygen evolved in photosynthesis. Most unicellular cyanobacteria temporally separate the two incompatible activities by fixing N-2 only at night, but, surprisingly, UCYN-A appears to fix N-2 during the day. The goal of this study was to determine how the unicellular UCYN-A strain coordinates N-2 fixation and general metabolism compared to other marine cyanobacteria. We found that UCYN-A has distinct daily cycles of many genes despite the fact that it lacks two of the three circadian clock genes found in most cyanobacteria. We also found that the transcription patterns in UCYN-A are more similar to those in marine cyanobacteria that are capable of aerobic N-2 fixation in the light, such as Trichodesmium and heterocyst-forming cyanobacteria, than to those in Crocosphaera or Cyanothece species, which are more closely related to unicellular marine cyanobacteria evolutionarily. Our findings suggest that the symbiotic interaction has resulted in a shift of transcriptional regulation to coordinate UCYN-A metabolism with that of the phototrophic eukaryotic host, thus allowing efficient coupling of N-2 fixation (by the cyanobacterium) to the energy obtained from photosynthesis (by the eukaryotic unicellular alga) in the light. IMPORTANCE The symbiotic N-2-fixing cyanobacterium UCYN-A, which is closely related to Braarudosphaera bigelowii, and its eukaryotic algal host have been shown to be globally distributed and important in open-ocean N-2 fixation. These unique cyanobacteria have reduced metabolic capabilities, even lacking genes for oxygenic photosynthesis and carbon fixation. Cyanobacteria generally use energy from photosynthesis for nitrogen fixation but require mechanisms for avoiding inactivation of the oxygen-sensitive nitrogenase enzyme by ambient oxygen (O-2) or the O-2 evolved through photosynthesis. This study showed that symbiosis between the N-2-fixing cyanobacterium UCYN-A and its eukaryotic algal host has led to adaptation of its daily gene expression pattern in order to enable daytime aerobic N-2 fixation, which is likely more energetically efficient than fixing N-2 at night, as found in other unicellular marine cyanobacteria.

The combination of taxa and size classes of phytoplankton that coexist at any location affects the structure of the marine food web and the magnitude of carbon fluxes to the deep ocean. But what controls the patterns of this community structure across environmental gradients remains unclear. Here, we focus on the North East Pacific Transition Zone, a similar to 10 degrees region of latitude straddling warm, nutrient-poor subtropical and cold, nutrient-rich subpolar gyres. Data from three cruises to the region revealed intricate patterns of phytoplankton community structure: poleward increases in the number of cell size classes; increasing biomass of picoeukaryotes and diatoms; decreases in diazotrophs and Prochlorococcus; and both increases and decreases in Synechococcus. These patterns can only be partially explained by existing theories. Using data, theory, and numerical simulations, we show that the patterns of plankton distributions across the transition zone are the result of gradients in nutrient supply rates, which control a range of complex biotic interactions. We examine how interactions such as size-specific grazing, multiple trophic strategies, shared grazing between several phytoplankton size classes and heterotrophic bacteria, and competition for multiple resources can individually explain aspects of the observed community structure. However, it is the combination of all these interactions together that is needed to explain the bulk compositional patterns in phytoplankton across the North East Pacific Transition Zone. The synthesis of multiple mechanisms is essential for us to begin to understand the shaping of community structure over large environmental gradients.

In aquatic environments the availability of nitrogen (N) generally limits primary production. N₂-fixing prokaryotes (diazotrophs) can convert N₂ gas into ammonium and provide significant input of N into the oceans. Cyanobacteria are thought to be the main N₂-fixers but diazotrophs also include a wide range of heterotrophic bacteria. However, their activity and regulation in the water column is largely unknown.

In this thesis the distribution, diversity, abundance, and activity of marine and estuarine heterotrophic diazotrophs was investigated. With molecular methods targeting the nifH gene, encoding the nitrogenase enzyme for N₂ fixation, it was shown that diverse nifH genes affiliating with heterotrophic bacteria were ubiquitous in surface waters from ten marine locations world-wide and the estuarine Baltic Sea. Through enrichment cultures of Baltic Sea surface water in anaerobic N-free medium, heterotrophic N₂ fixation was induced showing that there was a functional N₂-fixing community present and isolates of heterotrophic diazotrophs were obtained. In Sargasso Sea surface waters, transcripts of nifH related to heterotrophic bacteria were detected indicating heterotrophic N₂-fixing activity.

Nitrogenase expression is thought to be highly regulated by the availability of inorganic N and the presence of oxygen. Low oxygen zones within the water column can be found in association with plankton. The presence of diazotrophs as symbionts of heterotrophic dinoflagellates was investigated and nifH genes related to heterotrophic diazotrophs rather than the cyanobacterial symbionts were found, suggesting that a symbiotic co-existence prevailed. Oxic-anoxic interfaces could also be potential sites for heterotrophic N₂ fixation. The Baltic Sea contains large areas of anoxic bottom water. At the chemocline and in anoxic deep water heterotrophic diazotrophs were diverse, abundant and active. These findings extend the currently known regime of N₂ fixation to also include ammonium-rich anaerobic waters.

The results of this thesis suggest that heterotrophic diazotrophs are diverse and widely distributed in marine and estuarine waters and that they can also be active. However, limits in the knowledge on their physiology and factors which regulate their N₂ fixation activity currently prevent an evaluation of their importance in the global marine N budget.

Cyanobacteria are thought to be the main N(2)-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations world-wide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N(2) fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.

The Baltic Sea receives large nitrogen inputs by diazotrophic (N-2-fixing) heterocystous cyanobacteria but the significance of heterotrophic N-2 fixation has not been studied. Here, the diversity, abundance and transcription of the nifH fragment of the nitrogenase enzyme in two basins of the Baltic Sea proper was examined. N-2 fixation was measured at the surface (5 m) and in anoxic water (200 m). Vertical sampling profiles of >10 and <10 mu m size fractions were collected in 2007, 2008 and 2011 at the Gotland Deep and in 2011 in the Bornholm Basin. Both of these stations are characterized by permanently anoxic bottom water. The 454-pyrosequencing nifH analysis revealed a diverse assemblage of nifH genes related to alpha-, beta- and gammaproteobacteria (nifH cluster I) and anaerobic bacteria (nifH cluster III) at and below the chemocline. Abundances of genes and transcripts of seven diazotrophic phylotypes were investigated using quantitative polymerase chain reaction revealing abundances of heterotrophic nifH phylotypes of up to 2.1 x 10(7) nifH copies l(-1). Abundant nifH transcripts (up to 3.2 x 10(4) transcripts l(-1)) within nifH cluster III and co-occurring N-2 fixation (0.44 +/- 0.26 nmol l(-1) day(-1)) in deep water suggests that heterotrophic diazotrophs are fixing N2 in anoxic ammonium-rich waters. Our results reveal that N-2 fixation in the Baltic Sea is not limited to illuminated N-deplete surface waters and suggest that N-2 fixation could also be of importance in other suboxic regions of the world's oceans.

The wide distribution of diverse nitrogenase (nifH) genes affiliated with those of heterotrophic bacteria in marine and estuarine waters indicates ubiquity and an ecologically relevant role for heterotrophic N-2-fixers (diazotrophs) in aquatic nitrogen (N) cycling. However, the lack of cultivated representatives currently precludes an evaluation of their N-2-fixing capacity. In this study, microoxic or anoxic N-free media were inoculated with estuarine Baltic Sea surface water to select for N-2-fixers. After visible growth and isolation of single colonies on oxic plates or in anoxic agar tubes, nifH gene amplicons were obtained from 64 strains and nitrogenase activity, applying the acetylene reduction assay, was confirmed for 40 strains. Two strains, one Gammaproteobacterium affiliated with Pseudomonas and one Alphaproteobacterium affiliated with Rhodopseudomonas were shown to represent established members of the indigenous diazotrophic community in the Baltic Sea, with abundances of up to 7.9x10(4) and 4.7x10(4)nifH copies l(-1) respectively. This study reports media for successful isolation of heterotrophic diazotrophs. The applied methodology and the obtained strains will facilitate future identification of factors controlling heterotrophic diazotrophic activity in aquatic environments, which is a prerequisite for understanding and evaluating their ecology and contribution to N cycling at local and regional scales.

Heterotrophic dinoflagellates bearing unicellular cyanobacterial symbionts are common within the order Dinophysiales. However, the ecological role of these symbionts is unclear. Due to the occurrence of such consortia in oceanic waters characterized by low nitrogen concentrations, we hypothesized that the symbionts fix gaseous nitrogen (N-2). Individual heterotrophic dinoflagellates containing cyanobacterial symbionts were isolated from the open Indian Ocean and off Western Australia, and characterized using light microscopy, transmission electron microscopy (TEM), and nitrogenase (nifH) gene amplification, cloning, and sequencing. Cyanobacteria, heterotrophic bacteria and eukaryotic algae were recognized as symbionts of the heterotrophic dinoflagellates. nifH gene sequences were obtained from 23 of 37 (62%) specimens of dinoflagellates (Ornithocercus spp. and Amphisolenia spp.). Interestingly, only 2 specimens contained cyanobacterial nifH sequences, while 21 specimens contained nifH genes related to heterotrophic bacteria. Of the 137 nifH sequences obtained 68% were most similar to Alpha-, Beta-, and Gammaproteobacteria, 8% clustered with anaerobic bacteria, and 5% were related to second alternative nitrogenases (anfH). Twelve sequences from 5 host cells formed a discrete cluster which may represent a not yet classified nifH cluster. Eight dinoflagellates contained only 1 type of nifH sequence (>99% sequence identity) but overall the putative N-2-fixing symbionts did not appear host specific and mixed assemblages were often found in single host cells. This study provides the first insights into the nifH diversity of dinoflagellate symbionts and suggests a symbiotic co-existence of non-diazotrophic cyanobacteria and N-2-fixing heterotrophic bacteria in heterotrophic dinoflagellates.

Photosynthetic picoeukaryotes are significant contributors to marine primary productivity. Associations between marine bacterioplankton and picoeukaryotes frequently occur and can have large biogeochemical impacts. We used flow cytometry to sort cells from seawater to identify non-eukaryotic phylotypes that are associated with photosynthetic picoeukaryotes. Samples were collected at the Santa Cruz wharf on Monterey Bay, CA, USA during summer and fall, 2014. The phylogeny of associated microbes was assessed through 16S rRNA gene amplicon clone and Illumina MiSeq libraries. The most frequently detected bacterioplankton phyla within the photosynthetic picoeukaryote sorts were Proteobacteria (Alphaproteobacteria and Gammaproteobacteria) and Bacteroidetes. Intriguingly, the presence of free-living bacterial genera in the photosynthetic picoeukaryote sorts could suggest that some of the photosynthetic picoeukaryotes were mixotrophs. However, the occurrence of bacterial sequences, which were not prevalent in the corresponding bulk seawater samples, indicates that there was also a selection for specific OTUs in association with photosynthetic picoeukaryotes suggesting specific functional associations. The results show that diverse bacterial phylotypes are found in association with photosynthetic picoeukaryotes. Taxonomic identification of these associations is a prerequisite for further characterizing and to elucidate their metabolic pathways and ecological functions.

Cyanobacterial nitrogen-fixers (diazotrophs) play a key role in biogeochemical cycling of carbon and nitrogen in the ocean. In recent years, the unusual symbiotic diazotrophic cyanobacterium Atelocyanobacterium thalassa (UCYN-A) has been recognized as one of the major diazotrophs in the tropical and subtropical oceans. In this review, we summarize what is currently known about the geographic distribution of UCYN-A, as well as the environmental factors that govern its distribution. In addition, by compiling UCYN-A nifH sequences from the GenBank no. database as well as those from nifH gene amplicon next generation sequencing studies, we present an in-depth analysis of the distribution of defined UCYN-A sublineages (UCYN-A1, UCYN-A2 and UCYN-A3) and identify a novel sublineage, UCYN-A4, which may be significant in some environments. Each UCYN-A sublineage exhibited a remarkable global distribution pattern and several UCYN-A sublineages frequently co-occurred within the same sample, suggesting that if they represent different ecotypes they have overlapping niches. Recently, single cell visualization techniques using specific probes targeting UCYN-A1 and UCYN-A2 and their respective associated eukaryotic partner cells showed that the size of the consortia and the number of UCYN-A cells differed between these 2 sublineages. Combined, the results highlight that UCYN-A sublineages likely have different physiological requirements, which need to be accounted for in future studies. Furthermore, based on our increasing knowledge of the diversity of the UCYN-A lineage, we discuss some of the limitations of currently used cultivation-independent molecular techniques for the identification and quantification of UCYN-A.

Sinking particles transport carbon and nutrients from the surface ocean into the deep sea and are considered hot spots for bacterial diversity and activity. In the oligotrophic oceans, nitrogen (N-2)-fixing organisms (diazotrophs) are an important source of new N but the extent to which these organisms are present and exported on sinking particles is not well known. Sinking particles were collected every 6 h over a 2-day period using net traps deployed at 150 m in the North Pacific Subtropical Gyre. The bacterial community and composition of diazotrophs associated with individual and bulk sinking particles was assessed using 16S rRNA and nifH gene amplicon sequencing. The bacterial community composition in bulk particles remained remarkably consistent throughout time and space while large variations of individually picked particles were observed. This difference suggests that unique biogeochemical conditions within individual particles may offer distinct ecological niches for specialized bacterial taxa. Compared to surrounding seawater, particle samples were enriched in different size classes of globally significant N-2-fixing cyanobacteria including Trichodesmium, symbionts of diatoms, and the unicellular cyanobacteria Crocosphaera and UCYN-A. The particles also contained nifH gene sequences of diverse non-cyanobacterial diazotrophs suggesting that particles could be loci for N-2 fixation by heterotrophic bacteria. The results demonstrate that diverse diazotrophs were present on particles and that new N may thereby be directly exported from surface waters on sinking particles.

Close associations between single-celled marine organisms can have a central role in biogeochemical processes and are of great interest for understanding the evolution of organisms. The global significance of such associations raises the question of whether unidentified associations are yet to be discovered. In this study, fluorescence-activated cell sorted photosynthetic picoeukayote (PPE) populations and single cells were analysed by sequencing of 16S rRNA genes in the oligotrophic North Pacific Subtropical Gyre. Samples were collected during two cruises, spanning depths near the deep chlorophyll maximum, where the abundance of PPEs was highest. The association between the widespread and significant nitrogen (N-2)-fixing cyanobacterium, UCYN-A and its prymnesiophyte host was prevalent in both population and single-cell sorts. Several bacterial sequences, affiliating with previously described symbiotic taxa were detected but their detection was rare and not well replicated, precluding identification of novel tightly linked species-specific associations. Similarly, no enrichment of dominant seawater taxa such as Prochlorococcus, SAR11 or Synechococcus was observed suggesting that these were not systematically ingested by the PPE in this study. The results indicate that apart from the UCYN-A symbiosis, similar tight species-specific associations with PPEs are unusual in the oligotrophic ocean.

Heterocystous filamentous cyanobacteria are regarded as the main N-2-fixing organisms (diazotrophs) in the Baltic Sea. However, some studies indicate that picoplankton may also be important. The aim of this study was to examine the composition of putative diazotrophs in the picoplankton (< 3 mu m) and to identify links to environmental factors. Nitrogenase (nifH) genes were amplified from community DNA by nested PCR, followed by cloning and sequencing. Clone libraries from nine environmental samples collected from the central Baltic Sea (April-October 2003, 3 m depth) and a negative control yielded a total of 433 sequences with an average clone library coverage of 92%. The sequences fell within nifH Clusters I, II and III and formed 15 distinct groups (> 96% amino acid similarity). Most of the sequences (77%) fell into nifH Cluster I (cyanobacteria and alpha-, beta- and gamma-Proteobacteria). However, only 26 sequences were related to cyanobacteria (e. g. Pseudanabaena) and among these no unicellular phylotypes were found. Sequences clustering with alternative nitrogenases (anfH) and Archaea were found in one sample while sequences related to anaerobic phylotypes were found in six samples distributed throughout the season. The identified phylogenetic groups showed covariance with several environmental factors but no strong links could be established. This suggests a variable and complex regulation of diazotrophic groups within Baltic Sea picoplankton.

The planktonic realm from bacteria to zooplankton provides the baseline for pelagic aquatic food webs. However, multiple trophic levels are seldomly included in time series studies, hampering a holistic understanding of the influence of seasonal dynamics and species interactions on food web structure and biogeochemical cycles. Here, we investigated plankton community composition, focusing on bacterio-, phyto- and large mesozooplankton, and how biotic and abiotic factors correlate at the Linnaeus Microbial Observatory (LMO) station in the Baltic Sea from 2011 to 2018. Plankton communities structures showed pronounced dynamic shifts with recurring patterns. Summarizing the parts of the planktonic microbial food web studied here to total carbon, a picture emerges with phytoplankton consistently contributing > 39% while bacterio- and large mesozooplankton contributed ~ 30% and ~ 7%, respectively, during summer. Cyanophyceae, Actinobacteria, Bacteroidetes, and Proteobacteria were important groups among the prokaryotes. Importantly, Dinophyceae, and not Bacillariophyceae, dominated the autotrophic spring bloom whereas Litostomatea (ciliates) and Appendicularia contributed significantly to the consumer entities together with the more traditionally observed mesozooplankton, Copepoda and Cladocera. Our findings of seasonality in both plankton composition and carbon stocks emphasize the importance of time series analyses of food web structure for characterizing the regulation of biogeochemical cycles and appropriately constraining ecosystem models. 

The number of marine environments known to harbor dinitrogen (N-2)-fixing (diazotrophic) microorganisms is increasing, prompting a reassessment of the biogeography of marine diazotrophs and N(2)fixation rates (NFRs). Here, we investigate the diversity, abundance, and activity of diazotrophic microorganisms in the North Pacific Subtropical Gyre (NPSG), a diazotrophic habitat, and the North Pacific Transition Zone (NPTZ), a region characterized by strong physical, chemical, and biological gradients. Samples were collected on two springtime meridional cruises during 2016 and 2017, spanning from 23.5 degrees N to 41.4 degrees N along 158 degrees W. We observed an abrupt decrease in diazotrophic abundances near the southern edge of the NPTZ, which coincided with a salinity front and with a similar to 10-fold increase inSynechococcusabundance, but without a concomitant change in phosphate or nitrate concentrations. In NPSG waters south of this diazotrophic boundary,nifHgenes and NFRs were consistently detected and diazotrophic communities were dominated by UCYN-A, an uncultivated, symbiotic cyanobacterium (2.8 x 10(3)to 1.0 x 10(6)nifHgene copies L-1). There was a significant positive relationship between quantitative polymerase chain reaction-derived UCYN-AnifHgene abundances and community NFRs in the NPSG, suggesting a large contribution of UCYN-A to community NFRs. In the NPTZ waters to the north, NFRs were low or undetected andnifHgenes were rare, with the few detected sequences represented by UCYN-A and noncyanobacterial diazotrophs. The patterns we observed in UCYN-A abundance in the context of local biogeochemistry suggest that the environmental controls of this organism may differ from those of cultivated marine cyanobacterial diazotrophs.

Micronutrients such as vitamins are transferred from lower to higher trophic levels, but no general ecological concept describes the factors regulating this process. Here, we investigated thiamin (thiamine, vitamin B1), which is an example of a metabolically important water-soluble micronutrient. Thiamin is produced by organisms such as bacteria and phytoplankton, and all consumers, such as zooplankton and fish, rely on a continuous intake of thiamin through their diet and possibly from de novo-synthesized thiamin by gut microbiota. A deficiency in thiamin negatively affects reproduction in fish and bird populations worldwide. The aim of this study was to quantify thiamin transfer in a planktonic food web in response to thiamin and/or nutrient addition, using an outdoor mesocosm system (an approximately 1.9 m3 bag submerged in sea water). These estimates were then compared with literature data on thiamin concentrations at different trophic levels. The results showed that thiamin was rapidly taken up by phytoplankton in both the ambient and nutrient-amended treatments. However, large differences in thiamin concentrations in phytoplankton did not lead to any significant changes in community composition or abundance. Nitrogen addition led to changes in the abundance and community composition of picoplankton and phytoplankton but there were no additional major effects of thiamin addition. Differences in thiamin concentrations in phytoplankton were not detected at the next trophic level in zooplankton. Although the concentrations did not change, a greater abundance of some zooplankton taxa were developed in the thiamin treatments. Comparing the mesocosm results with literature data demonstrated a gradual reduction in thiamin concentrations along the food chain, with six percent of the concentration in producers occurring in top consumers (i.e., piscivorous fish). Overall, these observations illustrate the concept of trophic dilution of micronutrients where concentrations decrease along the food web from phytoplankton via zooplankton and planktivorous fish to piscivorous fish.

Phytoplankton comprises a large fraction of the vertical carbon flux to deep water via the sinking of particulate organic matter (POM). However, despite the importance of phytoplankton in the coupling of benthic-pelagic productivity, the extent to which its deposition in the sediment affects bacterial dynamics at the water-sediment interface is poorly understood. Here, we conducted a microcosm experiment in which varying mixtures of diatom and cyanobacteria, representing phytoplankton-derived POM of differing quality, served as inputs to sediment cores. Characterization of 16S rRNA gene of the bacterial communities at the water-sediment interface showed that bacterial alpha-diversity was not affected by POM addition, while bacterial beta-diversity changed significantly along the POM quality gradient, with the variation driven by changes in relative abundance rather than in taxon replacement. Analysing individual taxa abundances across the POM gradient revealed two distinct bacterial responses, in which taxa within either diatom- or cyanobacteria-favoured groups were more phylogenetically closely related to one another than other taxa found in the water. Moreover, there was little overlap in taxon identity between sediment and water communities, suggesting the minor role played by sediment bacteria in influencing the observed changes in bacterial communities in the overlying water. Together, these results showed that variability in phytoplankton-originated POM can impact bacterial dynamics at the water-sediment interface. Our findings highlight the importance of considering the potential interactions between phytoplankton and bacteria in benthic-pelagic coupling in efforts to understand the structure and function of bacterial communities under a changing climate.

Picocyanobacteria are widespread and globally significant primary producers. In brackish waters, picocyanobacterial populations are composed of diverse species with both single-cell and colony-forming lifestyles. Compared to their marine counterparts, brackish picocyanobacteria are less well characterized and the focus of research has been weighted toward single-cell picocyanobacteria. Here, we investigate the uptake dynamics of single and colony-forming picocyanobacteria using incubations with dual carbon-13 and inorganic (ammonium and nitrate) or organic (urea and amino acids) nitrogen-15 sources during August and September 2020 in the central Baltic Sea. Phytoplankton community and group-specific uptake rates were obtained using an elemental analyzer isotope ratio mass spectrometer (EA-IRMS) and nano secondary-ion mass spectrometry (NanoSIMS). Picocyanobacteria contributed greater than one third of the ammonium, urea, amino acids, and inorganic carbon community uptake/fixation in September but < 10% in August when phytoplankton biomass was higher. Overall, single-cell ammonium and urea uptake rates were significantly higher for single-celled compared to colonial picocyanobacteria. In a 6-yr offshore central Baltic Sea time series (2015-2020), summer abundances of colonial picocyanobacteria reached up to 10(5) cells mL(-1) and represented > 5% of the average phytoplankton biomass, suggesting that they are periodically important for the ecosystem. Colonial strain identification was not distinguishable using 16S rRNA gene amplicon data, highlighting a need for refined tools for identification of colonial forms. This study shows the significance of single-celled brackish picocyanobacteria to nutrient cycling and the importance of considering uptake and lifestyle strategies when assessing the role of picocyanobacteria in aquatic ecosystems.

The picophytoplankton Synechococcus is a globally abundant autotroph that contributes significantly to primary production in the oceans and coastal areas. These cyanobacteria constitute a diverse genus of organisms that have developed independent niche spaces throughout aquatic environments. Here, we use the 16S V3-V4 rRNA gene region and flow cytometry to explore the diversity of Synechococcus within the picophytoplankton community in the Gullmar Fjord, on the west coast of Sweden. We conducted a station-based 1-year time series and two transect studies of the fjord. Our analysis revealed that within the large number of Synechococcus amplicon sequence variants (ASVs; 239 in total), prevalent ASVs phylogenetically clustered with clade representatives in both marine subcluster 5.1 and 5.2. The near-surface composition of ASVs shifted from spring to summer, when a 5.1 subcluster dominated community developed along with elevated Synechococcus abundances up to 9.3 x 10(4) cells ml(-1). This seasonal dominance by subcluster 5.1 was observed over the length of the fjord (25 km), where shifts in community composition were associated with increasing depth. Unexpectedly, the community shift was not associated with changes in salinity. Synechococcus abundance dynamics also differed from that of the photosynthetic picoeukaryote community. These results highlight how seasonal variations in environmental conditions influence the dynamics of Synechococcus clades in a high latitude threshold fjord.

The study evaluated removal of industrial CO2 from cement flue gas using algal cultivation. Local polycultures were grown in an up-scaled outdoor photobioreactor over 5 years in northern Europe. Algal biomass was harvested 2–3 times per week and the closed panel system was re-filled with seawater amended with nutrients. Flue gas was fed to the photobioreactor circulatory system in one direction or re-circulated. Removal efficiency of CO2 averaged 9 % in non-recirculation and 17 % in re-circulation modes and reached 20–60 % under best cultivation conditions. Recovery of carbon into algal biomass reached up to 10 g m2d−1 in non-recirculation mode. Biomass performance was explained by circulation mode and shift of polyculture traits. Stability of biomass quality was shown over seasons, with higher relative content of protein in autumn. Toxic elements in biomass were below legal thresholds for upcycling. The study shows feasibility of algal solutions for conversion of waste, applied in temperate climate.

Microalgal solutions to clean waste streams and produce biomass were evaluated in Nordic conditions during winter, spring, and autumn in Southeast Sweden. The study investigated nitrogen (N) removal, biomass quality, and safety by treating industrial leachate water with a polyculture of local microalgae and bacteria in open raceway ponds, supplied with industrial CO2 effluent. Total N (TN) removal was higher in spring (1.5 g-2d-1), due to beneficial light conditions compared to winter and autumn (0.1 and 0.09 g-2d-1). Light, TN, and N species influenced the microalgal community (dominated by Chlorophyta), while the bacterial community remained stable throughout seasons with a large proportion of cyanobacteria. Winter conditions promoted biomass protein (19.6-26.7%) whereas lipids and carbohydrates were highest during spring (11.4-18.4 and 15.4-19.8%). Biomass toxin and metal content were below safety levels for fodder, but due to the potential presence of toxic strains, biofuels or fertilizer could be suitable applications for the algal biomass.Practitioner points Microalgal removal of nitrogen from leachate water was evaluated in Nordic conditions during winter, spring, and autumn. Total nitrogen removal was highest in spring (1.5 g-2d-1), due to beneficial light conditions for autotrophic growth. Use of local polyculture made the cultivation more stable on a seasonal (light) and short-term (N-species changes) scale. Toxic elements in produced algal biomass were below legal thresholds for upcycling. The study investigated nitrogen removal, biomass quality, and safety by treating industrial leachate water with a polyculture of local microalgae and bacteria in open raceway ponds, supplied with industrial CO2 effluent. Nitrogen removal by the polyculture was highest in spring and the biomass biochemical composition changed with season. image

Functionally uniform monocultures have remained the paradigm in microalgal cultivation despite the apparent challenges to avoid invasions by other microorganisms. A mixed microbial consortium approach has the potential to optimize and maintain biomass production despite of seasonal changes and to be more resilient toward contaminations. Here we present a 3-year outdoor production of mixed consortia of locally adapted microalgae and bacteria in cold temperate latitude. Microalgal consortia were cultivated in flat panel photobioreactors using brackish Baltic Sea water and CO2 from a cement factory (Degerhamn, Cementa AB, Heidelberg Cement Group) as a sustainable CO2 source. To evaluate the ability of the microbial consortia to maintain stable biomass production while exposed to seasonal changes in both light and temperature, we tracked changes in the microbial community using molecular methods (16S and 18S rDNA amplicon sequencing) and monitored the biomass production and quality (lipid, protein, and carbohydrate content) over 3 years. Despite changes in environmental conditions, the mixed consortia maintained stable biomass production by alternating between two different predominant green microalgae (Monoraphidium and Mychonastes) with complementary tolerance to temperature. The bacterial population was few taxa co-occured over time and the composition did not have any connection to the shifts in microalgal taxa. We propose that a locally adapted and mixed microalgal consortia, with complementary traits, can be useful for optimizing yield of commercial scale microalgal cultivation.

Coastal ecosystems deteriorate globally due to human-induced stress factors, like nutrient loading and pollution. Bacteria are critical to marine ecosystems, e.g., by regulating nutrient cycles, synthesizing vitamins, or degrading pollutants, thereby providing essential ecosystem services ultimately affecting economic activities. Yet, until now bacteria are overlooked both as mediators and indicators of ecosystem health, mainly due to methodological limitations in assessing bacterial ecosystem functions. However, these limitations are largely overcome by the advances in molecular biology and bioinformatics methods for characterizing the genetics that underlie functional traits of key bacterial populations - "key" in providing important ecosystem services, being abundant, or by possessing high metabolic rates. It is therefore timely to analyze and define the functional responses of bacteria to human-induced effects on coastal ecosystem health. We posit that categorizing the responses of key marine bacterial populations to changes in environmental conditions through modern microbial oceanography methods will allow establishing the nascent field of genetic counselling for our coastal waters. This requires systematic field studies of linkages between functional traits of key bacterial populations and their ecosystem functions in coastal seas, complemented with systematic experimental analyses of the responses to different stressors. Research and training in environmental management along with dissemination of results and dialogue with societal actors are equally important to ensure the role of bacteria is understood as fundamentally important for coastal ecosystems. Using the responses of microorganisms as a tool to develop genetic counselling for coastal ecosystems can ultimately allow for integrating bacteria as indicators of environmental change.

Marine waters are generally considered to be nitrogen (N) limited and are therefore favourable environments for diazotrophs, i.e. organisms converting atmospheric N-2 into ammonium or nitrogen oxides available for growth. In some regions, this import of N supports up to half of the primary productivity. Diazotrophic Cyanobacteria appear to be the major contributors to marine N-2 fixation in surface waters, whereas the contribution of heterotrophic or chemoautotrophic diazotrophs to this process is usually regarded inconsequential. Culture-independent studies reveal that non-cyanobacterial diazotrophs are diverse, widely distributed, and actively expressing the nitrogenase gene in marine and estuarine environments. The detection of nifH genes and nifH transcripts, even in N-replete marine waters, suggests that N-2 fixation is an ecologically important process throughout the oceans. Because this process is highly sensitive to and inhibited by molecular oxygen (O-2), diazotrophy requires efficient scavenging of intracellular O-2 or growth in environments with low ambient O-2 concentration. Particles with interior low-O-2 micro-zones and oceanic oxygen minimum zones are just 2 potential habitats suitable for N-2 fixation by non-cyanobacterial diazotrophs. Our ignorance about the regulation of N-2 fixation by non-Cyanobacteria in their natural marine environments currently prevents an evaluation of their importance in marine N cycling and budgets. A review of the molecular data on distribution and expression of nifH genes in non-Cyanobacteria suggests that further study of the role of these Bacteria in N cycling at local, regional and global scales is needed.

Phytoplankton and bacteria interactions have a significant role in aquatic ecosystem functioning. Associations can range from mutualistic to parasitic, shaping biogeochemical cycles and having a direct influence on phytoplankton growth. How variations in phenotype and sampling location, affect the phytoplankton microbiome is largely unknown. A high‐resolution characterization of the bacterial community in cultures of the dinoflagellate Alexandrium was performed on strains isolated from different geographical locations and at varying anthropogenic impact levels. Microbiomes of Baltic Sea Alexandrium ostenfeldii isolates were dominated by Betaproteobacteria and were consistent over phenotypic and genotypic Alexandrium strain variation, resulting in identification of an A. ostenfeldii core microbiome. Comparisons with in situ bacterial communities showed that taxa found in this A. ostenfeldii core were specifically associated to dinoflagellate dynamics in the Baltic Sea. Microbiomes of Alexandrium tamarense and minutum, isolated from the Mediterranean Sea, differed from those of A. ostenfeldii in bacterial diversity and composition but displayed high consistency, and a core set of bacterial taxa was identified. This indicates that Alexandrium isolates with diverse phenotypes host predictable, species‐specific, core microbiomes reflecting the abiotic conditions from which they were isolated. These findings enable in‐depth studies of potential interactions occurring between Alexandrium and specific bacterial taxa.

To better predict the consequences of environmental change on aquatic microbial ecosystems it is important to understand what enables community resilience. The mechanisms by which a microbial community maintain its overall function, for example, the cycling of carbon, when exposed to a stressor, can be explored by considering three concepts: biotic interactions, functional adaptations, and community structure. Interactions between species are traditionally considered as, e.g., mutualistic, parasitic, or neutral but are here broadly defined as either coexistence or competition, while functions relate to their metabolism (e.g., autotrophy or heterotrophy) and roles in ecosystem functioning (e.g., oxygen production, organic matter degradation). The term structure here align with species richness and diversity, where a more diverse community is though to exhibit a broader functional capacity than a less diverse community. These concepts have here been combined with ecological theories commonly used in resilience studies, i.e., adaptive cycles, panarchy, and cross-scale resilience, that describe how the status and behavior at one trophic level impact that of surrounding levels. This allows us to explore the resilience of a marine microbial community, cultivated in an outdoor photobioreactor, when exposed to a naturally occurring seasonal stress. The culture was monitored for 6weeks during which it was exposed to two different temperature regimes (21 +/- 2 and 11 +/- 1 degrees C). Samples were taken for metatranscriptomic analysis, in order to assess the regulation of carbon uptake and utilization, and for amplicon (18S and 16S rRNA gene) sequencing, to characterize the community structure of both autotrophs (dominated by the green microalgae Mychonastes) and heterotrophs (associated bacterioplankton). Differential gene expression analyses suggested that community function at warm temperatures was based on concomitant utilization of inorganic and organic carbon assigned to autotrophs and heterotrophs, while at colder temperatures, the uptake of organic carbon was performed primarily by autotrophs. Upon the shift from high to low temperature, community interactions shifted from coexistence to competition for organic carbon. Network analysis indicated that the community structure showed opposite trends for autotrophs and heterotrophs in having either high or low diversity. Despite an abrupt change of temperature, the microbial community as a whole responded in a way that maintained the overall level of diversity and function within and across autotrophic and heterotrophic levels. This is in line with cross-scale resilience theory describing how ecosystems may balance functional overlaps within and functional redundancy between levels in order to be resilient to environmental change (such as temperature).

Eutrophication coupled to climate change disturbs the balance between competition and coexistence in microbial communities including the partitioning of organic and inorganic nutrients between phytoplankton and bacteria. Competition for inorganic nutrients has been regarded as one of the drivers affecting the productivity of the eutrophied coastal Baltic Sea. Yet, it is unknown at the molecular expression level how resources are competed for, by phytoplankton and bacteria, and what impact this competition has on the community composition. Here we use metatranscriptomics and amplicon sequencing and compare known metabolic pathways of both phytoplankton and bacteria co-occurring during a summer bloom in the archipelago of Åland in the Baltic Sea to examine phytoplankton bacteria resource partitioning. The expression of selected pathways of carbon (C), nitrogen (N), and phosphorus (P) metabolism varied over time, independently, for both phytoplankton and bacteria, indicating partitioning of the available organic and inorganic resources. This occurs regardless of eukaryotic plankton growth phase (exponential or stationary), based on expression data, and microbial community composition. Further, the availability of different nutrient resources affected the functional response by the bacteria, observed as minor compositional changes, at class level, in an otherwise taxonomically stable bacterial community. Resource partitioning and functional flexibility seem necessary in order to maintain phytoplankton-bacteria interactions at stable environmental conditions. More detailed knowledge of which organisms utilize certain nutrient species are important for more accurate projections of the fate of coastal waters.

Major advances in understanding the diversity, distribution, and activity of marine N-2-fixing microorganisms (diazotrophs) have been made in the past decades, however, large gaps in knowledge remain about the environmental controls on growth and mortality rates. In order to measure diazotroph net growth rates and microzooplankton grazing rates on diazotrophs, nutrient perturbation experiments and dilution grazing experiments were conducted using free-floating in situ incubation arrays in the vicinity of Station ALOHA in March 2016. Net growth rates for targeted diazotroph taxa as well as Prochlorococcus, Synechococcus and photosynthetic picoeukaryotes were determined under high (H) and low (L) nitrate:phosphate (NP) ratio conditions at four depths in the photic zone (25, 45, 75, and 100 m) using quantitative PCR and flow cytometry. Changes in the prokaryote community composition in response to HNP and LNP treatments were characterized using 16S rRNA variable region tag sequencing. Microzooplankton grazing rates on diazotrophs were measured using a modified dilution technique at two depths in the photic zone (15 and 125 m). Net growth rates for most of the targeted diazotrophs after 48 h were not stimulated as expected by LNP conditions, rather enhanced growth rates were often measured in HNP treatments. Interestingly, net growth rates of the uncultivated prymnesiophyte symbiont UCYN-Al were stimulated in HNP treatments at 75 and 100 m, suggesting that N used for growth was acquired through continuing to fix N-2 in the presence of nitrate. Net growth rates for UCYN-Al , UCYN-C, Crocosphaera sp. (UCYN-B) and the diatom symbiont Richelia (associated with Rhizosolenia) were uniformly high at 45 m (up to 1.6 +/- 0.5 d(-1)), implying that all were growing optimally at the onset of the experiment at that depth. Differences in microzooplankton grazing rates on UCYN-Al and UCYN-C in 15 m waters indicate that the grazer assemblage preyed preferentially on UCYN-Al. Deeper in the water column (125 m), both diazotrophs were grazed at substantial rates, suggesting grazing pressure may increase with depth in the photic zone. Constraining in situ diazotroph growth and mortality rates are important steps for improving parameterization for diazotrophs in global ecosystem models.

A recently described symbiosis between the metabolically streamlined nitrogen-fixing cyanobacterium UCYN-A and a single-celled eukaryote prymnesiophyte alga is widely distributed throughout tropical and subtropical marine waters, and is thought to contribute significantly to nitrogen fixation in these regions. Several UCYN-A sublineages have been defined based on UCYN-A nitrogenase (nifH) sequences. Due to the low abundances of UCYN-A in the global oceans, currently existing molecular techniques are limited for detecting and quantifying these organisms. A targeted approach is needed to adequately characterize the diversity of this important marine cyanobacterium, and to advance understanding of its ecological importance. We present findings on the distribution of UCYN-A sublineages based on high throughput sequencing of UCYN-A nifH PCR amplicons from 78 samples distributed throughout many major oceanic provinces. These UCYN-A nifH fragments were used to define oligotypes, alternative taxonomic units defined by nucleotide positions with high variability. The data set was dominated by a single oligotype associated with the UCYN-A1 sublineage, consistent with previous observations of relatively high abundances in tropical and subtropical regions. However, this analysis also revealed for the first time the widespread distribution of the UCYN-A3 sublineage in oligotrophic waters. Furthermore, distinct assemblages of UCYN-A oligotypes were found in oligotrophic and coastally influenced waters. This unique data set provides a framework for determining the environmental controls on UCYN-A distributions and the ecological importance of the different sublineages.

In nature, the number of genome or chromosome copies within cells (ploidy) can vary between species and environmental conditions, potentially influencing how organisms adapt to changing environments. Although ploidy levels cannot be easily determined by standard genome sequencing, understanding ploidy is crucial for the quantitative interpretation of molecular data. Cyanobacteria are known to contain haploid, oligoploid, and polyploid species. The smallest cyanobacteria, picocyanobacteria (less than 2 μm in diameter), have a widespread distribution ranging from marine to freshwater environments, contributing significantly to global primary production. In this study, we determined the ploidy level of genetically and physiologically diverse brackish picocyanobacteria isolated from the Baltic Sea using a qPCR assay targeting the rbcL gene. The strains contained one to four genome copies per cell. The ploidy level was not linked with phylogeny based on the identity of the 16S rRNA gene. The variation of ploidy among the brackish strains was lower compared to what has been reported for freshwater strains and was more similar to what has been reported for marine strains. The potential ecological advantage of polyploidy among picocyanobacteria has yet to be described. Our study highlights the importance of considering ploidy to interpret the abundance and adaptation of brackish picocyanobacteria.

Nitrogen fixation — the reduction of dinitrogen (N₂) gas to biologically available nitrogen (N) — is an important source of N for terrestrial and aquatic ecosystems. In terrestrial environments, N₂-fixing symbioses involve multicellular plants, but in the marine environment these symbioses occur with unicellular planktonic algae. An unusual symbiosis between an uncultivated unicellular cyanobacterium (UCYN-A) and a haptophyte picoplankton alga was recently discovered in oligotrophic oceans. UCYN-A has a highly reduced genome, and exchanges fixed N for fixed carbon with its host. This symbiosis bears some resemblance to symbioses found in freshwater ecosystems. UCYN-A shares many core genes with the ‘spheroid bodies’ of Epithemia turgida and the endosymbionts of the amoeba Paulinella chromatophora. UCYN-A is widely distributed, and has diversified into a number of sublineages that could be ecotypes. Many questions remain regarding the physical and genetic mechanisms of the association, but UCYN-A is an intriguing model for contemplating the evolution of N₂-fixing organelles.