Despite the worldwide occurrence of marine hypoxic regions, benthic nitrogen (N) cycling within these areas is poorly understood and it is generally assumed that these areas represent zones of intense fixed N loss from the marine system. Sulfate reduction can be an important process for organic matter degradation in sediments beneath hypoxic waters and many sulfate-reducing bacteria (SRB) have the genetic potential to fix molecular N (N₂). Therefore, SRB may supply fixed N to these systems, countering some of the N lost via microbial processes, such as denitrification and anaerobic ammonium oxidation. The objective of this study was to evaluate if N₂ fixation, possibly by SRB, plays a role in N cycling within the seasonally hypoxic sediments from the Eckernförde Bay, Baltic Sea. Monthly samplings were performed over the course of one year to measure nitrogenase activity (NA) and sulfate reduction rates, to determine the seasonal variations in bioturbation (bioirrigation) activity and important benthic geochemical profiles, such as sulfur and N compounds, and to monitor changes in water column temperature and oxygen concentrations. Additionally, at several time points, the active N-fixing community was examined via molecular tools. Integrated rates of N₂ fixation (approximated from NA) and sulfate reduction showed a similar seasonality pattern, with highest rates occurring in August (approx. 22 and 880 nmol cm⁻³ d⁻¹ of N and SO₄²⁻, respectively) and October (approx. 22 and 1300 nmol cm⁻³ d⁻¹ of N and SO₄²⁻ respectively), and lowest rates occurring in February (approx. 8 and 32 nmol cm⁻³ d⁻¹ of N and SO₄²⁻, respectively). These rate changes were positively correlated with bottom water temperatures and previous reported plankton bloom activities, and negatively correlated with bottom water oxygen concentrations. Other variables that also appeared to play a role in rate determination were bioturbation, bubble irrigation and winter storm events. Molecular analysis demonstrated the presence of nifH sequences related to two known N₂ fixing SRB, namely Desulfovibrio vulgaris and Desulfonema limicola, supporting the hypothesis that some of the nitrogenase activity detected may be attributed to SRB. Overall, our data show that Eckernförde Bay represents a complex ecosystem where numerous environmental variables combine to influence benthic microbial activities involving N and sulfur cycling

Foraminifera are ubiquitous marine protists with an important role in the benthic carbon cycle. However, morphological observations often fail to resolve their exact taxonomic placement and there is a lack of field studies on their particular trophic preferences. Here, we propose the application of metabarcoding as a tool for the elucidation of the in situ feeding behavior of benthic foraminifera, while also allowing the correct taxonomic assignment of the feeder, using the V9 region of the 18S (small subunit; SSU) rRNA gene. Living foraminiferal specimens were collected from two intertidal mudflats of the Wadden Sea and DNA was extracted from foraminiferal individuals and from the surrounding sediments. Molecular analysis allowed us to confirm that our foraminiferal specimens belong to three genetic types: Ammonia sp. T6, Elphidium sp. S5 and Haynesina sp. S16. Foraminiferal intracellular eukaryote communities reflected to an extent those of the surrounding sediments but at different relative abundances. Unlike sediment eukaryote communities, which were largely determined by the sampling site, foraminiferal intracellular eukaryote communities were driven by foraminiferal species, followed by sediment depth. Our data suggests that Ammonia sp. T6 can predate on metazoan classes, whereas Elphidium sp. S5 and Haynesina sp. S16 are more likely to ingest diatoms. These observations, alongside the use of metabarcoding in similar ecological studies, significantly contribute to our overall understanding of the ecological roles of these protists in intertidal benthic environments and their position and function in the benthic food webs.

Benthic foraminifera are known to play an important role in marine carbon and nitrogen cycles. Here, we report an enrichment of sulphur cycle -associated bacteria inside intertidal benthic foraminifera ( Ammonia sp. (T6), Haynesina sp. (S16) and Elphidium sp. (S5)), using a metabarcoding approach targeting the 16S rRNA and aprA -genes. The most abundant intracellular bacterial groups included the genus Sulfurovum and the order Desulfobacterales. The bacterial 16S OTUs are likely to originate from the sediment bacterial communities, as the taxa found inside the foraminifera were also present in the sediment. The fact that 16S rRNA and aprA –gene derived intracellular bacterial OTUs were species-specific and significantly different from the ambient sediment community implies that bacterivory is an unlikely scenario, as benthic foraminifera are known to digest bacteria only randomly. Furthermore, these foraminiferal species are known to prefer other food sources than bacteria. The detection of sulphur-cycle related bacterial genes in this study suggests a putative role for these bacteria in the metabolism of the foraminiferal host. Future investigation into environmental conditions under which transcription of S-cycle genes are activated would enable assessment of their role and the potential foraminiferal/endobiont contribution to the sulphur-cycle.

Metabarcoding is a method that combines high-throughput DNA sequencing and DNA based identification. Previously, this method has been successfully used to target spatial variation of eukaryote communities in marine sediments, however, the temporal changes in these communities remain understudied. Here, we follow the temporal changes of the eukaryote communities in Baltic Sea surface sediments collected from two coastal localities during three seasons of two consecutive years. Our study reveals that the structure of the sediment eukaryotic ecosystem was primarily driven by annual and seasonal changes in prevailing environmental conditions, whereas spatial variation was a less significant factor in explaining the variance in eukaryotic communities over time. Therefore, our data suggests that shifts in regional climate regime or large-scale changes in the environment are the overdriving factors in shaping the coastal eukaryotic sediment ecosystems rather than small-scale changes in local environmental conditions or heterogeneity in ecosystem structure. More studies targeting temporal changes are needed to further understand the long-term trends in ecosystem stability and response to climate change. Furthermore, this work contributes to the recent efforts in developing metabarcoding applications for environmental biomonitoring, proving a comprehensive option for traditional monitoring approaches.

A B S T R A C TThe ultrastructure of the living foraminiferan, Ammonia sp. (phylotype unknown), collected from surficial and deeper, subsurface (anoxic) sediments from the Dutch Wadden Sea, was examined to provide information on the physiology of the foraminiferal cell and its adaptive strategies to low-oxygen conditions. The observed changes in cell ultrastructure under anoxia were further compared with the cell ultrastructure of Ammonia sp. (phylotype T6), from oxic and anoxic incubation experiments. The ultrastructural evidence indicates that under low-oxygen conditions Ammonia spp. may accumulate lipid droplets. In addition, the size of the lipid droplets may increase with the duration of anoxic conditions, becoming over 5 μm in size, while the remaining cytosol of the foraminiferan become less electron dense. In some specimens, lipid droplets were also found in the space between the plasma membrane and the organic lining. We expect that the apparent increase in the number and size of the lipid droplets is indicative of a stress response of the foraminifera to the adverse anoxic conditions. Other ultrastructural changes in response to anoxia include the presence of intact bacteria and electron dense opaque bodies within the foraminiferal cytosol, and a possible thickening of the organic lining. The role of the bacteria remains enigmatic but they may be linked to foraminiferal dormancy in anoxia.