Stefan Bertilsson

Peatlands are major global mercury (Hg) reservoirs and are highly sensitive to warming, yet climate effects on microbial controls of neurotoxic methylmercury (MeHg) formation remain unclear. Here, we combined global synthesis with new measurements to assess climate-associated variation in net MeHg production. Metagenomes from 144 peat soils revealed higher relative abundance of the Hg-methylation gene hgcA but lower relative abundance of the demethylation gene merB at high latitudes, consistent with their opposing correlations with mean annual temperature. The ratio of nitrogen-fixation to denitrification genes ( nifH / nosZ ) covaried with hgcA and merB , suggesting links between nitrogen cycling and Hg transformation. Genome-resolved analyses further showed expanded denitrification pathways in hgcA -bearing taxa from warmer peatlands. Together, these findings indicate that warmer climates may be associated with microbial community restructuring and lower net MeHg production. A space-for-time framework therefore suggests reduced MeHg yields under future warming, informing Hg-risk assessment under the Minamata Convention.

Microbial communities in peatlands vary with depth, but most studies have been limited in spatial extent or replication, constraining assessment of general community patterns. We characterized microbial community structure and metabolic potential across replicated depth profiles in seven boreal peatlands in Sweden. Combining 16S rRNA gene sequencing and shotgun metagenomics, we identified recurring vertical stratification across sites. Microbial richness declined with depth, and core taxa including Rice Cluster II, Terriglobales, Subgroup 2, Roseiarcus, Methylocystis, and Candidatus Solibacter were shared across locations. Functional gene profiles revealed depth-related reorganization of carbon-cycling potential, with hydrolytic and fermentative functions relatively more abundant near the surface and acetogenic and methanogenic pathways more prominent in deeper anoxic horizons. Gene-centric analyses indicated an important contribution of Rice Cluster II-related methanogens to hydrogenotrophic methanogenesis, while co-occurrence networks identified a depth-conserved syntrophic module centered on this lineage. Environmental variables showed depth-specific associations with microbial communities. Despite geographic separation and site-specific variation among peatlands, recurring depth-resolved microbial assemblages and functional profiles were observed, suggesting that common environmental constraints associated with peat development contribute to broad patterns of microbial community organization and carbon-cycling potential.

Microeukaryotes are abundant, diverse, and morphologically and functionally complex. They represent one of the largest groups of primary producers and are involved in major ecosystem functions such as nutrient transformations and maintenance of food webs. Although ~74,000 species of microeukaryotes are estimated to exist, very few are available in culture and only ~3000 are represented by reference genomes and transcriptomes. This limited representation significantly hinders the study of microeukaryotes in their natural environments. Single-cell transcriptomics with sequencing of full-length transcripts has potential to bypass this limitation. In this study, we employ Smart-seq3xpress for taxonomic identification and functional characterization of non-model mixotrophic freshwater microeukaryotes in a culture-free and reference-free manner, while also enabling inferences to be made for their prokaryotic associations. Computational analyses revealed 22 distinct microeukaryote taxa upon sequencing of 1520 randomly sampled cells, with assemblies of 12 good quality de novo partial transcriptomes, followed by functional annotation using sequence and structure homology. Gene expression analysis of the transcriptome for the most abundant microeukaryote in the sample, Rhodomonas, revealed a transcriptomic dip a few hours after experimental shading, followed by a gradual recovery in the next few days. Compared to samples in illuminated conditions, there was widespread downregulation of photosynthesis-, lysosome-, and carbon metabolism-related pathways in the shade, but the prevailing associations with prokaryotic lineages was unaffected by the light conditions. The different microeukaryotes, all present in the same environmental sample, featured distinct prokaryotic associates that were consistent across illumination conditions. This reference-free and culture-free study of 22 different microeukaryotic taxa sets the stage for high-throughput single cell transcriptomics to study microeukaryotic diversity in complex natural ecosystems, for their population-level taxonomic identity, sub-population-level transcriptomic profiles and metabolic states, and individual-level prokaryotic associations.

Subterranean estuaries (STEs) are key bioreactors regulating the quantity and chemical composition of groundwater‐derived nitrogen (N) reaching coastal ecosystems. Yet, the microbial controls on N‐cycling within these groundwater–seawater mixing zones remain poorly understood. We investigated the spatio‐temporal variations in microbial communities and their N‐cycling potential within an alluvial Mediterranean STE with high N concentration. We explored changes in microbial abundance, heterotrophic activity, taxonomic composition, and the abundance of N‐cycling genes across groundwater samples collected at several depths and distances from the shoreline in winter and summer. Microbial abundance, activity, and diversity varied strongly across hydrochemical zones according to physicochemistry and aquifer depth but showed limited seasonality. Functional predictions suggested a complex, spatially structured suite of N pathways encoded by diverse taxa occupying different STE zones, and quantitative‐PCR revealed niche partitioning between ammonia‐oxidizing archaea, prevalent in fresh‐groundwater, and bacterial denitrifiers enriched in deep‐saline layers. Multiple linear model predictions showed a stronger fit for NO ₂ ⁻ and NH ₄ ⁺ concentrations when using microbial properties than when using environmental variables, highlighting their importance for understanding N cycling in STEs. Our results suggest that the functional potential of the STE microbiome is complex and spatially structured across hydrochemical zones, explaining spatial variations in STE N‐cycling.

The hypersaline soils of the Odiel Saltmarshes Natural Area in Southwest Spain harbor highly diverse microbial communities adapted to extreme conditions. However, their genomic diversity remains largely unexplored. In addition to high salinity, these soils are contaminated with heavy metals, creating a hostile environment of great interest for studying extremophilic microorganisms and their metabolic adaptations. This study aims to characterize the uncovered prokaryotic taxa as Candidatus species inhabiting the hypersaline soils of the Odiel Saltmarshes, based on their metagenomic assembled genomic sequences. The reconstructed genomes were assessed for quality based on completeness and contamination thresholds and subsequently taxonomically classified. Comparative genomic analysis of six high-quality MAGs revealed key metabolic traits related to survival under extreme salinity and heavy metal conditions. The findings provide new insights about microbial diversity of hypersaline environments and expand the catalog of known prokaryotic genomes. Detailed characterization of six novel Candidatus taxa highlights the unique adaptations of these microorganisms, enhancing our understanding of life in extreme habitats.

The continental subsurface hosts energy-constrained groundwaters with a high diversity of ecologically elusive microorganisms adapted to the prevailing low-energy conditions. This study explored potential interactions among microbes using anaerobic enrichment incubations with three types of groundwater of contrasting hydrochemistry from the Äspö Hard Rock Laboratory, Sweden. Removing cells larger than 0.45 µm from the inoculum resulted in incubations enriched in populations characterized by very small genomes, including Patescibacteria, Nanobdellota, and Omnitrophota. These incubations had a higher diversity than non-fractionated incubations. However, cell numbers and community structure of the fractionated incubations did not change over an incubation period up to four months, despite high microbial diversity and experimental amendments with either simple (acetate) or more complex (cell lysate) carbon sources. In addition, network analysis on the groundwaters revealed multiple co-occurrences between populations affiliated with the Patescibacteria and the Desulfobacterota. Overall, these findings support that a considerable part of microbial diversity has a small cell size in these low energy groundwaters and strong co-occurrences among populations as an important survival strategy.

Microbial methane generation (methanogenesis) is an important metabolic process in the terrestrial deep biosphere and is an analog to early Earth as it is proposed to be one of the most ancient metabolisms on Earth. Signs of methanogenesis in meteorite impact craters are of particular interest in this respect as these settings are proposed hot spots for deep microbial colonization of the upper crust. Yet, reports of active deep rock-hosted methanogenesis are scarce, particularly for methylotrophic methanogenesis, while reports from terrestrial meteorite impact craters are completely lacking. Here, we used indigenous communities in cultures enriched from 400-m deep fluids to confirm and characterize active methane production from several carbon donors, including indigenous oil, in a terrestrial impact crater at Siljan, Sweden. Metagenomic and metatranscriptomic data of the methane-producing cultures revealed a consortium dominated by Acetobacterium sp. KB-1 and Candidatus Methanogranum gryphiswaldense, mediating methanogenesis solely via the methyl-reduction pathway, and resulting in a δ ¹³ C _{methanol-methane} isotope enrichment of up to 98.6‰. These results provide insights into methylotrophic methanogenesis in deep subsurface environments in general, and in particular in fractured meteorite impact structures. IMPORTANCE: This study revealed that microbes enriched from groundwater in a 380-m deep borehole within the Siljan meteorite impact crater in Sweden were capable of producing methane, a key greenhouse gas. This is especially significant because it is the first proof of active methanogens in an impact crater and showing a specific pathway of methane production—methylotrophic methanogenesis—is present in the deep terrestrial subsurface, an environment that is typically hard to study. These findings shed light on life in extreme conditions on Earth and show that meteorite craters can be biological hotspots, rich with ancient life processes.

While the influence of salinity on microbial diversity is well documented in marine and brackish ecosystems, the impact of different dissolved inorganic ion types remains largely unexplored. In this study, we assessed how ionic composition shapes planktonic bacterial community structure in inland saline aquatic habitats, compared to the effects of salinity alone, spatial factors, and other environmental variables. We collected and analyzed 16S rRNA gene amplicon datasets from freshwater to hypersaline aquatic environments worldwide (375 samples from 130 lakes). The composition of major ions explained more variability in bacterioplankton structure than bulk salinity. Taxa contributing the most to the observed dissimilarity between communities included lineages characteristic of specific habitat types, such as Actinobacteria acI in freshwater, Halomonadaceae in saline waters, or Nitriliruptorales in soda‐ and soda‐saline systems. Many of these indicator lineages for specific habitat types were monophyletic, further underpinning ionic composition as a crucial eco‐evolutionary driver of aquatic microbial diversity.

With climate change-induced sea ice decline in the Arctic Ocean, nitrogen is expected to become an increasingly important determinant of primary productivity. Nitrogen fixation is the conversion of molecular nitrogen to bioavailable ammonium by microorganisms called diazotrophs. Here, we report nitrogen fixation rates, diazotroph composition, and expression under different stages of declining sea ice in the Central Arctic Ocean (multiyear ice, five stations) and the Eurasian Arctic (marginal ice zone, seven stations). Nitrogen fixation in the Central Arctic Ocean was positively correlated with primary production, ranging from 0.4 ± 0.1 to 2.5 ± 0.87 nmol N L⁻¹ d⁻¹. Along two transects across the marginal ice zone, nitrogen fixation varied between days and ice regime from below detection up to 5.3 ± 3.65 nmol N L⁻¹ d⁻¹ associated with an ice-edge phytoplankton bloom. We show nitrogen fixation in sea ice-covered waters of the Arctic Ocean and provide insight into present and active non-cyanobacterial diazotrophs in the region.