Daniel P. R. Herlemann

Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea

Salinity is a major factor controlling the distribution of biota in aquatic systems, and most aquatic multicellular organisms are either adapted to life in saltwater or freshwater conditions. Consequently, the saltwater–freshwater mixing zones in coastal or estuarine areas are characterized by limited faunal and floral diversity. Although changes in diversity and decline in species richness in brackish waters is well documented in aquatic ecology, it is unknown to what extent this applies to bacterial communities. Here, we report a first detailed bacterial inventory from vertical profiles of 60 sampling stations distributed along the salinity gradient of the Baltic Sea, one of worlds largest brackish water environments, generated using 454 pyrosequencing of partial (400 bp) 16S rRNA genes. Within the salinity gradient, bacterial community composition altered at broad and finer-scale phylogenetic levels. Analogous to faunal communities within brackish conditions, we identified a bacterial brackish water community comprising a diverse combination of freshwater and marine groups, along with populations unique to this environment. As water residence times in the Baltic Sea exceed 3 years, the observed bacterial community cannot be the result of mixing of fresh water and saltwater, but our study represents the first detailed description of an autochthonous brackish microbiome. In contrast to the decline in the diversity of multicellular organisms, reduced bacterial diversity at brackish conditions could not be established. It is possible that the rapid adaptation rate of bacteria has enabled a variety of lineages to fill what for higher organisms remains a challenging and relatively unoccupied ecological niche.

Metagenomic De Novo Assembly of an Aquatic Representative of the Verrucomicrobial Class Spartobacteria

ABSTRACT The verrucomicrobial subdivision 2 class Spartobacteria is one of the most abundant bacterial lineages in soil and has recently also been found to be ubiquitous in aquatic environments. A 16S rRNA gene study from samples spanning the entire salinity range of the Baltic Sea indicated that, in the pelagic brackish water, a phylotype of the Spartobacteria is one of the dominating bacteria during summer. Phylogenetic analyses of related 16S rRNA genes indicate that a purely aquatic lineage within the Spartobacteria exists. Since no aquatic representative from the Spartobacteria has been cultured or sequenced, the metabolic capacity and ecological role of this lineage are yet unknown. In this study, we reconstructed the genome and metabolic potential of the abundant Baltic Sea Spartobacteria phylotype by metagenomics. Binning of genome fragments by nucleotide composition and a self-organizing map recovered the near-complete genome of the organism, the gene content of which suggests an aerobic heterotrophic metabolism. Notably, we found 23 glycoside hydrolases that likely allow the use of a variety of carbohydrates, like cellulose, mannan, xylan, chitin, and starch, as carbon sources. In addition, a complete pathway for sulfate utilization was found, indicating catabolic processing of sulfated polysaccharides, commonly found in aquatic phytoplankton. The high frequency of glycoside hydrolase genes implies an important role of this organism in the aquatic carbon cycle. Spatiotemporal data of the phylotype’s distribution within the Baltic Sea indicate a connection to Cyanobacteria that may be the main source of the polysaccharide substrates. IMPORTANCE The ecosystem roles of many phylogenetic lineages are not yet well understood. One such lineage is the class Spartobacteria within the Verrucomicrobia that, despite being abundant in soil and aquatic systems, is relatively poorly studied. Here we circumvented the difficulties of growing aquatic Verrucomicrobia by applying shotgun metagenomic sequencing on a water sample from the Baltic Sea. By using a method based on sequence signatures, we were able to in silico isolate genome fragments belonging to a phylotype of the Spartobacteria. The genome, which represents the first aquatic representative of this clade, encodes a diversity of glycoside hydrolases that likely allow degradation of various complex carbohydrates. Since the phylotype cooccurs with Cyanobacteria, these may be the primary producers of the carbohydrate substrates. The phylotype, which is highly abundant in the Baltic Sea during summer, may thus play an important role in the carbon cycle of this ecosystem. The ecosystem roles of many phylogenetic lineages are not yet well understood. One such lineage is the class Spartobacteria within the Verrucomicrobia that, despite being abundant in soil and aquatic systems, is relatively poorly studied. Here we circumvented the difficulties of growing aquatic Verrucomicrobia by applying shotgun metagenomic sequencing on a water sample from the Baltic Sea. By using a method based on sequence signatures, we were able to in silico isolate genome fragments belonging to a phylotype of the Spartobacteria. The genome, which represents the first aquatic representative of this clade, encodes a diversity of glycoside hydrolases that likely allow degradation of various complex carbohydrates. Since the phylotype cooccurs with Cyanobacteria, these may be the primary producers of the carbohydrate substrates. The phylotype, which is highly abundant in the Baltic Sea during summer, may thus play an important role in the carbon cycle of this ecosystem.

Particle-Associated Differ from Free-Living Bacteria in Surface Waters of the Baltic Sea.

Many studies on bacterial community composition (BCC) do not distinguish between particle-associated (PA) and free-living (FL) bacteria or neglect the PA fraction by pre-filtration removing most particles. Although temporal and spatial gradients in environmental variables are known to shape BCC, it remains unclear how and to what extent PA and FL bacterial diversity responds to such environmental changes. To elucidate the BCC of both bacterial fractions related to different environmental settings, we studied surface samples of three Baltic Sea stations (marine, mesohaline, and oligohaline) in two different seasons (summer and fall/winter). Amplicon sequencing of the 16 S rRNA gene revealed significant differences in BCC of both bacterial fractions among stations and seasons, with a particularly high number of PA operational taxonomic units (OTUs at genus-level) at the marine station in both seasons. “Shannon and Simpson indices” showed a higher diversity of PA than FL bacteria at the marine station in both seasons and at the oligohaline station in fall/winter. In general, a high fraction of bacterial OTUs was found exclusively in the PA fraction (52% of total OTUs). These findings indicate that PA bacteria significantly contribute to overall bacterial richness and that they differ from FL bacteria. Therefore, to gain a deeper understanding on diversity and dynamics of aquatic bacteria, PA and FL bacteria should be generally studied independently.

Uncoupling of bacterial and terrigenous dissolved organic matter dynamics in decomposition experiments.

The biodegradability of terrigenous dissolved organic matter (tDOM) exported to the sea has a major impact on the global carbon cycle, but our understanding of tDOM bioavailability is fragmentary. In this study, the effects of preparative tDOM isolation on microbial decomposition were investigated in incubation experiments consisting of mesocosms containing mesohaline water from the Baltic Sea. Dissolved organic carbon (DOC) consumption, molecular DOM composition, bacterial activities, and shifts in bacterial community structure were compared between mesocosms supplemented with riverine tDOM, either as filtered, particle-free river water or as a concentrate obtained by lyophilization/tangential ultrafiltration, and those containing only Baltic Sea water or river water. As shown using ultra-high-resolution mass spectrometry (15 Tesla Fourier-transform ion cyclotron resonance mass spectrometry, FT-ICR-MS) covering approximately 4600 different DOM compounds, the three DOM preparation protocols resulted in distinct patterns of molecular DOM composition. However, despite DOC losses of 4–16% and considerable bacterial production, there was no significant change in DOM composition during the 28-day experiment. Moreover, tDOM addition affected neither DOC degradation nor bacterial dynamics significantly, regardless of the tDOM preparation. This result suggested that the introduced tDOM was largely not bioavailable, at least on the temporal scale of our experiment, and that the observed bacterial activity and DOC decomposition mainly reflected the degradation of unknown, labile, colloidal and low-molecular weight DOM, both of which escape the analytical window of FT-ICR-MS. In contrast to the different tDOM preparations, the initial bacterial inoculum and batch culture conditions determined bacterial community succession and superseded the effects of tDOM addition. The uncoupling of tDOM and bacterial dynamics suggests that mesohaline bacterial communities cannot efficiently utilize tDOM and that in subarctic estuaries other factors are responsible for the removal of imported tDOM.

Phylogenetic Signals of Salinity and Season in Bacterial Community Composition Across the Salinity Gradient of the Baltic Sea.

Understanding the key processes that control bacterial community composition has enabled predictions of bacterial distribution and function within ecosystems. In this study, we used the Baltic Sea as a model system to quantify the phylogenetic signal of salinity and season with respect to bacterioplankton community composition. The abundances of 16S rRNA gene amplicon sequencing reads were analyzed from samples obtained from similar geographic locations in July and February along a brackish to marine salinity gradient in the Baltic Sea. While there was no distinct pattern of bacterial richness at different salinities, the number of bacterial phylotypes in winter was significantly higher than in summer. Bacterial community composition in brackish vs. marine conditions, and in July vs. February was significantly different. Non-metric multidimensional scaling showed that bacterial community composition was primarily separated according to salinity and secondly according to seasonal differences at all taxonomic ranks tested. Similarly, quantitative phylogenetic clustering implicated a phylogenetic signal for both salinity and seasonality. Our results suggest that global patterns of bacterial community composition with respect to salinity and season are the result of phylogenetically clustered ecological preferences with stronger imprints from salinity.

Distribution of the verrucomicrobial clade Spartobacteria along a salinity gradient in the Baltic Sea

A recent pyrosequencing study along the whole Baltic Sea salinity transect identified members of the Verrucomicrobia class Spartobacteria as an important component of Baltic Sea bacterioplankton. In this study, catalysed reporter deposition-fluorescence in situ hybridization was used for cellular quantification. The published probes VER47 and SPA714 were optimized for samples from the Baltic Sea and a new, specific probe (SPA476) was used to quantify the dominant spartobacterial lineage ‘LD29’. The results confirmed that in the brackish surface waters of the Baltic Sea Spartobacteria comprise an important component, constituting up to 12% of all bacteria. The positive correlation and physical association of Spartobacteria with phytoplankton suggest their involvement in the utilization of phytoplankton-derived organic matter in the Baltic Sea.

Mucosa‐attached bacterial community in Crohn's disease coheres with the clinical disease activity index

In inflammatory bowel diseases (IBD), microbial communities often become imbalanced suggesting abnormal microbial-gut interactions. In this study, we analysed the mucosa-attached gut microbiota from 26 Crohns disease (CD) patients using 16S rRNA gene amplicon sequencing. The samples were stratified according to their disease activity (Crohns disease activity index, CDAI). The different disease activity categories had a comparable bacterial richness. Bacterial communities of patients in remission and intermediate CDAI (0-220) were relatively similar and dominated by the genus Bacteroides (>40%). The bacterial composition of patients assigned to a high CDAI category was dominated by Pelomonas (25%) and Flavobacterium (13%) but had a low relative abundance of Bacteroidetes (4%). This indicates the presence of specific abundant bacterial taxa at different CDAI levels. In addition, bacterial communities were also significantly influenced when a tumour necrosis factor (TNF)-α inhibitor was applied or by the local mucosal inflammation level. As a consequence, a shift of the microbial composition may also indicate a change of the disease activity in CD patients.

Composition and Transformation of Dissolved Organic Matter in the Baltic Sea

The processing of terrestrial dissolved organic matter (DOM) in coastal shelf seas is an important part of the global carbon cycle, yet, it is still not well understood. One of the largest brackish shelf seas, the Baltic Sea in northern Europe, is characterized by high freshwater input from sub-arctic rivers and limited water exchange with the Atlantic Ocean via the North Sea. We studied the molecular and isotopic composition and turnover of solid-phase extractable (SPE) DOM and its transformation along the salinity and redox continuum of the Baltic Sea during spring and autumn. We applied ultrahigh-resolution mass spectrometry and other geochemical and biological approaches. Our data demonstrate a large influx of terrestrial riverine DOM, especially into the northern part of the Baltic Sea. The DOM composition in the central Baltic Sea changed seasonally and was mainly related to autochthonous production by phytoplankton in spring. Especially in the northern, river-dominated basins, a major fraction of riverine DOM was removed, likely by bio- and photo-degradation. We estimate that the removal rate of terrestrial DOM in the Baltic Sea (Bothnian Bay to the Danish Straits/Kattegat area) is 1.6 – 1.9 Tg C per year which is 43 to 51% of the total riverine input. The export of terrestrial DOM from the Danish Straits/Kattegat area towards the North Sea is 1.8 – 2.1 Tg C per year. Due to the long residence time of terrestrial DOM in the Baltic Sea (total of ca. 12 years), seasonal variations caused by bio- and photo-transformations and riverine discharge are dampened, resulting in a relatively invariant DOM molecular and isotopic signature exported to the North Sea. In the deep stagnant basins of the Baltic Sea, the DOM composition and dissolved organic nitrogen concentrations changed seasonally, likely because of vertical particle transport and subsequent degradation releasing DOM. DOM in the deep anoxic basins was also enriched in sulfur-containing organic molecules, pointing to abiotic sulfurization of DOM under sulfidic conditions.

Differential responses of marine, mesohaline, and oligohaline bacterial communities to the addition of terrigenous carbon

In response to global warming, increasing quantities of tDOM are transported through estuaries from land to the sea. In this study, we investigated microbial responses to increased tDOM concentrations in three salinity regimes (salinity: 32, 7 and 3) characteristic of the Baltic Sea. Mesocosm experiments performed in May and November revealed low (0-6%) dissolved organic carbon (DOC) utilisation. Molecular DOM analyses using ultrahigh-resolution mass spectrometry identified the terrigenous signal in the tDOM manipulation, but the molecular changes in DOM levels over the course of the experiment were subtle. However, tDOM had significant stimulatory effects on bacterial production in the oligohaline mesocosms. The shift in the bacterial community composition was especially prominent in the tDOM-amended marine and mesohaline mesocosms, but not in the oligohaline mesocosms after 7 and 11 days of incubation. These results suggested the inherent ability of oligohaline bacterial communities to adapt to high tDOM concentrations and therefore to use tDOM. The higher rates of bacterial activity and DOC removal in mesocosms containing UV-pretreated tDOM supported the increased bioavailability of photoinduced, modified tDOM. The overall low rates of microbial tDOM utilisation highlights the importance of abiotic factors in determining the distribution and dynamics of tDOM in estuaries.

Zonation of bacterioplankton communities along aging upwelled water in the northern Benguela upwelling

Upwelling areas are shaped by enhanced primary production in surface waters, accompanied by a well-investigated planktonic succession. Although bacteria play an important role in biogeochemical cycles of upwelling systems, little is known about bacterial community composition and its development during upwelling events. The aim of this study was to investigate the succession of bacterial assemblages in aging upwelled water of the Benguela upwelling from coastal to offshore sites. Water from the upper mixed layer at 12 stations was sampled along two transects from the origin of the upwelling to a distance of 220 km. 16S rRNA gene amplicon sequencing was then used in a bacterial diversity analysis and major bacterial taxa were quantified by catalyzed reporter deposition-fluorescence in situ hybridization. Additionally, bacterial cell numbers and bacterial production were assessed. Community statistical analysis revealed a reproducible zonation along the two transects, with four clusters of significantly different microbial assemblages. Clustering was mainly driven by phytoplankton composition and abundance. Similar to the temporal succession that occurs during phytoplankton blooms in temperate coastal waters, operational taxonomic units (OTUs) affiliated with Bacteroidetes and Gammaproteobacteria were dominant during algal blooming whereas “Pelagibacterales” were highly abundant in regions with low algal abundance. The most dominant heterotrophic OTU (9% of all reads) was affiliated with “Pelagibacterales” and showed a strong negative correlation with phytoplankton. By contrast, the second most abundant heterotrophic OTU (6% of all reads) was affiliated with the phylum Verrucomicrobia and correlated positively with phytoplankton. Together with the close relation of bacterial production and phytoplankton abundance, our results showed that bacterial community dynamics is strongly driven by the development and composition of the phytoplankton community.