Taxonomic and Functional Analyses of Marine Microbial Polysaccharide Utilisation

Marine primary production accounts for half of the Earth' s carbon fixation and therefore has a significant impact on the global carbon cycle. However, although marine primary producers fix such a significant fraction of carbon, their biomass makes up only a small fraction of the total organic carbon pool. This is due to the extremely high turnover of phytoplankton and phytoplankton-derived organic matter within the oceans. This turnover is mediated by marine heterotrophic microorganisms (Bacteria and Archaea). Marine microorganisms therefore significantly affect the global carbon cycle. The objectives of this thesis were to investigate how the taxonomic and functional diversity of marine microorganisms affects the bacterially mediated carbon turnover in the Atlantic Ocean. In chapter 1 and 2 I developed methods which enable a shipboard high-throughput analysis of microbial diversity and abundance. These methods were developed to overcome the time delay between sampling and results and enable a comprehensive interpretation of the microbial community composition even in remote sampling sites. In chapter 3 I explored the biogeographical distribution patterns of the free-living (FL) and particle-associated (PA) bacterial communities across different provinces of the Atlantic Ocean. The FL and PA bacterial community compositions were more similar under copiotrophic condition and more dissimilar under oligotrophic conditions. I could associate these results to the relative age of the available particles as well as the availability of organic matter. In Chapter 4 I investigated alternative substrate uptake mechanisms in marine bacteria. Using fluorescently labelled polysaccharides (FLA-PS) and super-resolution structured illumination microscopy I could show that a significant fraction of marine bacteria use a a selfisha substrate utilisation mechanism. I combined this analysis with fluorescence in situ hybridization (FISH) to taxonomically identify the organisms as belonging to the Bacteroidetes, Planctomycetes and Gammaproteobacteria. The discovery of a widespread alternative substrate utilisation mechanism significantly affects our global estimates of carbon turnover by marine bacteria. Finally, in Chapter 5 I investigated the extracellular hydrolysis rates and bacterial community dynamics within fluorescently labelled polysaccharide incubations. These analyses lead to the identification of the dominant microorganisms associated with the hydrolysis of polysaccharides in the marine environment. The work done in this thesis has furthered our understanding of the activity and distribution patterns of bacterial polysaccharide utilisation mechanisms across the Atlantic Ocean. Additionally, the activity of the individual mechanisms could be associated with specific microbial groups, thereby linking the taxonomy and function of the dominant marine polysaccharide degrading organisms. This study will enable us to make better predictions of the impact which marine microorganisms have on global biogeochemical cycles

Single cell fluorescence imaging of glycan uptake by intestinal bacteria

Microbes in the intestines of mammals degrade dietary glycans for energy and growth. The pathways required for polysaccharide utilization are functionally diverse; moreover, they are unequally dispersed between bacterial genomes. Hence, assigning metabolic phenotypes to genotypes remains a challenge in microbiome research. Here we demonstrate that glycan uptake in gut bacteria can be visualized with fluorescent glycan conjugates (FGCs) using epifluorescence microscopy. Yeast α-mannan and rhamnogalacturonan-II, two structurally distinct glycans from the cell walls of yeast and plants, respectively, were fluorescently labeled and fed to Bacteroides thetaiotaomicron VPI-5482. Wild-type cells rapidly consumed the FGCs and became fluorescent; whereas, strains that had deleted pathways for glycan degradation and transport were non-fluorescent. Uptake of FGCs, therefore, is direct evidence of genetic function and provides a direct method to assess specific glycan metabolism in intestinal bacteria at the single cell level.</p

Taxonomische und Funktionelle Analysen der Marinen Mikrobiellen Polysaccharidverwendung

Marine primary production accounts for half of the Earth' s carbon fixation and therefore has a significant impact on the global carbon cycle. However, although marine primary producers fix such a significant fraction of carbon, their biomass makes up only a small fraction of the total organic carbon pool. This is due to the extremely high turnover of phytoplankton and phytoplankton-derived organic matter within the oceans. This turnover is mediated by marine heterotrophic microorganisms (Bacteria and Archaea). Marine microorganisms therefore significantly affect the global carbon cycle. The objectives of this thesis were to investigate how the taxonomic and functional diversity of marine microorganisms affects the bacterially mediated carbon turnover in the Atlantic Ocean. In chapter 1 and 2 I developed methods which enable a shipboard high-throughput analysis of microbial diversity and abundance. These methods were developed to overcome the time delay between sampling and results and enable a comprehensive interpretation of the microbial community composition even in remote sampling sites. In chapter 3 I explored the biogeographical distribution patterns of the free-living (FL) and particle-associated (PA) bacterial communities across different provinces of the Atlantic Ocean. The FL and PA bacterial community compositions were more similar under copiotrophic condition and more dissimilar under oligotrophic conditions. I could associate these results to the relative age of the available particles as well as the availability of organic matter. In Chapter 4 I investigated alternative substrate uptake mechanisms in marine bacteria. Using fluorescently labelled polysaccharides (FLA-PS) and super-resolution structured illumination microscopy I could show that a significant fraction of marine bacteria use a a selfisha substrate utilisation mechanism. I combined this analysis with fluorescence in situ hybridization (FISH) to taxonomically identify the organisms as belonging to the Bacteroidetes, Planctomycetes and Gammaproteobacteria. The discovery of a widespread alternative substrate utilisation mechanism significantly affects our global estimates of carbon turnover by marine bacteria. Finally, in Chapter 5 I investigated the extracellular hydrolysis rates and bacterial community dynamics within fluorescently labelled polysaccharide incubations. These analyses lead to the identification of the dominant microorganisms associated with the hydrolysis of polysaccharides in the marine environment. The work done in this thesis has furthered our understanding of the activity and distribution patterns of bacterial polysaccharide utilisation mechanisms across the Atlantic Ocean. Additionally, the activity of the individual mechanisms could be associated with specific microbial groups, thereby linking the taxonomy and function of the dominant marine polysaccharide degrading organisms. This study will enable us to make better predictions of the impact which marine microorganisms have on global biogeochemical cycles

Short term changes in polysaccharide utilisation during a spring phytoplankton bloom on Helgoland

Spring phytoplankton blooms contribute significantly to global marine primary production. A large fraction of the bloom derived organic matter is available to heterotrophic bacteria in the form of polysaccharides. We analyzed changes in the modes of polysaccharide utilization (selfish uptake and extracellular hydrolysis) during a spring phytoplankton bloom using fluorescently labelled polysaccharide incubations coupled with 16s rRNA sequencing and fluorescence in situ hybridization. We found that in the early bloom phases there was high selfish activity of simple polysaccharides (laminarin) and low extracellular hydrolysis rates of a limited range of polysaccharides. During the course of the bloom both the selfish uptake and extracellular hydrolysis rates increased but only for a limited range of substrates. At the late bloom phase a wide range of substrate was extracellularly hydrolyzed and the level of selfish uptake decreased. We found that during a spring phytoplankton bloom the mode of substrate utilization depended on both the substrates structural complexity and the composition of the heterotrophic community related to the bloom phase

A biogeographical study of microbial substrate utilisation in the Atlantic Ocean

A large fraction of the organic matter fixed in the oceans is transformed and remineralised by marine heterotrophic microorganisms. They, therefore, play a critical role in the marine carbon cycle. In this study, we set out to identify the roles played by individual heterotrophic bacteria in the degradation of high molecular weight polysaccharides. At five sites in the Atlantic Ocean, we investigated the processing of organic matter in microbial communities by tracking the changes in community composition (fluorescence in situ hybridisation (FISH), 16S rRNA tag sequencing) in substrate incubation using a defined concentration of a known fluorescently labelled polysaccharide (FLA-laminarin, FLA-xylan, and FLA-chondroitin sulfate). Additionally, we tracked the dynamics of substrate processing (selfish uptake and extracellular hydrolysis) within the microbial communities between sites. We found that the same substrate was processed in different ways by different members of a pelagic microbial community which points to significant follow-on effects for carbon cycling

Microbial community composition and polysaccharide processing potential in the South Pacific Gyre

The South Pacific Gyre (SPG) covers 10% of the ocean's surface and is considered a marine biological desert. However, recent investigations have shown that primary production occurs throughout its deep euphotic zone and that this fuels the regeneration of nutrients and the recycling of organic matter. We set out to investigate the SPG's microbial communities' heterotrophic capability to utilize polysaccharides, an important marine organic matter component. Using fluorescently labeled polysaccharide (FLA-PS) incubations (Reintjes, et al., 2017), we analyzed the initial step of organic matter degradation by measuring both the rate of external hydrolysis and the rate of direct uptake of polysaccharides by marine microorganisms. Furthermore, we investigated the change in bacterial abundance and diversity during the FLA-PS incubations using direct cell counts and 16S rRNA sequencing. The presented dataset contains the microbial diversity, total cellular abundance, and direct FLA-PS uptake results generated during the FLA-PS incubations performed with six polysaccharides (laminarin, xylan, chondroitin sulfate, arabinogalactan, fucoidan, and pullulan) over 18 days. The incubations were performed with seawater from the epipelagic and bathypelagic (75 m, 160 m, 1250 m, and 2800 m) in the central gyre, and seawater from the epipelagic (75 m) at two stations adjacent to the gyre. Our study found that the SPG's microbial community showed remarkably high extracellular enzyme activities, and a considerable fraction of the microorganisms were capable of the direct uptake (selfish-uptake) of FLA-PS. Interestingly, a wide variety of bacteria were capable of cycling HMW organic matter using distinct polysaccharide processing mechanisms in the SPG. This research shows that the SPG features not only organisms capable of existing on the fine edge of minimal substrate concentrations but also those capable of taking advantage of abrupt changes in physical conditions and substrate availabilit

On-board sequencing of the microbial community of the South Pacific Gyre

Fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of the seven most abundant bacterial clades (determined by 16S rRNA tag sequencing) of the South Pacific Gyre (SPG). Seawater samples were collected aboard the RV Sonne SO-245 "UltraPac"cruise from Antofagasta, Chile (17.12.2015) to Wellington, New Zealand (28.01.2016). A total of 15 stations were sampled at multiple depths from surface (20 m) to ~5000 m. Total cell counts (TCC, by DAPI staining) and FISH were carried out as described in Bennke et al. (2016). DAPI and FISH stained cells were visualised and counted automatically using a fully automated image acquisition and cell enumeration system (Bennke et al., 2016). The cellular abundance of SAR11 clade, Prochlorococcus, AEGEAN-169 marine group, SAR86, SAR202, SAR324 and SAR406 were enumerated and are shown here in total (cell ml-1) and relative abundance (% TCC). For this study, a new probe specific for the AEGEAN-169 clade was designed and tested, based on the latest SILVA 16S rRNA database (refnr 128). We found that the microbial community within the SPG was highly similar to that of other oceanic gyres and showed a pronounced vertical distribution pattern. Two major differences which we observed, in comparison to previous studies of both the SPG and other oceanic Gyres, was a high abundance of the AEGEAN-169 marine group, a sister group of the SAR11 clade, and a low abundance of Prochlorococcus specifically in the surface waters of the central gyre

Microbial community composition and bacterioplankton at time series station Helgoland Roads, North Sea

A process of global importance in carbon cycling is the remineralization of algae biomass by heterotrophic bacteria, most notably during massive marine algae blooms. Such blooms can trigger secondary blooms of planktonic bacteria that consist of swift successions of distinct bacterial clades, most prominently members of the Flavobacteriia, Gammaproteobacteria and the alphaproteobacterial Roseobacter clade. This study explores such successions during spring phytoplankton blooms in the southern North Sea (German Bight) for four consecutive years. The surface water samples were taken at Helgoland Island about 40 km offshore in the southeastern North Sea in the German Bight at the station 'Kabeltonne' (54° 11.3' N, 7° 54.0' E) between the main island and the minor island, Düne (German for 'dune') using small research vessels (http://www.awi.de/en/expedition/ships/more-ships.html). Water depths at this site fluctuate from 6 to 10 m over the tidal cycle. Samples were processed as described previously (Teeling et al., 2012; doi:10.7554/eLife.11888.001) in the laboratory of the Biological Station Helgoland within less than two hours after sampling. Assessment of absolute cell numbers and bacterioplankton community composition was carried out as described previously (Thiele et al., 2011; doi:10.1016/B978-0-444-53199-5.00056-7). To obtain total cell numbers, DNA of formaldehyde fixed cells filtered on 0.2 mm pore sized filters was stained with 4',6-diamidino-2-phenylindole (DAPI). Fluorescently labeled cells were subsequently counted on filter sections using an epifluores-cence microscope. Likewise, bacterioplankton community composition was assessed by catalyzedreporter deposition fluorescence in situ hybridization (CARD-FISH) of formaldehyde fixed cells on 0.2 mm pore sized filters