Amann, Rudolf
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Amann, Rudolf
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Amann, Rudolf
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Amann, Rudi
Amann, Rudolph
Aman, Rudolf
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Item-typ:Veröffentlichung, Systematische Optimierung für den Nachweis einzelner Gene in Mikroorganismen durch Fluoreszenz In Situ Hybridisierung (GENEFISH)(2015-12-08); ; ; Culture-independent techniques have played an increasingly important role in microbial ecology. Fluorescence in situ Hybridization (FISH) is a well-established method for the identification and quantification of microorganisms. Among the FISH-based techniques, geneFISH allows the linkage of particular genes to the microbial identity at the single cell level. The protocol involves ribosomal RNA-targeted Catalyzed Amplification Reported Deposition (CARD) for cell identification. For gene detection, digoxigenin-labeled polynucleotide probes are applied, to which specific antibodies conjugated with horseradish-peroxidases can bind. So far, low gene detection efficiencies have prevented a quantitative determination of the cell fraction carrying the target gene. Additionally, the CARD step hinders the sub-cellular signal quantification, increases DNA degradation and cell damage and makes the protocol long and labor intensive. Therefore, the main aim of this thesis was to systematically improve the geneFISH protocol by increasing the gene detection efficiency, simplifying the method and achieving a quantification of gene number per cell.Dissertation407 133 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Hochparallele Analyse natürlicher mikrobieller Lebensgemeinschaften durch oberflächengebundene Oligonukleotidsonden(2003-07-04); ; ; DNA microarrays represent a high throughput format for the application of nucleic acid probes by reverse hybridization and, therefore, provide a powerful tool for the parallel detection of microorganisms. However, their application in molecular ecology is still hampered by different methodological limitations. In this study, various parameters affecting a surface-mediated hybridization were systematically evaluated with a model system composed of 6 bacterial strains and 20 oligonucleotide probes targeting the 16S rRNA. With adequate hybridization conditions, false-positive signals could be almost completely prevented resulting in clear data interpretation. For microarray analysis of mixed microbial communities, a straightforward protocol for direct chemical labeling of extracted rRNA was established to avoid potential PCR-biases. In samples of different complexity, including an oligotrophic marine system, major bacterial populations (approx. >5%) could specifically be detected by microarray hybridization, as determined by parallel fluorescence in situ hybridization (FISH). However, quantitative FISH data suggested that current microarray analysis provides, at best, semiquantitative information on community composition. Application of phylogenetically redundant and/or nested probe sets is a prerequisite for the reliable hybridization-based detection of microorganisms. A potential reservoir of additional probe target sites is represented by the 23S rRNA. In this study, almost complete 23S rRNA gene sequences were obtained from 11 alpha-proteobacteria isolated from North Sea bacterioplankton. Comparative sequence analysis revealed consistent tree topologies for the alpha-proteobacteria based on their 16S and 23S rRNA genes. The suitability of both markers for the design of specific oligonucleotide probes was systematically evaluated in silico. The overall number of phylogenetic redundant probes available could significantly be increased by also targeting the 23S rRNA.Dissertation270 102 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Mikrobielle Aktivität in dem huminstoffreichen Moorsee Große Fuchskuhle(2005-06-17); ; ; Dystrophic lakes are characterized by a large pool of dissolved organic carbon (DOC) with a high portion of humic substances. These high molecular weight substances can be utilized by microorganisms releasing extracellular enzymes such as peroxidases, resulting in the formation of unstable radicals. Most organisms known for the production of extracellular peroxidases were found in terrestrial environments whereas investigations on these enzymes in the aquatic environment are rather rare. For investigations on microbial activities in a humic rich environment Lake Große Fuchskuhle has been chosen. This lake was artificially subdivided into four compartments which subsequently developed divergent chemical and microbiological characteristics. Thus, the DOC pool differed remarkably between the compartments. Due to a strong impact of the catchment area at one side of the lake two compartments are rich in humic substances whereas the other two compartments have a lower concentration of humic substances but a higher concentration of polysaccharides. Only few studies focus on the hydrogeology of lakes and the catchment area with respect to the genesis. In this thesis results are presented from field investigations on the water balance and on chemical fluxes between Lake Große Fuchskuhle and the catchment area. During high groundwater inflow into one compartment higher concentrations of DOC, nitrogen and phosphorus were detected. These findings then raised the questions i) whether specific bacterial groups rapidly respond to changes in DOC availability and ii) after their potential to degrade humic substances by extracellular enzymes. Seasonal changes of microbial community composition were studied in a humic rich and a humic poor compartment. These changes were compared to community shifts induced during short-term enrichment experiments. In these experiments beta-proteobacteria were enriched whereas Actinobacteria declined during the incubations. However, in Lake Große Fuchskuhle the beta-proteobacteria and Actinobacteria appeared in high numbers. In the humic rich compartment the abundance of beta-proteobacteria (beta II cluster) was significantly higher than in the humic poor compartment. The abundance of Actinobacteria was similar in both compartments. The ratio between the bacteria of the beta II cluster and the Actinobacteria showed a high saisonal fluctuation between the compartments. This indicates differences in their ecological role which is supported by the rapidly response of the beta-proteobacteria and a decline of the Actinobacteria to the changed growth conditions in the enrichment experiment. Measurements of extracellular peroxidase activity indicated seasonal differences between the humic rich and humic poor compartment. The activity of extracellular peroxidases was higher in the humic rich than in the humic poor compartment, with highest values during fall and winter which coincided with a high abundance of Actinobacteria.Dissertation308 119 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Der bakterielle Schwefelkreislaufim Benguela Auftriebssystem(2009-09-22); ; ; Coastal upwelling regions are economically important as they contribute to ~50% of the world's fisheries landings. High primary production and extensive mineralization of organic matter often lead to oxygen minimum zones (OMZ) and the transient formation of hydrogen sulfide with severe consequences for coastal ecosystems such as the Benguela Upwelling System (BAG). However, the involved microorganisms catalyzing the cycling of sulfur cycle in the water column are still unknown. In this thesis, the microbial community structure in the BAG was studied in a high spatiotemporal resolution by the 16S rRNA approach to identify potential key players of the sulfur cycle.In a sulfidic lens covering 7,000 km2 on the Namibian Shelf both 16S rRNA gene libraries and FISH identified two discrete populations of Gamma- and Epsilonproteobacteria that accounted for up to 20% of the bacterioplankton. Their cell numbers increased from the nitrate-sulfide transition zone to the sediment that indicated a chemolithotrophic oxidation of sulphide with nitrate. Indeed, one population was closely related to gammaproteobacterial sulfur oxidizing endosymbionts (GSO), whereas the second population affiliated with the sulfur oxidizing Arcobacter sulfidicus suggesting their sulfur oxidizing potential. In addition, the congruent phylogenies of 16S rRNA and of the diagnostic marker genes aprA and dsrA strongly confirmed the sulfur oxidizing potential of the GSO cluster. Apparently both populations can efficiently oxidize sulfide, which is a so far unique example for a physiologically-constrained bacterial group catalyzing a large-scale detoxification process in the open ocean.Besides these organisms, in particular the high dsrA diversity indicated other, still unknown sulfur oxidizers in the water column. Sulfate reducing bacteria were too low to account for the observed sulfide formation. Additional abundant, usually aerobic or phototrophic organisms like the SAR11 cluster and the Roseobacterio lineage also occurred in the OMZ, but their role remains unclear. The occurrence of the GSO cluster in other OMZ such as the Peruvian upwelling system (PUS) may reflect yet unrecognized sulfur cycling in various coastal habitats. Interestingly, the comparative analysis of microbial diversity patterns of the BAG and PUS consistently displayed the highest diversity in the OMZ that suggested similar factors streamlining the community structure.Dissertation437 193 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Molecular characterization of microbial populations in methane-rich marine habitats(2009-09-18); ; ; The anaerobic oxidation of methane (AOM) with sulfate is a globally important microbial process, catalyzed by syntrophic consortia of marine anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). The main objective of this thesis was to gain further insights into AOM habitats and communities by using biogeochemical and molecular techniques.One chapter of this thesis provides first insights into the abundance and distribution of ANME and SRB at cold seep sites from the coastal margin off New Zealand, far away from all known ANME habitats. Another part describes new insights into the abundance and distribution of ANME and SRB in microbial reefs from the Black Sea shelf, the most intensively studied ANME habitats. Chloroflexi related cells were identified as additional key reef bacteria accounting for up to 28% of all cells. Another chapter deals with the identification and quantification of Verrucomicrobia in a humic lake. Further, this thesis gives insights into the quantification of ANME by real time PCR and ANME-3 genomic analysis after cell sorting by flow cytometry.Dissertation276 175 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Polysaccharide utilization loci and associated genes in marine Bacteroidetes - compositional diversity and ecological relevance(2019-03-06); ; ; The synthesis of marine organic carbon compounds by photosynthetic macroalgae, microalgae (phytoplankton) and bacteria provide a basis for life in the ocean. In marine surface waters this primary production is largely dominated by microalgae and is especially pronounced during spring phytoplankton blooms. During and after these often diatom-dominated blooms, increased amounts of organic matter are released into the surrounding waters. Here, the organic matter, rich in polysaccharides, can trigger blooms of heterotrophic bacteria. Marine members of the Bacteroidetes are consistently found related to such bloom events. These bacteria are regularly detected as the first responders to thrive after phytoplankton spring blooms in temperate coastal regions and are often equipped with a variety of polysaccharide utilization gene clusters. These gene clusters, termed polysaccharide utilization loci (PULs), encode enzymes for the extracellular hydrolysis of polysaccharides and the subsequent uptake of oligosaccharides into the periplasm, where they are shielded from competing bacteria. This mechanism allows for rapid uptake and substrate hoarding, and thus could be one reason why Bacteroidetes are often seen as the first responders of the bacterioplankton community. The investigation of the so far largely unknown diversity and the ecological relevance of PULs in marine Bacteroidetes was the major goal of the work presented here. We could show that genomes of Bacteroidetes isolates from the North Sea, with free-living to micro- and macro-algae associated lifestyles, harboured a variety of these loci predicted to target in total 18 different substrate classes. Overall PUL repertoires of these isolates showed considerable intra-genus and inter-genus, variations suggesting that Bacteroidetes species harbour distinct glycan niches, independent of their phylogenetic relationships. By investigating the PUL repertoires of uncultured free-living Bacteroidetes during three consecutive years of spring phytoplankton blooms at the North Sea island of Helgoland, I could further reveal that the set of targeted substrates during these bloom events was dominated by only five of the substrate classes targeted by the isolates. These were the diatom storage polysaccharide laminarin, alpha-glucans, alginates, as well as substrates rich in alpha-mannans and sulfated xylans. In addition to this constrained set of substrate classes targeted by the free-living Bacteroidetes community, I could show that the species diversity during these blooms was limited and dominated by only 27 abundant and recurrent species that carried a limited number of abundant PULs. The majority of these PULs were targeting laminarin and alpha-glucan substrates, which were likely targeted during the entire time of the blooms. The less frequent PULs, targeting alpha-mannans and sulfated xylans, were predominantly detected during mid- and late- bloom phases, suggesting a relevance of these two substrate classes in the later phases of phytoplankton blooms. Overall these findings highlight the recurrence of a few specialized Bacteroidetes species and the environmental relevance of specific polysaccharide substrate classes during spring phytoplankton blooms. However, for some of these substrate classes the origin, structural details and their abundance during blooms are as yet largely unknown. To further shed light on the polysaccharide niches of abundant key-players, these findings can serve as a guide for future laboratory studies.Dissertation419 183 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Spatiotemporal dynamics of the bacterial community in the German Bight(2015-11-06); ; ; Microbes comprise the most abundant and diverse group of organisms on earth, contribute substantially to every conceivable biogeochemical cycle and thus, are fundamental to ecosystem functioning. Research on marine microbial communities has proven the existence of biogeographic patterns, but the mechanisms that shape the microbial assemblages are still not understood properly, as most of the studies revealed different driving forces for different temporal and spatial scales and individual habitats. However, to predict and estimate potential ecological impacts that arise from microbial community variation, it is of utmost importance to unveil the mechanisms that are generating and maintaining community assembly. This thesis aimed at providing detailed insight into the spatiotemporal variation of bacterioplankton communities in the German Bight, to deconvolute spatial and temporal signals and to identify the main regulating forces driving these dynamics.Dissertation292 129 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Bacterial niche differentiation in Arctic sandy surface sediments(2025-02-11); ; ; ; Permeable sandy sediments cover at least half of the continental margins and are regions of high biogeochemical activity. Central to remineralization processes in sediments are heterotrophic microbial communities, which possess a vast array of metabolic capabilities that allow them to use diverse substrates, ranging from complex organic matter to simple sugars and amino acids. These benthic microorganisms colonize surfaces of sand grains, as well as the interstitial porewater. This thesis aims to improve our knowledge of carbon cycling by benthic bacterial communities using a novel fractionation method which separates cells in sandy surface sediments based on their attachment to the grains. The work focuses on high latitude environments, specifically Isfjorden, Svalbard (78°N), which experiences extreme seasonality in primary productivity with prolonged periods of continuous daylight followed by a rapid transition to months of continuous darkness. Collecting undisturbed sediment samples is the critical first step in disentangling niches and microhabitats within surface sediments. Thus, in this thesis a new small and lightweight sediment sampler, the Ellrott grab, was developed, which was designed specifically for sampling undisturbed sandy surface sediments with the porewater and overlying seawater (Chapter 2). By comparing oxygen profiles measured in situ with those in cores subsampled from the grab, I showed that the Ellrott grab causes minimal disturbance to the sample. Furthermore, a novel fractionation method that separates microorganisms in the porewater (PW) from those loosely attached (LA) and firmly attached (FA) to the grains was developed (Chapter 3). The PW and LA cells, which comprised 3% and 8-13% of cells, respectively, were significantly enriched in aerobic heterotrophs and had faster per-cell oxygen consumption and laminarin hydrolysis rates compared to the FA fraction. In contrast, the FA fraction (84-89% of cells) was significantly enriched in anaerobes such as sulfate reducers. I hypothesize that these fractions occupy distinct niches in surface sediments: the FA fraction likely consists of cells colonizing protected areas on the grain, but are more diffusion-limited. On the other hand, the PW and LA fractions are less resource-limited but abrasion and grazing may keep their cell numbers low. These differences in composition and activity could point towards distinct contributions by these fractions to benthic carbon cycling. Next, the composition and activities of the sediment fractions across polar day and polar night in Isfjorden were compared (Chapter 4). During polar day, taxa specialized in degrading high molecular weight organic matter increased in relative abundance in the PW and LA fractions. These two fractions also showed increased laminarin hydrolysis rates. While the FA fraction remained more stable, I observed an increase in sulfate reducers in this fraction during polar night. Across seasons, Woeseia and Maribacter remained abundant in the FA fraction. I propose a partitioning of the benthic bacterial community into seasonal and stable microbial guilds, wherein the PW and LA fractions comprise the seasonally responsive communities. On the other hand, the FA fraction comprises the more stable community, potentially utilizing constantly available or less labile substrates. The hydrolysis and uptake of mucin, an animal-derived glycoprotein, by the fractions across seasons was the focus of Chapter 5. During polar day, maximum extracellular hydrolysis rates in the PW and LA increased, while the maximum rate did not fluctuate in the FA. Selfish uptake (i.e., taking up oligosaccharides into the periplasm), was mainly mediated by Verrucomicrobiota and Planctomycetota, and was restricted to a maximum of 2% of the microbial community. In Chapter 6, I contextualize my findings from this thesis by presenting macro-scale observations from the station in Isfjorden. Then, I synthesize the key findings presented across the chapters, and provide a cohesive overview on how this work advances our understanding of carbon cycling processes in sandy surface sediments. Finally, I propose potential directions for future research, identifying approaches that can further investigate the remaining open questions and new hypotheses generated from this thesis.Dissertation184 110 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Bacterial community in the intertidal sediments populated by Arenicola marina, a terminal restriction fragment length polymorphisms study(2008-04-25); ; ; Sediments offer microorganisms an unexplored numbers of niches with the opportunity to evolve specialized microbial communities. The small size of microbial niches in biogeochemical gradients in sediments called for a high resolution study of the populations. We applied a genetic fingerprint method, the terminal restriction fragment polymorphism (T-RFLP), to characterize the diversity of the 16S rRNA gene present in thin sediment layers at which one TRF represents one operational taxonomic unit (OTU). A partial gene amplification and restriction enzyme digestion of the amplicon allows the detection of about 150 different fragments in an intensity range of 100 to 10000 relative fluorescence units as a picture of the richness and evenness of the bacterial community.The T-RFLP method was established for intertidal soft sediments from the Wadden Sea, the North Sea. The variations in the results were correlated to variations in individual steps of the method protocol. Restriction enzyme digest and digest analysis on a capillary sequencer correlated with a dissimilarity of about 20% and 10% in the obtained replicate datasets describing one pooled amplicon from one DNA sample after binning with a fixed window size of 0.5 and 1 base pair, respectively. The biases in individual PCR reactions did not increase the dissimilarity after performing independent T-RFLP analyses from one DNA sample. The dissimilarity was partly caused by an imperfect binning. Working with a high resolution window size of 0.5 bp, no starting point (50.25, 50.20, 50.30 and 50.65 bp) gave a perfect binning result. Some of identical TRFs were always binned into two different TRFs, thus creating an additional OTU. A window size of 1 bp with starting point 50.50 bp gave similar dissimilarities. Although our results may require an improved binning technique to utilize the full biodiversity information in the profiles, the current T-RFLP technique clearly detected the biological variation in adjacent small sediment layers and can be used to characterize the microbial community in individual sediment layers. Eukaryotes offer and create a number of niches. The lugworm Arenicola marina is a bioturbator in marine intertidal sediments. The T-RFLP method was applied to investigate the bacterial community in the burrow of the lugworm A. marina. The U-shaped burrow is divided into three compartments: the vertical head shaft tube through which the surface sediment is sinking down and ingested by the lugworm, the horizontal gallery tube at where the lugworm relatively stays permanent inside the sediment and the vertical tail shaft tube through which the lugworm does defecation by moving backward until the tail reaches sediment surface and ejects characteristic fecal cast on the sediment surface. In the bulk sediment surrounding the U-shaped burrow, the sediment contained a number of different bacterial communities changing with depth. On the basis of an aerobic layer, a redox potential discontinuity (RDP) layer and an anoxic layer, the decreasing and the increasing TRFs over depth may represent surface and subsurface layer bacteria respectively at 0-2 cm and 2-10 cm depth. T-RFLP data suggested that the RDP layer is at 3-5 cm sediment depth, because the unique TRFs of the surface layer and subsurface layers were not found at this depth and the change of abundance of TRFs was fast. The T-RFLP analyses clearly grouped the microbial population in the head shaft tube with sediment surface populations. The tail shaft tube was populated by different populations; close to the surface dominated by the surface bacteria and below 3 cm dominated by the subsurface bacteria. The populations in the gallery tube were similar to those in the head and tail shaft tube. The richness in the gallery tube was the lowest but had the highest evenness. T-RFLP analyses of two mm-thick sediment layers from areas with A. marina and without A. marina also revealed a strong depth-dependence of the surface bacterial community composition. According to the T-RFLP analyses, the presence or absence of A. marina had no clear detectable influence on the microbial populations in the top two centimeter of sediment. Most likely, the increase presence of other burrowing animals in the A. marina exclusion areas seems to form highly similar biogeochemical environments for the development of bacterial communities.Dissertation319 87 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Polysaccharide utilization by bacteria associated with micro- and macroalgae(2024-05-02); ; ; ; Algae play a crucial role in marine carbon cycling. This involves various polysaccharides of different functions to which algae convert a significant proportion of their photoassimilated organic carbon. Suitable conditions provided, microalgae (phytoplankton) can proliferate very fast. During such blooms, substantial quantities of dissolved and particulate polysaccharide-rich organic matter are released to the environment, both by exudation of living and by decomposition of dead algal cells. While research on the community and functional dynamics of free-living bacteria during phytoplankton blooms is extensive, knowledge about particle-attached bacteria, especially regarding taxonomic composition and associated gene functions, is limited. Like planktonic microalgae, sessile macroalgae are also rich in polysaccharides and harbor bacteria with significant potential for polysaccharide degradation, particularly for complex polysaccharides. However, research on macroalgal phycosphere microbes so far primarily focused on community composition studies via 16S rRNA gene amplicon sequencing and (meta-)genomic analysis of single algal species or a limited number of samples. This thesis provides a comprehensive exploration of microalgae-associated microbial communities during phytoplankton blooms and of macroalgae-associated phycophere communities, both with a focus on polysaccharide utilization functions. Chapter I establishes the theoretical foundation of this thesis. It starts out with a review of the critical roles that micro- and macroalgae play within the marine carbon budgets. It then narrows down to explore phytoplankton blooms, key events in marine carbon cycling marked by rapid carbon fixation, remineralization, and release. The studies of marine particles are summarized. Afterwards, common polysaccharides of micro- and macroalgae are introduced, as well as the mechanisms of bacterial polysaccharide utilization. Then research methodologies in microbial ecology are outlined with a focus on the analysis of omics data, which form the core methodologies in the two subsequent chapters, and finally the chapter closes with a summary of the research questions that this thesis aims to address. In Chapter II, I present a study in which I explored dynamic shifts in community composition and polysaccharide degradation functions of particle-attached bacteria throughout an extensive phytoplankton bloom in contrast to those of free-living bacteria. Bloom progression was captured using microscopic, chlorophyll a and 18S rRNA gene amplicon data at high temporal resolution. I identified abundant bacterial clades using corresponding high-resolution 16S rRNA gene amplicon sequencing across three filter size fractions during the bloom. Through metagenome data analysis using both short- and long-read sequencing, I then identified and explored abundant polysaccharide degrading bacteria. Additionally, metaproteome data were analyzed to correlate bacterial proteins with the breakdown of algal glycans. The combined results indicated that, similar to their free-living counterparts, particle-attached polysaccharide-degrading bacteria targeted soluble and structurally simple polysaccharides such as laminarin. However, they also possessed abundant genes dedicated to the degradation of insoluble and structurally complex polysaccharides, setting them apart in their functional capabilities. As detailed in Chapter III, I had a major part in analyzing the composition and polysaccharide utilization functions of macroalgal phycosphere bacteria on four types of macroalgae across the four seasons. Comparisons were made with microbial communities in surrounding seawater and sediment, revealing fourteen core genera consistently present on all algae. Metagenome analysis focused on polysaccharide degradation and secondary metabolite production, while cultivation techniques yielded pure isolates for draft genome sequencing. A high cultivability of macroalgal phycosphere bacteria enabled sequencing of numerous isolates, offering insights into a manifold of polysaccharide utilization loci and their possible polysaccharide substrates. Chapter IV presents a comparative analysis of particle-attached bacteria originating from microalgae versus those from macroalgal phycospheres, a pioneering endeavor not previously undertaken. This way, I identified dominant bacteria shared between both studied communities, alongside unique clades specific to each community. Furthermore, the advantages of utilizing PacBio metagenome sequencing, drafting genome sequences of cultivable strains, and the application of multiple databases for functional annotation are discussed, including insights to enhance the study of particle-attached bacteria and isolated strains. The thesis then closes with an outlook with a proposal for future projects.Dissertation844 200