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Microbial insights into ocean alkalinity enhancement: Bacterial community risk assessment and the benefit of increasing research on carbonic anhydrase

Dominik Antoni — Thu, 11 Dec 2025 12:52:01 +0100

Climate change driven by anthropogenic CO₂ emissions requires effective mitigation strategies. Negative emission technologies (NETs), particularly ocean alkalinity enhancement (OAE), are promising because they increase ocean alkalinity and promote CO₂ sequestration. This dissertation examines how marine molecular biology can help assess ecological risks and the overall efficacy of OAE. It presents two risk assessments on bacterial community responses to alkalinity exposure and develops a framework for a novel biological proxy for monitoring, reporting, and verification (MRV) in OAE. Chapter 1 provides a general introduction. Chapter 2 investigates how gradually increased alkalinity affects pelagic bacterial communities using a mesocosm experiment with 16S rRNA gene sequencing and flow cytometry. Results show high structural resilience, but quantitative shifts in bacterial abundance linked to phytoplankton dynamics indicate indirect ecological effects of OAE. Chapter 3 expands this work by comparing two OAE strategies: olivine dissolution and direct dissolved alkalinity addition. A mesocosm experiment assessed microbial responses in seawater and oyster gills (Ostrea edulis). Olivine increased pollution-tolerant and biofilm-forming taxa, while dissolved alkalinity caused minimal change. These findings suggest that dissolved alkalinity below 500 µmol L⁻¹ is a relatively safe OAE approach. Chapter 4 proposes carbonic anhydrase (CA), a key enzyme in marine carbon cycling, as a biological proxy for evaluating OAE performance. Structured hypotheses outline how CA expression and activity assays could support future OAE MRV systems. The chapter recommends shifting resources from broad bacterial community assessments toward investigating how alkalization affects CA.

Unveiling Small Microplastics from European Waters to the Arctic: Surface Water to Deep Sediment and Reflections on Data Representativeness

Fangzhu Wu — Tue, 10 Dec 2024 12:52:31 +0100

Since the invention of the first synthetic polymer in 1907, plastics have revolutionized industries but have also caused significant environmental challenges. Over 170 trillion plastic particles are estimated to float in the world's oceans. Once in the marine environment, these plastics fragment into smaller particles (microplastics, MPs, <5 mm) under environmental forces, contaminating ecosystems worldwide, including the remote Arctic. This thesis investigates the Norwegian Coastal Current (NCC), a key transport route for MPs from northern Europe to the Arctic Ocean. Using novel sampling devices and advanced micro-Fourier transform infrared microscopy (μFTIR), small MPs (SMPs, 11–300 μm) were analyzed in seawater and sediments, providing the first detailed assessment of their spatial and temporal distribution in the NCC. The results reveal the prevalence of SMPs from surface seawater to deep sediments, including layers deposited before the advent of plastics. Key findings include a relatively homogeneous horizontal distribution of SMPs in surface and subsurface seawater and significant variability in sediment concentrations (54–12491 MP kg⁻¹) across cores. SMP accumulation trends in post-1950 sediment layers varied, challenging their reliability as markers of the Anthropocene. A total of 21 polymer types were identified, with smaller size classes dominating, highlighting their ecological significance. Further analysis of data representativeness revealed significant variability in MP concentrations and polymer diversity across stations, emphasizing the need for standardized protocols to ensure reliable data. Despite being based on a single research cruise, this study provides a valuable snapshot of SMP distribution in the NCC. The findings critically evaluate current MP research practices and highlight the need for robust methodologies to improve the reliability of future studies.

Microbial physiology of nitric oxide-transforming microorganisms

Paloma Garrido Amador — Tue, 19 Nov 2024 14:48:15 +0100

Nitric oxide (NO) is a small gaseous molecule with important functions in cell biology and atmospheric chemistry owed to its unique physical and chemical properties. Since its relevance in biology was established, research on NO has focused primarily on its roles as signaling molecule, cytotoxin, and metabolic intermediate. Indeed, as a free radical and highly reactive compound, NO is as a potent toxin that can inhibit microbial growth, however it also has a central position in the microbial nitrogen cycle as a key intermediate in processes such as denitrification, aerobic ammonia oxidation, anaerobic ammonium oxidation, and nitrite-dependent anaerobic methane oxidation. Additionally, NO is a very energy-rich molecule with a high redox potential (NO/N2O; E0’ = +1.175 V) and it may have played a key role in the evolution of life on early Earth and the bioenergetic pathways related to modern denitrification and aerobic respiration. During recent years, we have been presented with new roles of NO in the nitrogen cycle. It appears as if the focus of NO research has slowly started to change its course as we begin to recognize its potential as direct substrate for microbial growth. Given its important roles in past and present microbial life, we believe that there must be a plethora of microorganisms that are capable of growing on NO conversions. Therefore, the main goal of my PhD project was to challenge our understanding of NO as mere toxin and intermediate, and investigate its potential as direct energy source for microbial life, whether it is through known or novel biochemical reactions, and the microorganisms that use it for this purpose, using a combination of continuous and batch incubations, physiological experiments, and multi-OMIC analyses.

Physiology and genomics of new marine methane-oxidizing bacteria

Margarita Kniaziuk — Tue, 22 Oct 2024 11:18:52 +0200

Methane is the most abundant hydrocarbon on Earth, and plays a vital role in the global carbon cycle. In marine ecosystems, large quantities of produced methane are oxidized by methane-oxidizing microorganisms before it reaches the atmosphere. Aerobic methanotrophic bacteria, which consume methane in the upper oxic layers of marine sediments and the water column, represent the final oceanic methane filter. To date, the majority of marine aerobic methanotrophs remains uncultivated, with currently only nine formally described cultures. This hinders our understanding of their physiology that ultimately controls their activity and affects the dispersal of these methanotrophs in nature. In order to fill this gap in knowledge, the present work was focused on the isolation and characterization of marine methanotrophs from the North Sea and the Western Scheldt estuary sediments. The obtained methanotrophic cultures were investigated in physiological tests, and their metabolism was reconstructed based on high quality genomes. The isolation of four new methanotrophic species affiliated to the genera Methyloprofundus and Methylomarinum allowed to determine specific ecophysiological preferences and key conserved and distinct features within these genera. The isolate of Methylomarinum sarcina B3 exhibited a sarcina-like cell organization, which has not been previously reported for any marine methanotroph. The unusual Embden-Meyerhof-Parnas pathway identified in the new Methyloprofundus spp. could potentially serve as an alternative to the canonical glycolytic route. Finally, the discovery of a new putative nitrate reductase in Methyloprofundus spp. could have important implications for the understanding of the diversity of bacterial nitrate reductases and anaerobic respiration. Altogether, this work has advanced the characterization of the Methyloprofundus and Methylomarinum genera and laid the foundation for future research in microbial carbon and nitrogen metabolism.

Marine microbes on the map: Defining spatial scales of functional microbial biogeography in the ocean

Cora Hörstmann — Mon, 30 May 2022 12:10:22 +0200

Marine microorganisms have markedly great functional and phylogenetic diversity and sustain major elemental cycles, including those of carbon and nitrogen. However, a major challenge in microbial observation is that the spatial scales of microbial biodiversity patterns and microbial activity differentially change within their physical oceanographic environment, which requires sampling across multiple scales. In this thesis, I applied a combination of metabarcoding (16S and 18S rRNA gene sequencing) and stable isotope C and N2 fixation measurements of surface ocean samples (0 - 40m) against the backdrop of chemical (dissolved inorganic nutrients, particulate organic matter) and physical (temperature, salinity, and surface currents) environmental variables in the Atlantic, Indian and the Arctic Ocean. I demonstrate how functional activity can be decoupled from phylogenetic diversity. I show that beta diversity patterns generally reflect ocean provinces and can also be used to refine oceanographic boundaries. In a pan-Arctic study, I show how microbial communities disperse and form regional and within-fjord signals, with different co-occurrence patterns between fjords with and without marine-terminating glaciers. The presented calculations of a productivity-specific length scale can help identify sample patchiness and scale sample diversity in relation to marine ecosystem structure. In order to harmonize research in meta-analyses and across global scales, we provided perspectives on best practices in method documentation. In conclusion, my work helps to better understand pelagic microbial ecosystems, taking into account the patchiness and ecosystem boundaries and their impact on productivity and food web interactions that are typically overlooked in marine microbial ecology. The presented approaches will support mapping microbiomes to relevant oceanographic scales and have potential implications for researching, observing, and monitoring marine ecosystem structures.

The Impact of Environmental Changes on the Microbial Community Dynamics and Abundance of Pathogenic Vibrio species in Coastal Ecosystems

Candice Thorstenson — Wed, 31 Mar 2021 09:59:15 +0200

Coastal ecosystems are highly dynamic regions and extremely important to the livelihoods and health of the people living in coastal regions. Tourism, the aquaculture industry, and human health are all directly tied to the microbial community in these regions. Furthermore, many key aquaculture stocks, such as oysters, may serve as vectors for a variety of foodborne diseases in humans, both at the harvesting area and when imported far inland. Rising sea surface temperatures in the North Atlantic and North Sea have been heavily implicated in the spread and rising abundance of the human pathogenic Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in North America and Northern Europe. This study investigates how environmental changes may impact the growth and behavior of these key Vibrio species within the context of the surrounding microbial community. The genetic accessibility of V. cholerae, V. parahaemolyticus, and V. vulnificus environmental strains was initially assessed, and this work resulted in the development of new genetic tools and a rapid fluorescent protein-labelling protocol which worked universally in all tested strains. In a small-scale microcosm investigation, red fluorescent protein-labelled Vibrio clones were co-incubated with a native microbial community under three discrete temperatures. These microcosm experiments indicated that increased temperatures alone do not confer greater competitive fitness to these Vibrio species. Finally, through environmental field sampling for pathogenic Vibrio, two discrete pathogenic Vibrio populations in the North Sea and the North Atlantic, where identified and further characterized, allowing for future speculation on the role these species may play in their respective regions.

Potentially pathogenic Vibrio species in the German Bight, North Sea

Sidika Hackbusch — Mon, 30 Nov 2020 16:10:08 +0100

Vibrio spp. are ubiquitous bacteria, common to estuaries and coasts. V.parahaemolyticus, V.vulnificus and V.cholerae are the main water-related pathogenic species, able to cause serious gastroenteritis, wound infections or septicaemia. Infection cases have become more frequent in northern temperate waters, attributed to climate change related events. Little is known about pathogenic Vibrio spp. in the German Bight. Occurrence, abundance and pathogenicity of V.parahaemolyticus, V.vulnificus and V.cholerae were investigated in a salinity gradient of the German Bight over 14 months. Seasonal patterns with increased abundances during summer were detected, while extended periods of warm seawater coincided with prolonged Vibrio spp. occurrences in the German Bight. Temperature and nitrite were the significant factors explaining variations in Vibrio spp. abundances. This study revealed that environmental human pathogenic Vibrio spp. comprise multiple virulence-associated genes in the German Bight, especially in estuarine regions. Pathogen growth potentials of clinically relevant V.vulnificus and V.cholerae strains were investigated in the German Bight to determine the extent of in vitro growth in a broad range of physico-chemical conditions of surface waters originating from a salinity gradient. Those strains, despite revealing different growth patterns, are capable of growth in most seawater samples under ambient physicochemical conditions. Growth kinetics showed strong temperature dependency when grown in seawater, while no salinity dependency was detected. Potentially pathogenic V.parahaemolyticus was detected on marine microplastics for the first time, evidencing that microplastics can serve as additional surfaces for attachment and function as vectors for the enrichment and dispersal. Given the future predictions of climate variability, a species-specific monitoring and risk assessment regarding potentially pathogenic Vibrio spp. in the German Bight is crucial.

Viruses in the North Sea: viromics and prophage genomics

Alexa Garin Fernandez — Thu, 14 Nov 2019 14:34:09 +0100

Despite their small size, viruses have an enormous influence on microbial population dynamics, due to lysis and horizontal gene transfer. Due the high abundance of their hosts, bacteriophages or phages comprise the majority of viruses and provide the largest reservoirs of unexplored genetic diversity in marine environments. The rise of Next Generation Sequencing (NGS) techniques brings new opportunities to investigate the marine virus community. However, there is no current statutory pipeline applied in marine phage ecology. Therefore, this thesis proposes a virus-specific pipeline based on the integration of existing tools and state of the art techniques. The developed pipeline was applied to accomplish the two research aims of this thesis: (1) to analyze the virus community in the North Sea with viromics(virus metagenomics), and (2) to characterize lysogenic phages from potentially pathogenic Vibrio species. The results of the first part of this thesis show that the virus community is dominated by phages and they are not evenly distributed throughout the North Sea. In general, the coastal virus community was genetically more diverse than the open sea community. The influence of riverine inflow and currents affects the genetic virus diversity with the community carrying genes from a variety of metabolic pathways and other functions. In the second part, lysogenic Vibrio phages from the North Sea were induced (ca. 40 % of tested isolates) and four phage genomes were characterized. The phages from V. parahaemolyticus (2 tailed phages, 1 filamentous phage) and V. cholerae (1 tailed phage) can integrate into their host genome and might have a role in pathogenicity. This thesis represents an exemplary study of the virus community in the North Sea, with special emphasis on the marine phages. The settled virus-specific pipeline the obtained insights will contribute to extend the study of the virus diversity dynamics in other marine areas to characterize novel phage groups.

Insights into the interaction of Marinobacter adhaerens with the diatom Thalassiosira weissflogii by comparative mutant analysis

Veronika Will — Tue, 09 Jul 2019 16:08:16 +0200

Interactions between diatoms and heterotrophic bacteria play an important role in the marine biological pump. Diatoms form marine snow aggregates that are used by heterotrophic bacteria as rich nutrient sources. A model system consisting of the γ-proteobacterium, Marinobacter adhaerens HP15, and the diatom, Thalassiosira weissflogii, was used to study diatom-bacteria interactions on a molecular level using various mutants of M. adhaerens HP15. The assessment of the zinc-sensitive mutant HP15 ΔczcCBA.1/2 in diatom co-cultures with and without zinc stress showed that heavy metal resistance helps bacteria to colonize aggregates. Furthermore, addition of ZnSO4 increased the release of exopolymers by M. adhaerens HP15 and led to a higher rate of bacterial aggregate colonization by both, the wild-type and the mutant. Co-cultivation of the non-motile mutant ΔfliC and the non-chemotactic mutant ΔcheA showed that at high exopolymer concentrations motility is more important for bacterial attachment to aggregates than chemotaxis. Proteomic analysis suggested that amino acids present in these aggregates are the preferred nutrient source for M. adhaerens HP15. Amino acid quantification confirmed the presence of especially branched chain amino acids in the attached fractions of co-cultures suggesting that the diatom contributes to the amino acid pool therein. The role of branched chain amino acid uptake for M. adhaerens HP15 was studied with the mutant ΔlivK. M. adhaerens HP15 has five livK genes that are differentially expressed during in vitro growth. The studied livK gene plays only a minor role during in vitro growth despite its previously suggested importance for amino acid uptake in vivo, hinting towards specialized gene expression under different growth conditions. Overall, this study gives important insights into diatom-bacteria interactions on a molecular level and in terms of exchanged nutrients, dynamics of exopolymer release and bacterial aggregate colonization.

On the feasibility to engage heterogeneous communities in data gathering, sharing and enrichment

Julia Schnetzer — Fri, 12 Aug 2016 10:52:54 +0200

Marine microbes play critical roles in the well being of the planet Earth and all its inhabitants. Not only do they influence chemical cycles, the marine food chain, but also the whole atmosphere and climate of our planet. However, the field of marine microbiology is still in its infancy and there is much more waiting to be explored. Here, I present a new approach to investigate global marine microbial diversity and function on a single day of the year, the 21st of June 2014/2015: the Ocean Sampling Day (OSD). The collection of a simultaneous, global dataset, required marine researchers, worldwide, to be connected. The aim was not only to create a snap shot of the marine microbial diversity fixed in time, but also to raise awareness amongst the general public of the important role these tiny organisms play in our daily lives. Therefore, professional scientists as well as the non-scientific public were invited to join the corresponding citizen science project, MyOSD. They supported OSD by providing oceanographic measurements and even microbial samples. Data collected by citizen scientists were validated and show that citizen science can contribute valuable data to marine research. A special focus was set on additional environmental measurements such as water temperature. This contextual data is important for the interpretation of microbial diversity in any given sample; however, it is still not common practice in marine microbial research to measure or report contextual data; OSD aims to make scientists more aware of this problem. Extracting contextual data after a dataset or article has been published, is onerous work. Hence, I present two new tools to extract environmental information and geographic locations from scientific literature. The text mining tool, ENVIRONMENTS, automatically annotates scientific text with terms from the Environmental Ontology (EnvO). The PubMap application utilizes the power of the crowd to enable the creation of a manually curated database of georeferenced scientific publications. Overall, this thesis shows that enabling collaboration within the scientific community as well as the non-scientific public, leads to achievements not only in gathering of new datasets, but also in enhancing present and historic scientific literature.

Identification and Characterization of Vibrio species

René Erler — Fri, 04 Mar 2016 13:02:37 +0100

Potentially pathogenic Vibrio spp. in the marine environment are regarded to as emerging health risk in Europe, particularly infections caused by V. parahaemolyticus, V. vulnificus and non-O1/non-O139 V. cholerae strains. Thus, the inclusion of Vibrio spp. in surveillance programs and studies about the distribution of Vibrio spp. are urgently needed. This thesis contributes both: the evaluation of Matrix-assisted laser / desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as tool for species identification in environmental samples and the analysis of spatial patterns of Vibrio spp. populations in the North and Baltic Seas. Regarding MALDI-TOF MS, the specific reference database VibrioBase was developed. VibrioBase reveals Vibrio spp. identification agreements of 96.9% compared to the approved rpoB-sequence-based identification method as well as higher identifications scores and a better discrimination for closely related Vibrio spp. compared to the general BiotyperTM database of the manufacturer. Thus, the cost-effective and rapid MALDI-TOF MS method achieves accurate Vibrio spp. identification when used in combination with VibrioBase. In addition, single mass peaks were used to allow species identifications based on sensitive peaks (SIBOPS). The SIBOPS approach achieves 98% identification agreements with the conventional MALDI-TOF MS / VibrioBase system and is independend of the respective mass spectrometric eqipment and software tools. Furthermore, mass spectrometric biomarkers were found, that might be used for the direct identification of Vibrio spp. in environmental samples. Using ERIC-PCR genotyping, general patterns were not observed for the distribution of V. cholerae, V. parahaemolyticus and V. vulnificus in the North and Baltic Seas. Particularly the latter two species were separated into distinct regions, which are however not linked to geographical aspects and, to a minor extent, are asscociated with environmental parameters such as salinity. In consequence, MALDI-TOF MS was confirmed as a promising tool for species identifications from environmental samples. VibrioBase and SIBOPS are important steps towards the implementation of Vibrio spp. in surveillance programs executed by health authorities. Future reseach has to be done to acquire knowledge about ecological aspects of Vibrio spp. in temperate waters, in particular the linkage to plankton species in the North and Baltic Sea and the influence of bacteriophages for shaping Vibrio spp. populations.

Ecological aspects of the marine planctomycete Rhodopirellula baltica SH1T : a whole genome array approach

Patricia Wecker — Tue, 19 Jan 2016 11:34:54 +0100

Since the first microbial genome has been finished in the year 1995 more than 800 sequenced microorganisms are publicly available and many more are on their way. Bioinformatic analysis of the sequence information is required to process this huge amount of data and to generate sound hypotheses in terms of physiology and ecology of an organism as well as to efficiently screen for candidate genes for biotechnological relevant enzymes.However, high-throughput lab-methods are more than ever required for the subsequent verification of initial bioinformatic findings and hypotheses. Post-genomic approaches like DNA microarrays have revolutionised molecular biology by enabling a new dimension of parallelism in expression profiling. Eight years ago, the Max Planck Institute for Marine Microbiology has initiated a genomics project for the complete genome sequencing and analysis of environmental relevant marine bacteria. Rhodopirellula baltica was selected as a marine representative of the globally distributed and environmentally important bacterial phylum Planctomycetales. It is a model organism for aerobic carbohydrate degradation in marine systems, where polysaccharides represent the dominant components of biomass. The complete genome sequence of R. baltica has been determined and functional annotation was performed. However, pure in silico predictions left about 55% of the genes without functional assignment. The availability of the whole genome sequence has already stimulated a set of follow-up studies ranging from proteomics to protein crystallization as well as biotechnological applications. This work presents the establishment of, and the results gained with, the first whole genome microarray for a Planctomycete. It shows that massive expression profiling under defined environmental conditions can take the challenge to bridge the gap between classical studies on the cell biology and physiology of Planctomycetes and their active genetic repertoire.

Interaction of the Marine Bacterium Marinobacter adhaerens HP15 with the Diatom Thalassiosira weissflogii Analyzed by Proteomics Approaches

Antje Stahl — Mon, 18 Jan 2016 11:33:17 +0100

Bacteria and microalgae conduct close mutualistic interactions that might impact metabolic and physiological features in both partners. As microalgae play major roles in global primary production and carbon cycling in marine systems, knowledge about such interactions is directly linked to an understanding of large-scale carbon and nutrient turnover processes. The impact of bacteria that colonize microalgae has been the focus of a number of studies. However, an understanding of the basis of such interactions at an advanced molecular level, including the identification of ‘traded items’ between the interacting partners, remained scarce. In the present study, we analyzed a specific beneficial bacteria-diatom interaction by application of proteomics approaches. A bilateral model system formerly established for the analysis of such interactions was used, consisting of the marine γ-proteobacterium Marinobacter adhaerens HP15 and the ubiquitously occurring centric diatom Thalassiosira weissflogii. The present study focused on the proteome of M. adhaerens HP15. Alterations in the bacterial proteome during co-cultivation with the diatom in comparison to a reference treatment were identified. Results imply that free amino acids are probable those traded items provided by the diatom, taken up by the bacterium, and potentially used as carbon and nitrogen source. Furthermore, proteomic results indicated a favorable supply with nutrients, particularly with nitrogen. For prospective studies, we suggest including the analysis of the diatom’s proteome and recommend the application of proteomics in combination with metabolomic approaches. The thesis additionally dealt with aspects of chemotaxis, combining chemosensing and motility, in the context of bacterial colonization of marine aggregates and microalgae. As a further topic, features of heavy metal resistance in M. adhaerens HP15 and other members of the genus Marinobacter were analyzed.

Identification of bacterial genes required for diatom-bacteria interactions : Gene expression analysis in Marinobacter adhaerens HP15 interacting with Thalassosira weissflogii

Ingrid Lisbeth Torres Monroy — Thu, 07 Jan 2016 16:00:37 +0100

Aggregate formation in form of marine snow is an essential mechanism in the oceans that mediates the sinking of organic carbon to depth. Interactions between bacteria and diatom play an important role during this process by inducing secretion of different polymers, which increase the size of marine aggregates. Not much is known about the molecular mechanisms responsible for the diatom-bacteria interaction. To address this issue, a bilateral model system consisting of Marinobacter adhaerens HP15 and the diatom Thalassiosira weissflogii has been established. This bacterium specifically attaches to T. weissflogii cells thereby increasing its aggregation and inducing an increased formation of transparent exopolymeric particles. A genetic tool system was developed for M. adhaerens HP15, in which successful expression of reporter genes revealed useful tools for gene expression analyses. In addition, several bacterial genes potentially important during the interaction have been investigated. However, genes specifically expressed in vivo are still unknown. In this work we identified bacterial genes that are induced during the interaction with T. weissflogii by two different approaches. First, an In vivo expression technology (IVET) screening was conducted, constructing a promoter-trap vector containing a fusion between a promoterless selection marker gene and a reporter gene. Generation of a library of plasmids carrying genomic fragments upstream of the fusion and its subsequent transformation into a selection marker mutant allowed the selection of bacterial promoters specifically expressed during the interactions with T. weissflogii. Second, bacterial proteins expressed in response to the presence of T. weissflogii were identified by comparison of protein profiles of bacterial cultures incubated with or without diatom cells and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). By these approaches several genes expressed during the co-cultivation with diatoms were identified. Sequence analyses of these genes showed that they are required for central intracellular metabolism, cell envelope structure, nutrient scavenging, regulation, chemotaxis, secretion, stress response and DNA transfer. These genes may play an important role during the interaction between M. adhaerens HP15 and T. weissflogii. The results obtained in this study contribute to a better understanding of the molecular and biochemical mechanisms responsible for diatom-bacteria interactions. Additionally, the tight adherence (tad) gene locus present on the 187-kb HP15 plasmid and consisting of 9 genes (flp, rcpCA, tadZABCDG), was analyzed. This locus is found in several Gram-negative bacteria encoding for a type IVb fimbrial low-molecular-weight (Flp) pilus, which plays a role in adherence and biofilm formation. The identification of a constitutively active promoter upstream the flp gene of M. adhaerens HP15 suggested the tad locus is transcribed. A flp-rcpCA deletion mutant was generated and analyzed in terms of its motility and attachment to abiotic and biotic surfaces. Under the experimental conditions tested, the mutant did neither show a phenotype in terms of surface attachment nor motility. However, the preliminary results of the current study encourage an in-depth analysis of the role of the tad locus in M. adhaerens HP15.

A bilateral model system for the molecular investigation of diatom-bacteria interactions

Astrid Gärdes — Thu, 17 Dec 2015 16:08:21 +0100

Diatom blooms are frequently terminated by aggregation of algae and subsequent sedimentation in form of marine snow. Transparent exopolymer particles (TEP) are produced by phytoplankton or bacteria and play an important role in aggregate formation. This study was aimed at analyzing the interactions between bacteria and phytoplankton and their influences on TEP formation, aggregation dynamics, and sedimentation of phytoplankton. For this, an in vitro model system consisting of the diatom Thalassiosira weissflogii and the marine Gammaproteobacterium Marinobacter adhaerens sp. nov. HP15 was established and was analyzed in rolling tank experiments. From the results it could be concluded, that interaction of M. adhaerens with T. weissflogii increased aggregate formation and particle sinking. Detailed investigation of this interaction revealed differences in the response of T. weissflogii, towards bacteria, namely diatom growth and exopolymer production. A careful interpretation of these results suggested a close and specific interaction of HP15 with the diatom in a mutualistic relationship. The type of interaction shifted to commensalism during nutrient stress conditions. Depending on the nature of M. adhaerens - T. weissflogii interactions the extent and quality of algal exudation in form of TEP dramatically changed and consequently influenced aggregate formation. Another part of this thesis focused on the chemotaxonomic and genomic characterization of M. adhaerens as the bacterial counterpart of the established model system. In this context the bacterial genome sequence was determined and a genetic system for molecular work has been established. A site-directed deletion mutation of the flagellin-encoding gene fliC of HP15 was generated and confirmed the suitability of M. adhaerens as a genetically accessible model organism. With this bilateral model system being generated, a species-specific interaction can now be intensively and mechanistically studied in depth.

A Bacteria-Plant Model System to Study Nitrogen Fixation in Mangrove Ecosystems

Maria Gabriela Alfaro Espinoza — Fri, 11 Dec 2015 13:50:33 +0100

Mangrove ecosystems are highly productive and rich in organic matter. However, they are considered low-nutrient environments, being nitrogen one of the main nutrients limiting mangrove growth. Nitrogen-fixing bacteria are one of the main inputs of nitrogen to these forests. High nitrogen fixation rates have been detected in mangrove sediments and roots. Moreover, studies indicated that the relationship between diazotrophs and mangroves might be mutualistic. Several diazotrophs from mangrove roots and the associated rhizosphere have been isolated or identify by phylogenetic studies of the nitrogenase-coding nifH gene. However, our knowledge about the molecular signals and cellular mechanisms that govern diazotroph-mangrove interactions is scarce. Thus, in this thesis a diazotroph-mangrove model system was established to better understand the importance of this interaction for the ecosystem and how environmental changes could impact this organismal interplay. For this, nitrogen-fixing bacteria were isolated from mangrove roots. Moreover, root colonization pattern of the selected nitrogen fixer and the impact of some of the environmental factors that could affect its nitrogen fixation were investigated. The first result showed that the diazotroph M. mangrovicola Gal22 was a prominent candidate for the establishment of the model system. This bacterium was shown to possess all features and requirements needed for being a representative model organism. Thus a detail phenotypic and genotypic characterization of strain Gal22 was conducted. A strong mangrove root colonization was observed by M. mangrovicola in the absence of carbon and nitrogen sources, suggesting that root exudates were instrumental in establishing the interaction. Nitrogen fixation by this diazotroph was increased in the presence of the roots, supporting the above results. Finally, changes in environmental factors such as temperature and salinity had a rather minor impact on nitrogen fixation by M. mangrov

Taxonomic and genetic analysis of Marinobacter sp. HP15 and its chemotactic behaviour towards the diatom Thalassiosira weissflogii

Eva Käppel — Fri, 04 Dec 2015 15:00:21 +0100

Aggregation of micro-algae, mainly of diatoms, is an important process in marine pelagic systems leading to the sinking of particulate organic matter in form of marine snow. This process has been studied extensively, but the specific role of heterotrophic bacteria, their genes, gene products, and secondary metabolite signals for this process has largely been neglected. A bilateral model system consisting of the diatom Thalassiosira weissflogii, and the bacterial strain, Marinobacter sp. HP15, was found suitable for an in-depth molecular analysis by attachment assays, TEP production determination, and aggregation experiments. Following the taxonomic and genomic description of the bacterial model strain, its genetic accessibility was demonstrated and the interaction has been studied by molecular and bioinformatics investigations. Chemotaxis- and motility-deficient mutants were generated and their phenotypes were described in appropriate assays in order to investigate the particular role(s) of these processes in diatom-bacteria interactions. HP15 was attracted to diatom homogenate in soft agar assay and chemotaxis-deficient mutants showed a decreased ability to attach to T. weissflogii supporting the importance of chemotaxis in diatom-bacteria interactions. These and future results based on the model system will improve our understanding of bacteria-phytoplankton interactions and how they influence the flux and cycling of carbon during marine snow formation.

Vibrio spp. in the German Bight

Sonja Oberbeckmann — Thu, 03 Dec 2015 17:35:45 +0100

Pathogenic Vibrio strains represent an increasing health issue in European waters, but profound investigations on Vibrio spp. are lacking, especially in northern European waters. This thesis represents a pilot study for the German Bight, investigating the Vibrio community extensively, with special emphasis on V. parahaemolyticus. Insights into abundance, community composition and response to environmental parameters of Vibrio spp. at Helgoland Roads (North Sea, Germany) were obtained. Using a cultivation-independent approach, Vibrio numbers up to 3.37 x 104 cells x mL 1 were detected at Helgoland Roads. Targeting solely potentially pathogenic Vibrio spp., numbers up to 4.3 x 103 N x L-1 (water, plankton) and 240 MPN x g 1 (shellfish) were quantified using a selective cultivation approach. In the course of this thesis, a broad range of microbiological and molecular biological methods have been evaluated regarding the usefulness for the investigation of Vibrio spp. and the potentially pathogenic species V. parahaemolyticus. A reliable, cost- and labor-effective approach was defined including (i) the sequencing of the rpoB gene (encoding the RNA polymerase β-subunit) to differentiate even closely related Vibrio species, (ii) the use of the toxR gene as specific marker for V. parahaemolyticus, (iii) the screening for the virulence-associated genes tdh and trh, and (iv) the performance of genomic fingerprinting using ERIC-PCR to identify the intraspecies variability of V. parahaemolyticus and its close relative V. alginolyticus at Helgoland Roads. Applying this polyphasic approach, we gathered insights into the Vibrio community at Helgoland Roads. Vibrio strains were isolated from seawater, plankton and shellfish and the species V. parahaemolyticus appeared to be the second most abundant species at Helgoland Roads, behind its close relative V. alginolyticus. None of the V. parahaemolyticus strains carried the virulence-associated genes tdh or trh, but several strains were proven positive for hemolytic activity. Based on genomic fingerprinting of V parahaemolyticus and V. alginolyticus strains, three distinct groups became evident. One group represented V. parahaemolyticus, the second one V. alginolyticus. The third group was located in between the two species, displaying the high genomic plasticity and intraspecific variability of Vibrio spp.. To be able to estimate the distribution and dynamics of Vibrio spp. in response to environmental conditions, we assessed the effects of environmental parameters on the Vibrio community. High temperature in combination with events of low salinity and high abundance of phytoplankton led to increased Vibrio numbers at Helgoland Roads. The influence of the parameters, however, varied between the seasons. The population of the potentially pathogenic species V. parahaemolyticus at Helgoland Roads appeared to be structured by a complex combination of parameters, including temperature, NO3 , salinity, dinoflagellates, SiO2, secchi depth and NO2. Changing environmental conditions might not only lead to increasing numbers of Vibrio spp. in the German Bight, but also to a community shift towards potentially pathogenic species such as V. parahaemolyticus. Being the first profound study on Vibrio spp. in the German Bight, this thesis represents an invaluable first step towards the understanding of this important microbial group, harboring the potential pathogen V. parahaemolyticus, in northern European waters.

Assessing the Genetic Accessibility of Rhodopirellula baltica SH 1 T

Basak Öztürk — Thu, 03 Dec 2015 16:08:23 +0100

The marine bacterium Rhodopirellula baltica SH 1 T is a member of the phylum Planctomycetes which are distinguished from other bacteria by their unique properties such as reproduction by budding, intracellular compartments and cell walls lacking murein. In this study, I have worked on two method development projects to gain insights into this unique bacterium. The main aim of my thesis was to develop methods to deliver DNA into Rhodopirellula baltica. A chemical transformation method where chromosomal DNA from chloramphenicol-resistant mutants is used to transform the wild-type competent cells was developed. Other transformation methods, as well as replication of broad host range plasmids and fluorescence-protein expression were explored. A protoplast-formation protocol using enzymatic treatment with lysozyme and osmotic pressure was developed, which can be used in future cell biology studies to further investigate the cell wall structure of Rhodopirellula baltica, as well as for DNA transfer by protoplast transformation. This work is the first demonstration of DNA transfer into Rhodopirellula baltica and one of the few examples of protoplast formation of a Gram-negative bacterium. My second project was to develop an amplification protocol for prokaryotic transcriptomes to be later used for next-generation sequencing. The method was applied to both Rhodopirellula baltica and bacterial communities from the North Sea surface water. The protocol has successfully amplified both the pure-culture transcriptome and the metatranscriptome. This protocol can be used to amplify prokaryotic cDNA for next-generation sequencing and microarray studies where sample size is limited.

Molecular and ecological analysis of cellular attachment and induction of transparent exopolymeric particle formation in diatom-bacteria interactions

Shalin Seebah — Thu, 19 Nov 2015 11:36:23 +0100

Transparent exopolymeric particles (TEP) and marine snow aggregates are vital components of the oceanic carbon cycle, leading to a substantial fraction of organic carbon sinking to depth or being provided for further recycling. Diatom-associated bacteria have been recently shown to directly impact TEP production and aggregate formation. However, very little is known about the molecular components that govern this interaction or the dynamics of TEP production and marine aggregate formation under changing environmental conditions. By combining molecular techniques and ecological experiments, we use an interdisciplinary approach to unveil hitherto unknown processes of diatom-bacteria interactions. As part of a collaborative and concerted effort, we initially established a genetically accessible bilateral model system consisting of the diatom Thalassiosira weissflogii and the marine gammaproteobacterium Marinobacter adhaerens HP15. Herein, we taxonomically established M. adhaerens HP15 as a novel member of the Marinobacter genus, revealed its genome sequence and established a genetic system to allow for the precise manipulation of this bacterium at the molecular level. In a second part, we used the established genetic toolbox to investigate the role of M. adhaerens HP15s motility during its interaction with the diatom. By generating M. adhaerens HP15 flagellum- and MSHA type IV pilus-deficient mutants, we demonstrate that a fullyfunctional flagellum is a pre-requisite for the bacterial attachment to both abiotic and diatom surfaces. We further show that the MSHA type-IV pilus is important for attachment, albeit to a lesser extent. Although both cellular appendages were shown to be crucial for attachment to diatom surfaces, this type of attachment was demonstrated to not be essential for inducing the formation of diatom-borne transparent exopolymeric particles (TEP). In the final part of this work, how TEP production dynamics and aggregate formation might be impacted in putative future oceanic scenarios was investigated. The results of our study suggest that the combined effect of ocean acidification and increased temperature might lead to a significant reduction in aggregate formation and sinking velocities of marine aggregates. We suggest that a combination of ocean acidification and global warming may severely impact the vertical transport of particulate organic matter in a future ocean.