Marine Microbiology
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.
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.
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.
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 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.
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.
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.
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.
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
(2016)
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.
