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The sequencing of the human genome in the early 2000s led to an increased interest in cheap and fast sequencing technologies. This interest culminated in the advent of next generation sequencing (NGS). A number of different NGS platforms have arisen since then all promising to do the same thing, i.e. produce large amounts of genetic information for relatively low costs compared to more traditional methods such as Sanger sequencing. The capabilities of NGS meant that researchers were no longer bound to species for which a lot of previous work had already been done (e.g. model organisms and humans) enabling a shift in research towards more novel and diverse species of interest. This capability has greatly benefitted many fields within the biological sciences, one of which being the field of evolutionary biology. Researchers have begun to move away from the study of laboratory model organisms to wild, natural populations and species which has greatly expanded our knowledge of evolution. NGS boasts a number of benefits over more traditional sequencing approaches. The main benefit comes from the capability to generate information for drastically more loci for a fraction of the cost. This is hugely beneficial to the study of wild animals as, even when large numbers of individuals are unobtainable, the amount of data produced still allows for accurate, reliable population and species level results from a small selection of individuals.
The use of NGS to study species for which little to no previous research has been carried out on and the production of novel evolutionary information and reference datasets for the greater scientific community were the focuses of this thesis. Two studies in this thesis focused on producing novel mitochondrial genomes from shotgun sequencing data through iterative mapping, bypassing the need for a close relative to serve as a reference sequence. These mitochondrial genomes were then used to infer species level relationships through phylogenetic analyses. The first of these studies involved reconstructing a complete mitochondrial genome of the bat eared fox (Otocyon megalotis). Phylogenetic analyses of the mitochondrial genome confidently placed the bat eared fox as sister to the clade consisting of the raccoon dog and true foxes within the canidae family. The next study also involved reconstructing a mitochondrial genome but in this case from the extinct Macrauchenia of South America. As this study utilised ancient DNA, it involved a lot of parameter testing, quality controls and strict thresholds to obtain a near complete mitochondrial genome devoid of contamination known to plague ancient DNA studies. Phylogenetic analyses confidently placed Macrauchenia as sister to all living representatives of Perissodactyla with a divergence time of ~66 million years ago. The third and final study of this thesis involved de novo assemblies of both nuclear and mitochondrial genomes from brown and striped hyena and focussed on demographic, genetic diversity and population genomic analyses within the brown hyena. Previous studies of the brown hyena hinted at very low levels of genomic diversity and, perhaps due to this, were unable to find any notable population structure across its range. By incorporating a large number of genetic loci, in the form of complete nuclear genomes, population structure within the brown hyena was uncovered. On top of this, genomic diversity levels were compared to a number of other species. Results showed the brown hyena to have the lowest genomic diversity out of all species included in the study which was perhaps caused by a continuous and ongoing decline in effective population size that started about one million years ago and dramatically accelerated towards the end of the Pleistocene.
The studies within this thesis show the power NGS sequencing has and its utility within evolutionary biology. The most notable capabilities outlined in this thesis involve the study of species for which no reference data is available and in the production of large amounts of data, providing evolutionary answers at the species and population level that data produced using more traditional techniques simply could not.
Plants are unable to move away from unwanted environments and therefore have to locally adapt to changing conditions. Arabidopsis thaliana (Arabidopsis), a model organism in plant biology, has been able to rapidly colonize a wide spectrum of environments with different biotic and abiotic challenges. In recent years, natural variation in Arabidopsis has shown to be an excellent resource to study genes underlying adaptive traits and hybridization’s impact on natural diversity. Studies on Arabidopsis hybrids have provided information on the genetic basis of hybrid incompatibilities and heterosis, as well as inheritance patterns in hybrids. However, previous studies have focused mainly on global accessions and yet much remains to be known about variation happening within a local growth habitat. In my PhD, I investigated the impact of heterozygosity at a local collection site of Arabidopsis and its role in local adaptation. I focused on two different projects, both including hybrids among Arabidopsis individuals collected around Tübingen in Southern Germany. The first project sought to understand the impact of hybridization on metabolism and growth within a local Arabidopsis collection site. For this, the inheritance patterns in primary and secondary metabolism, together with rosette size of full diallel crosses among seven parents originating from Southern Germany were analyzed. In comparison to primary metabolites, compounds from secondary metabolism were more variable and showed pronounced non-additive inheritance patterns. In addition, defense metabolites, mainly glucosinolates, displayed the highest degree of variation from the midparent values and were positively correlated with a proxy for plant size.
In the second project, the role of ACCELERATED CELL DEATH 6 (ACD6) in the defense response pathway of Arabidopsis necrotic hybrids was further characterized. Allelic interactions of ACD6 have been previously linked to hybrid necrosis, both among global and local Arabidopsis accessions. Hence, I characterized the early metabolic and ionic changes induced by ACD6, together with marker gene expression assays of physiological responses linked to its activation. An upregulation of simple sugars and metabolites linked to non-enzymatic antioxidants and the TCA cycle were detected, together with putrescine and acids linked to abiotic stress responses. Senescence was found to be induced earlier in necrotic hybrids and cytoplasmic calcium signaling was unaffected in response to temperature. In parallel, GFP-tagged constructs of ACD6 were developed.
This work therefore gave novel insights on the role of heterozygosity in natural variation and adaptation and expanded our current knowledge on the physiological and molecular responses associated with ACD6 activation.
The movement of organisms has formed our planet like few other processes. Movements shape populations, communities, entire ecosystems, and guarantee fundamental ecosystem functions and services, like seed dispersal and pollination. Global, regional and local anthropogenic impacts influence animal movements across ecosystems all around the world. In particular, land-use modification, like habitat loss and fragmentation disrupt movements between habitats with profound consequences, from increased disease transmissions to reduced species richness and abundance. However, neither the influence of anthropogenic change on animal movement processes nor the resulting effects on ecosystems are well understood. Therefore, we need a coherent understanding of organismal movement processes and their underlying mechanisms to predict and prevent altered animal movements and their consequences for ecosystem functions.
In this thesis I aim at understanding the influence of anthropogenically caused land-use change on animal movement processes and their underlying mechanisms. In particular, I am interested in the synergistic influence of large-scale landscape structure and fine-scale habitat features on basic-level movement behaviours (e.g. the daily amount of time spend running, foraging, and resting) and their emerging higher-level movements (home range formation). Based on my findings, I identify the likely consequences of altered animal movements that lead to the loss of species richness and abundances.
The study system of my thesis are hares in agricultural landscapes. European brown hares (Lepus europaeus) are perfectly suited to study animal movements in agricultural landscapes, as hares are hermerophiles and prefer open habitats. They have historically thrived in agricultural landscapes, but their numbers are in decline. Agricultural areas are undergoing strong land-use changes due to increasing food demand and fast developing agricultural technologies. They are already the largest land-use class, covering 38% of the world’s terrestrial surface. To consider the relevance of a given landscape structure for animal movement behaviour I selected two differently structured agricultural landscapes – a simple landscape in Northern Germany with large fields and few landscape elements (e.g. hedges and tree stands), and a complex landscape in Southern Germany with small fields and many landscape elements.
I applied GPS devices (hourly fixes) with internal high-resolution accelerometers (4 min samples) to track hares, receiving an almost continuous observation of the animals’ behaviours via acceleration analyses. I used the spatial and behavioural information in combination with remote sensing data (normalized difference vegetation index, or NDVI, a proxy for resource availability), generating an almost complete idea of what the animal was doing when, why and where. Apart from landscape structure (represented by the two differently structured study areas), I specifically tested whether the following fine-scale habitat features influence animal movements: resource, agricultural management events, habitat diversity, and habitat structure.
My results show that, irrespective of the movement process or mechanism and the type of fine-scale habitat features, landscape structure was the overarching variable influencing hare movement behaviour. High resource variability forces hares to enlarge their home ranges, but only in the simple and not in the complex landscape. Agricultural management events result in home range shifts in both landscapes, but force hares to increase their home ranges only in the simple landscape. Also the preference of habitat patches with low vegetation and the avoidance of high vegetation, was stronger in the simple landscape. High and dense crop fields restricted hare movements temporarily to very local and small habitat patch remnants. Such insuperable barriers can separate habitat patches that were previously connected by mobile links. Hence, the transport of nutrients and genetic material is temporarily disrupted. This mechanism is also working on a global scale, as human induced changes from habitat loss and fragmentation to expanding monocultures cause a reduction in animal movements worldwide.
The mechanisms behind those findings show that higher-level movements, like increasing home ranges, emerge from underlying basic-level movements, like the behavioural modes. An increasing landscape simplicity first acts on the behavioural modes, i.e. hares run and forage more, but have less time to rest. Hence, the emergence of increased home range sizes in simple landscapes is based on an increased proportion of time running and foraging, largely due to longer travelling times between distant habitats and scarce resource items in the landscape. This relationship was especially strong during the reproductive phase, demonstrating the importance of high-quality habitat for reproduction and the need to keep up self-maintenance first, in low quality areas. These changes in movement behaviour may release a cascade of processes that start with more time being allocated to running and foraging, resulting into an increased energy expenditure and may lead to a decline in individual fitness. A decrease in individual fitness and reproductive output will ultimately affect population viability leading to local extinctions.
In conclusion, I show that landscape structure has one of the most important effects on hare movement behaviour. Synergistic effects of landscape structure, and fine-scale habitat features, first affect and modify basic-level movement behaviours, that can scales up to altered higher-level movements and may even lead to the decline of species richness and abundances, and the disruption of ecosystem functions. Understanding the connection between movement mechanisms and processes can help to predict and prevent anthropogenically induced changes in movement behaviour. With regard to the paramount importance of landscape structure, I strongly recommend to decrease the size of agricultural fields and increase crop diversity. On the small-scale, conservation policies should assure the year round provision of areas with low vegetation height and high quality forage. This could be done by generating wildflower strips and additional (semi-) natural habitat patches. This will not only help to increase the populations of European brown hares and other farmland species, but also ensure and protects the continuity of mobile links and their intrinsic value for sustaining important ecosystem functions and services.
For more than two centuries, plant ecologists have aimed to understand how environmental gradients and biotic interactions shape the distribution and co-occurrence of plant species. In recent years, functional trait–based approaches have been increasingly used to predict patterns of species co-occurrence and species distributions along environmental gradients (trait–environment relationships). Functional traits are measurable properties at the individual level that correlate well with important processes. Thus, they allow us to identify general patterns by synthesizing studies across specific taxonomic compositions, thereby fostering our understanding of the underlying processes of species assembly. However, the importance of specific processes have been shown to be highly dependent on the spatial scale under consideration. In particular, it remains uncertain which mechanisms drive species assembly and allow for plant species coexistence at smaller, more local spatial scales. Furthermore, there is still no consensus on how particular environmental gradients affect the trait composition of plant communities. For example, increasing drought because of climate change is predicted to be a main threat to plant diversity, although it remains unclear which traits of species respond to increasing aridity. Similarly, there is conflicting evidence of how soil fertilization affects the traits related to establishment ability (e.g., seed mass). In this cumulative dissertation, I present three empirical trait-based studies that investigate specific research questions in order to improve our understanding of species distributions along environmental gradients.
In the first case study, I analyze how annual species assemble at the local scale and how environmental heterogeneity affects different facets of biodiversity—i.e. taxonomic, functional, and phylogenetic diversity—at different spatial scales. The study was conducted in a semi-arid environment at the transition zone between desert and Mediterranean ecosystems that features a sharp precipitation gradient (Israel). Different null model analyses revealed strong support for environmentally driven species assembly at the local scale, since species with similar traits tended to co-occur and shared high abundances within microsites (trait convergence). A phylogenetic approach, which assumes that closely related species are functionally more similar to each other than distantly related ones, partly supported these results. However, I observed that species abundances within microsites were, surprisingly, more evenly distributed across the phylogenetic tree than expected (phylogenetic overdispersion). Furthermore, I showed that environmental heterogeneity has a positive effect on diversity, which was higher on functional than on taxonomic diversity and increased with spatial scale. The results of this case study indicate that environmental heterogeneity may act as a stabilizing factor to maintain species diversity at local scales, since it influenced species distribution according to their traits and positively influenced diversity. All results were constant along the precipitation gradient.
In the second case study (same study system as case study one), I explore the trait responses of two Mediterranean annuals (Geropogon hybridus and Crupina crupinastrum) along a precipitation gradient that is comparable to the maximum changes in precipitation predicted to occur by the end of this century (i.e., −30%). The heterocarpic G. hybridus showed strong trends in seed traits, suggesting that dispersal ability increased with aridity. By contrast, the homocarpic C. crupinastrum showed only a decrease in plant height as aridity increased, while leaf traits of both species showed no consistent pattern along the precipitation gradient. Furthermore, variance decomposition of traits revealed that most of the trait variation observed in the study system was actually found within populations. I conclude that trait responses towards aridity are highly species-specific and that the amount of precipitation is not the most striking environmental factor at this particular scale.
In the third case study, I assess how soil fertilization mediates—directly by increased nutrient addition and indirectly by increased competition—the effect of seed mass on establishment ability. For this experiment, I used 22 species differing in seed mass from dry grasslands in northeastern Germany and analyzed the interacting effects of seed mass with nutrient availability and competition on four key components of seedling establishment: seedling emergence, time of seedling emergence, seedling survival, and seedling growth. (Time of) seedling emergence was not affected by seed mass. However, I observed that the positive effect of seed mass on seedling survival is lowered under conditions of high nutrient availability, whereas the positive effect of seed mass on seedling growth was only reduced by competition. Based on these findings, I developed a conceptual model of how seed mass should change along a soil fertility gradient in order to reconcile conflicting findings from the literature. In this model, seed mass shows a U-shaped pattern along the soil fertility gradient as a result of changing nutrient availability and competition.
Overall, the three case studies highlight the role of environmental factors on species distribution and co-occurrence. Moreover, the findings of this thesis indicate that spatial heterogeneity at local scales may act as a stabilizing factor that allows species with different traits to coexist. In the concluding discussion, I critically debate intraspecific trait variability in plant community ecology, the use of phylogenetic relationships and easily measured key functional traits as a proxy for species’ niches. Finally, I offer my outlook for the future of functional plant community research.
The highly conserved protein complex containing the Target of Rapamycin (TOR) kinase is known to integrate intra- and extra-cellular stimuli controlling nutrient allocation and cellular growth. This thesis describes three studies aimed to understand how TOR signaling pathway influences carbon and nitrogen metabolism in Chlamydomonas reinhardtii. The first study presents a time-resolved analysis of the molecular and physiological features across the diurnal cycle. The inhibition of TOR leads to 50% reduction in growth followed by nonlinear delays in the cell cycle progression. The metabolomics analysis showed that the growth repression is mainly driven by differential carbon partitioning between anabolic and catabolic processes. Furthermore, the high accumulation of nitrogen-containing compounds indicated that TOR kinase controls the carbon to nitrogen balance of the cell, which is responsible for biomass accumulation, growth and cell cycle progression. In the second study the cause of the high accumulation of amino acids is explained. For this purpose, the effect of TOR inhibition on Chlamydomonas was examined under different growth regimes using stable 13C- and 15N-isotope labeling. The data clearly showed that an increased nitrogen uptake is induced within minutes after the inhibition of TOR. Interestingly, this increased N-influx is accompanied by increased activities of nitrogen assimilating enzymes. Accordingly, it was concluded that TOR inhibition induces de-novo amino acid synthesis in Chlamydomonas. The recognition of this novel process opened an array of questions regarding potential links between central metabolism and TOR signaling. Therefore a detailed phosphoproteomics study was conducted to identify the potential substrates of TOR pathway regulating central metabolism. Interestingly, some of the key enzymes involved in carbon metabolism as well as amino acid synthesis exhibited significant changes in the phosphosite intensities immediately after TOR inhibition. Altogether, these studies provide a) detailed insights to metabolic response of Chlamydomonas to TOR inhibition, b) identification of a novel process causing rapid upshifts in amino acid levels upon TOR inhibition and c) finally highlight potential targets of TOR signaling regulating changes in central metabolism. Further biochemical and molecular investigations could confirm these observations and advance the understanding of growth signaling in microalgae.
Plant-derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae
Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects where tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harbouring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver / reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast, than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC - EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF – DNA-binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.
COMPASS: Rapid combinatorial optimization of biochemical pathways based on artificial transcription factors
We established a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) for controlling the expression of pathway genes, and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, we equipped COMPASS with multi-locus CRISPR/Cas9-mediated modification capacity. In its current realization, COMPASS allows combinatorial optimization of up to ten pathway genes, each transcriptionally controlled by nine different ATFs spanning a 10-fold difference in expression strength. The application of COMPASS was demonstrated by generating cell libraries producing beta-carotene and co-producing beta-ionone and biosensor-responsive naringenin. COMPASS will have many applications in other synthetic biology projects that require gene expression balancing.
CaPRedit: Genome editing using CRISPR-Cas9 and plant-derived transcriptional regulators for the redirection of flux through the FPP branch-point in yeast. Technologies developed over the past decade have made Saccharomyces cerevisiae a promising platform for production of different natural products. We developed CRISPR/Ca9- and plant derived regulator-mediated genome editing approach (CaPRedit) to greatly accelerate strain modification and to facilitate very low to very high expression of key enzymes using inducible regulators. CaPRedit can be implemented to enhance the production of yeast endogenous or heterologous metabolites in the yeast S. cerevisiae. The CaPRedit system aims to faciltiate modification of multiple targets within a complex metabolic pathway through providing new tools for increased expression of genes encoding rate-limiting enzymes, decreased expression of essential genes, and removed expression of competing pathways. This approach is based on CRISPR/Cas9-mediated one-step double-strand breaks to integrate modules containing IPTG-inducible plant-derived artificial transcription factor and promoter pair(s) in a desired locus or loci. Here, we used CaPRedit to redirect the yeast endogenous metabolic flux toward production of farnesyl diphosphate (FPP), a central precursor of nearly all yeast isoprenoid products, by overexpression of the enzymes lead to produce FPP from glutamate. We found significantly higher beta-carotene accumulation in the CaPRedit-mediated modified strain than in the wild type (WT) strain. More specifically, CaPRedit_FPP 1.0 strain was generated, in which three genes involved in FPP synthesis, tHMG1, ERG20, and GDH2, were inducibly overexpressed under the control of strong plant-derived ATFPs. The beta–carotene accumulated in CaPRedit_FPP 1.0 strain to a level 1.3-fold higher than the previously reported optimized strain that carries the same overexpressed genes (as well as additional genetic modifications to redirect yeast endogenous metabolism toward FPP production). Furthermore, the genetic modifications implemented in CaPRedit_FPP 1.0 strain resulted in only a very small growth defect (growth rate relative to the WT is ~ -0.03).
Monoclonal antibodies (mAbs) are an innovative group of drugs with increasing clinical importance in oncology, combining high specificity with generally low toxicity. There are, however, numerous challenges associated with the development of mAbs as therapeutics. Mechanistic understanding of factors that govern the pharmacokinetics (PK) of mAbs is critical for drug development and the optimisation of effective therapies; in particular, adequate dosing strategies can improve patient quality life and lower drug cost. Physiologically-based PK (PBPK) models offer a physiological and mechanistic framework, which is of advantage in the context of animal to human extrapolation. Unlike for small molecule drugs, however, there is no consensus on how to model mAb disposition in a PBPK context. Current PBPK models for mAb PK hugely vary in their representation of physiology and parameterisation. Their complexity poses a challenge for their applications, e.g., translating knowledge from animal species to humans.
In this thesis, we developed and validated a consensus PBPK model for mAb disposition taking into account recent insights into mAb distribution (antibody biodistribution coefficients and interstitial immunoglobulin G (IgG) pharmacokinetics) to predict tissue PK across several pre-clinical species and humans based on plasma data only. The model allows to a priori predict target-independent (unspecific) mAb disposition processes as well as mAb disposition in concentration ranges, for which the unspecific clearance (CL) dominates target-mediated CL processes. This is often the case for mAb therapies at steady state dosing.
The consensus PBPK model was then used and refined to address two important problems:
1) Immunodeficient mice are crucial models to evaluate mAb efficacy in cancer therapy. Protection from elimination by binding to the neonatal Fc receptor is known to be a major pathway influencing the unspecific CL of both, endogenous and therapeutic IgG. The concentration of endogenous IgG, however, is reduced in immunodeficient mouse models, and this effect on unspecific mAb CL is unknown, yet of great importance for the extrapolation to human in the context of mAb cancer therapy.
2) The distribution of mAbs into solid tumours is of great interest. To comprehensively investigate mAb distribution within tumour tissue and its implications for therapeutic efficacy, we extended the consensus PBPK model by a detailed tumour distribution model incorporating a cell-level model for mAb-target interaction. We studied the impact of variations in tumour microenvironment on therapeutic efficacy and explored the plausibility of different mechanisms of action in mAb cancer therapy.
The mathematical findings and observed phenomena shed new light on therapeutic utility and dosing regimens in mAb cancer treatment.
In this work, different strategies for the construction of biohybrid photoelectrodes are investigated and have been evaluated according to their intrinsic catalytic activity for the oxidation of the cofactor NADH or for the connection with the enzymes PQQ glucose dehydrogenase (PQQ-GDH), FAD-dependent glucose dehydrogenase (FAD-GDH) and fructose dehydrogenase (FDH). The light-controlled oxidation of NADH has been analyzed with InGaN/GaN nanowire-modified electrodes. Upon illumination with visible light the InGaN/GaN nanowires generate an anodic photocurrent, which increases in a concentration-dependent manner in the presence of NADH, thus allowing determination of the cofactor. Furthermore, different approaches for the connection of enzymes to quantum dot (QD)-modified electrodes via small redox molecules or redox polymers have been analyzed and discussed. First, interaction studies with diffusible redox mediators such as hexacyanoferrate(II) and ferrocenecarboxylic acid have been performed with CdSe/ZnS QD-modified gold electrodes to build up photoelectrochemical signal chains between QDs and the enzymes FDH and PQQ-GDH. In the presence of substrate and under illumination of the electrode, electrons are transferred from the enzyme via the redox mediators to the QDs. The resulting photocurrent is dependent on the substrate concentration and allows a quantification of the fructose and glucose content in solution. A first attempt with immobilized redox mediator, i.e. ferrocenecarboxylic acid chemically coupled to PQQ-GDH and attached to QD-modified gold electrodes, reveal the potential to build up photoelectrochemical signal chains even without diffusible redox mediators in solution. However, this approach results in a significant deteriorated photocurrent response compared to the situation with diffusing mediators. In order to improve the photoelectrochemical performance of such redox mediator-based, light-switchable signal chains, an osmium complex-containing redox polymer has been evaluated as electron relay for the electronic linkage between QDs and enzymes. The redox polymer allows the stable immobilization of the enzyme and the efficient wiring with the QD-modified electrode. In addition, a 3D inverse opal TiO2 (IO-TiO2) electrode has been used for the integration of PbS QDs, redox polymer and FAD-GDH in order to increase the electrode surface. This results in a significantly improved photocurrent response, a quite low onset potential for the substrate oxidation and a broader glucose detection range as compared to the approach with ferrocenecarboxylic acid and PQQ-GDH immobilized on CdSe/ZnS QD-modified gold electrodes. Furthermore, IO-TiO2 electrodes are used to integrate sulfonated polyanilines (PMSA1) and PQQ-GDH, and to investigate the direct interaction between the polymer and the enzyme for the light-switchable detection of glucose. While PMSA1 provides visible light excitation and ensures the efficient connection between the IO-TiO2 electrode and the biocatalytic entity, PQQ-GDH enables the oxidation of glucose. Here, the IO-TiO2 electrodes with pores of approximately 650 nm provide a suitable interface and morphology, which is required for a stable and functional assembly of the polymer and enzyme. The successful integration of the polymer and the enzyme can be confirmed by the formation of a glucose-dependent anodic photocurrent. In conclusion, this work provides insights into the design of photoelectrodes and presents different strategies for the efficient coupling of redox enzymes to photoactive entities, which allows for light-directed sensing and provides the basis for the generation of power from sun light and energy-rich compounds.
East Africa is a natural laboratory: Studying its unique geological and biological history can help us better inform our theories and models. Studying its present and future can help us protect its globally important biodiversity and ecosystem services. East African vegetation plays a central role in all these aspects, and this dissertation aims to quantify its dynamics through computer simulations.
Computer models help us recreate past settings, forecast into the future or conduct simulation experiments that we cannot otherwise perform in the field. But before all that, one needs to test their performance. The outputs that the model produced using the present day-inputs, agreed well with present-day observations of East African vegetation. Next, I simulated past vegetation for which we have fossil pollen data to compare. With computer models, we can fill the gaps of knowledge between sites where we have fossil pollen data from, and create a more complete picture of the past. Good level of agreement between model and pollen data where they overlapped in space further validated our model performance.
Once the model was tested and validated for the region, it became possible to probe one of the long standing questions regarding East African vegetation: How did East Africa lose its tropical forests? The present-day vegetation in the tropics is mainly characterized by continuous forests worldwide except in tropical East Africa, where forests only occur as patches. In a series of simulation experiments, I was able to show under which conditions these forest patches could have been connected and fragmented in the past. This study showed the sensitivity of East African vegetation to climate change and variability such as those expected under future climate change.
El Niño Southern Oscillation (ENSO) events that result from the fluctuations in temperature between the ocean and atmosphere, bring further variability to East African climate and are predicted to increase in intensity in the future. But climate models are still not good at capturing the pattens of these events. In a study where I quantified the influence of ENSO events on East African vegetation, I showed how different the future vegetation could be from what we currently predict with these climate models that lack accurate ENSO contribution. Consideration of these discrepancies is important for our future global carbon budget calculations and management decisions.
Biological invasions are the dispersal and following establishment of species outside their native habitat. Due to globalisation, connectivity of regions and climate changes the number of invasive species and their successful establishment is rising. The impact of these species is mostly negative, can induce community and habitat alterations, and is one main cause for biodiversity loss. This impact is particularly high and less researched in aquatic systems and microbial organisms and despite the high impact, the knowledge about overall mechanisms and specific factors affecting invasions are not fully understood. In general, the characteristics of the habitat, native community and invader determine the invasiveness.
In this thesis, I aimed to provide a better understanding of aquatic invasions focusing on the invader and its traits and identity. This thesis used a set of 12 strains of the invasive cyanobacterium <i>Cylindrospermopsis raciborskii</i> to examine the effect and impact of the invaders’ identity and genetic diversity. Further, the effect of timing on the invasion potential and success was determined, because aquatic systems in particular undergo seasonal fluctuations.
Most studies revealed a higher invasion success with increasing genetic diversity. Here, the increase of the genetic diversity, by either strain richness or phylogenetic dissimilarity, is not firstly driving the invasion, but the strain-identity. The high variability among the strains in traits important for invasions led to the highly varying strain-specific invasion success. This success was most dependent on nitrogen uptake and efficient resource use. The lower invasion success into communities comprising further N-fixing species indicates <i>C. raciborskii</i> can use this advantage only without the presence of competitive species. The relief of grazing pressure, which is suggested to be more important in aquatic invasions, was only promoting the invasion when unselective and larger consumers were present. High abundances of unselective consumers hampered the invasion success.
This indicates a more complex and temporal interplay of competitive and consumptive resistance mechanisms during the invasion process. Further, the fluctuation abundance and presence of competitors (= primary producers) and consumers (= zooplankton) in lakes can open certain ‘invasion windows’.
Remarkably, the composition of the resident community was also strain-specific affected and altered, independent of a high or low invasion success. Prior, this was only documented on the species level. Further, investigations on the population of invasive strains can reveal more about the invasion patterns and how multiple strain invasions change resident communities.
The present dissertation emphasises the importance of invader-addition experiments with a community context and the importance of the strain-level for microbial invasions and in general, e.g. for community assemblies and the outcome of experiments. The strain-specific community changes, also after days, may explain some sudden changes in communities, which have not been explained yet. This and further knowledge may also facilitate earlier and less cost-intensive management to step in, because these species are rarely tracked until they reach a high abundance or bloom, because of their small size.
Concluded for <i>C. raciborskii</i>, it shows that this species is no ‘generalistic’ invader and its invasion success depends more on the competitor presence than grazing pressure. This may explain its, still unknown, invasion pattern, as <i>C. raciborskii</i> is not found in all lakes of a region.
Plastic pollution is ubiquitous on the planet since several millions of tons of plastic waste enter aquatic ecosystems each year. Furthermore, the amount of plastic produced is expected to increase exponentially shortly. The heterogeneity of materials, additives and physical characteristics of plastics are typical of these emerging contaminants and affect their environmental fate in marine and freshwaters. Consequently, plastics can be found in the water column, sediments or littoral habitats of all aquatic ecosystems. Most of this plastic debris will fragment as a product of physical, chemical and biological forces, producing particles of small size. These particles (< 5mm) are known as “microplastics” (MP). Given their high surface-to-volume ratio, MP stimulate biofouling and the formation of biofilms in aquatic systems.
As a result of their unique structure and composition, the microbial communities in MP biofilms are referred to as the “Plastisphere.” While there is increasing data regarding the distinctive composition and structure of the microbial communities that form part of the plastisphere, scarce information exists regarding the activity of microorganisms in MP biofilms. This surface-attached lifestyle is often associated with the increase in horizontal gene transfer (HGT) among bacteria. Therefore, this type of microbial activity represents a relevant function worth to be analyzed in MP biofilms. The horizontal exchange of mobile genetic elements (MGEs) is an essential feature of bacteria. It accounts for the rapid evolution of these prokaryotes and their adaptation to a wide variety of environments. The process of HGT is also crucial for spreading antibiotic resistance and for the evolution of pathogens, as many MGEs are known to contain antibiotic resistance genes (ARGs) and genetic determinants of pathogenicity.
In general, the research presented in this Ph.D. thesis focuses on the analysis of HGT and heterotrophic activity in MP biofilms in aquatic ecosystems. The primary objective was to analyze the potential of gene exchange between MP bacterial communities vs. that of the surrounding water, including bacteria from natural aggregates. Moreover, the thesis addressed the potential of MP biofilms for the proliferation of biohazardous bacteria and MGEs from wastewater treatment plants (WWTPs) and associated with antibiotic resistance. Finally, it seeks to prove if the physiological profile of MP biofilms under different limnological conditions is divergent from that of the water communities. Accordingly, the thesis is composed of three independent studies published in peer-reviewed journals. The two laboratory studies were performed using both model and environmental microbial communities. In the field experiment, natural communities from freshwater ecosystems were examined.
In Chapter I, the inflow of treated wastewater into a temperate lake was simulated with a concentration gradient of MP particles. The effects of MP on the microbial community structure and the occurrence of integrase 1 (int 1) were followed. The int 1 is a marker associated with mobile genetic elements and known as a proxy for anthropogenic effects on the spread of antimicrobial resistance genes. During the experiment, the abundance of int1 increased in the plastisphere with increasing MP particle concentration, but not in the surrounding water. In addition, the microbial community on MP was more similar to the original wastewater community with increasing microplastic concentrations. Our results show that microplastic particles indeed promote persistence of standard indicators of microbial anthropogenic pollution in natural waters.
In Chapter II, the experiments aimed to compare the permissiveness of aquatic bacteria towards model antibiotic resistance plasmid pKJK5, between communities that form biofilms on MP vs. those that are free-living. The frequency of plasmid transfer in bacteria associated with MP was higher when compared to bacteria that are free-living or in natural aggregates. Moreover, comparison increased gene exchange occurred in a broad range of phylogenetically-diverse bacteria. The results indicate a different activity of HGT in MP biofilms, which could affect the ecology of aquatic microbial communities on a global scale and the spread of antibiotic resistance.
Finally, in Chapter III, physiological measurements were performed to assess whether microorganisms on MP had a different functional diversity from those in water. General heterotrophic activity such as oxygen consumption was compared in microcosm assays with and without MP, while diversity and richness of heterotrophic activities were calculated by using Biolog® EcoPlates. Three lakes with different nutrient statuses presented differences in MP-associated biomass build up. Functional diversity profiles of MP biofilms in all lakes differed from those of the communities in the surrounding water, but only in the oligo-mesotrophic lake MP biofilms had a higher functional richness compared to the ambient water. The results support that MP surfaces act as new niches for aquatic microorganisms and can affect global carbon dynamics of pelagic environments.
Overall, the experimental works presented in Chapters I and II support a scenario where MP pollution affects HGT dynamics among aquatic bacteria. Among the consequences of this alteration is an increase in the mobilization and transfer efficiency of ARGs. Moreover, it supposes that changes in HGT can affect the evolution of bacteria and the processing of organic matter, leading to different catabolic profiles such as demonstrated in Chapter III. The results are discussed in the context of the fate and magnitude of plastic pollution and the importance of HGT for bacterial evolution and the microbial loop, i.e., at the base of aquatic food webs. The thesis supports a relevant role of MP biofilm communities for the changes observed in the aquatic microbiome as a product of intense human intervention.
The continuously increasing pollution of aquatic environments with microplastics (plastic particles < 5 mm) is a global problem with potential implications for organisms of all trophic levels. For microorganisms, trillions of these floating microplastics particles represent a huge surface area for colonization. Due to the very low biodegradability, microplastics remain years to centuries in the environment and can be transported over thousands of kilometers together with the attached organisms. Since also pathogenic, invasive, or otherwise harmful species could be spread this way, it is essential to study microplastics-associated communities.
For this doctoral thesis, eukaryotic communities were analyzed for the first time on microplastics in brackish environments and compared to communities in the surrounding water and on the natural substrate wood. With Illumina MiSeq high-throughput sequencing, more than 500 different eukaryotic taxa were detected on the microplastics samples. Among them were various green algae, dinoflagellates, ciliates, fungi, fungal-like protists and small metazoans such as nematodes and rotifers. The most abundant organisms was a dinoflagellate of the genus Pfiesteria, which could include fish pathogenic and bloom forming toxigenic species. Network analyses revealed that there were numerous interaction possibilities among prokaryotes and eukaryotes in microplastics biofilms. Eukaryotic community compositions on microplastics differed significantly from those on wood and in water, and compositions were additionally distinct among the sampling locations. Furthermore, the biodiversity was clearly lower on microplastics in comparison to the diversity on wood or in the surrounding water.
In another experiment, a situation was simulated in which treated wastewater containing microplastics was introduced into a freshwater lake. With increasing microplastics concentrations, the resulting bacterial communities became more similar to those from the treated wastewater. Moreover, the abundance of integrase I increased together with rising concentrations of microplastics. Integrase I is often used as a marker for anthropogenic environmental pollution and is further linked to genes conferring, e.g., antibiotic resistance.
This dissertation gives detailed insights into the complexity of prokaryotic and eukaryotic communities on microplastics in brackish and freshwater systems. Even though microplastics provide novel microhabitats for various microbes, they might also transport toxigenic, pathogenic, antibiotic-resistant or parasitic organisms; meaning their colonization can pose potential threats to humans and the environment. Finally, this thesis explains the urgent need for more research as well as for strategies to minimize the global microplastic pollution.