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Pectinolytic enzymes of Aspergillus sojae ATCC 20235: The impact of bioprocessing strategy on solid-state production and downstream processing of polygalacturonase
No full textThe present study revealed the potential of Aspergillus sojae ATCC 20235 for pectinase production in solid-state fermentation (SSF). A microbial screening of various Aspergillus species for pectinase production in SSF identified A. sojae ATCC 20235 as potential production organism. Media design and optimization of solid-state process parameters traced the increase of polygalacturonase (PG) titers. Optimization was performed in several steps applying various statistical designs, which yielded with 909.5 ± 2.7 U/g PG activity in 10.9 times increased enzyme production after 8 days at 30 °C applying sugar beet pulp (30 %) in combination with wheat bran as medium, wetted at 160 % by 0.2 M HCl.
Development and implementation of a classical mutation and selection strategy for the improved production of pectinases resulted in the generation of mutant M3 after three cycles of repeated treatment by UV irradiation. Mutant M3 yielded 1.7 times increased PG activity in SSF. Scale up of the optimized SSF process for PG enzyme production was successfully demonstrated without loss in the total amount of enzyme at a rotating drum type solid-state bioreactor with a scaling factor of 100. Additionally, focus was also set on the enzyme recovery and purification. Optimizing the enzyme leaching process resulted in efficient PG recovery. PG purification by combination of ion exchange chromatography, size exclusion chromatography and hydrophobic interaction chromatography yielded in the isolation of the enzyme by means of a single band on the SDS-polyacrylamide gel with a molecular weight of about 40 kDa.
Commercial pectinase preparations are usually mixtures of various enzyme species. Mass spectrometric characterization of A. sojae proteins identified a broad spectrum of carbohydrate-active enzymes. The characterized enzyme extracts were tested in several application studies related to fruit juice production and winemaking, which indicated improved process performances by enzyme treatment
Rare earth elements as emerging contaminants in the Rhine River, Germany and its tributaries
No full textThe first comprehensive geochemical dataset of the Rhine River and its major tributaries is provided. The major ion chemistry of the Rhine River and its tributaries is characterized. Natural variation of rare earth elements (REE) with discharge and season, and the effect of catchment geology on the distribution of rare earth elements is discussed. The sources and quantities of known and newly discovered anthropogenic REE are examined.
While the presence of anthropogenic gadolinium (Gd) anomalies in the Rhine River has been reported previously, anthropogenic lanthanum (La) and anthropogenic samarium (Sm) anomalies are documented for the first time, both for the Rhine River and natural waters worldwide. Both anthropogenic La and Sm originate from the waste water effluent at a facility producing catalytic fluid cracking catalysts. Ultrafiltration results suggest that anthropogenic Gd is not particle-reactive and is exclusively present in the truly dissolved REE fraction, while the anthropogenic La and Sm are present in both the truly dissolved and the nanoparticulate phase.
In parts of the Rhine River, anthropogenic REE concentrations are an order of magnitude higher than background concentrations for three (La, Sm and Gd) of the fourteen REE. Due to the high toxicity of REE, this may pose a threat to the ecosystem, especially given the lack of studies that focus on low-dosage long-term exposure.
With the demand for REE and the number of REE applications rapidly increasing, more anthropogenic REE are expected to enter the environment in the near future. Within the short duration of this study alone, two anthropogenic REE anomalies have been documented. The current situation of REE contamination may well be only a prelude to what will soon happen with other emerging contaminants
Mapping the Genetic Diversity of Eukaryotic Protists in the Arctic Ocean
No full textThis doctoral thesis aimed at the establishment of molecular tools (ARISA and 454-pyrosequencing) for protist diversity assessments in polar regions and at the application of these tools for studying protist diversity in the Fram Strait and in the Central Arctic Ocean. In this thesis, three hypotheses were put forward: i) Molecular surveys of genetic protist diversity, obtained by 454-pyrosequencing, constitute an adequate tool for assessing natural protist diversity ii) Water masses in the Arctic Ocean and in the Fram Strait are characterized by distinct protist communities and iii) Complex hydrographical and environmental situations can be evaluated via genetic information.
A comprehensive study of protist diversity is required, because so far investigations were biased towards big size cells (≥2 µm). A correct identification of smaller cells is almost impossible due to the small size and lack of morphological markers. However, a proper survey of protist diversity demands the inclusion of all size classes. This is of particular relevance, considering the fact that small cells were observed to dominate protist assemblages at certain times, according to abiotic circumstances. The Arctic Ocean constitutes in two ways an important research area: on the one hand, it experiences intense variations in the light regime based on seasonality and sea ice; and on the other hand, it is assumed to be affected more severely by climate change than other world oceans. Since marine microorganisms are highly responsive to environmental forcing, changes will likely impact the protist community structures. Against the background of ongoing environmental changes in the Arctic, a study of protist diversity is further crucial in order to get a baseline for the assessment of future community structure changes.
i) The assay of the 454-pyrosequencing suitability was carried out by using different wide-employed methods for reconciliation, sharing the same (clone library and ARISA) and different drawbacks (light microscopy and HPLC). The use of 18S rRNA clone library sequencing thereby, aimed to compare but also to complement the 454-pyrosequencing data, because of the longer sequence lengths that allow a more comprehensive taxon detailed analysis. However, the clone library approach was not suitable neither for 454-pyrosequencing comparison nor for 454-pyrosequencing complementation. While one approach, comprising the picoplankton fraction, was biased against haptophytes, the other approach, comprising the whole size fraction was biased against diatoms. Moreover, despite the comparable high number of ~140 clones per library, the abundant biosphere of 454-pyrosequencing was not comprehensively recovered. Hence, a consult of previous data
for 454-pyrosequencing gathered by clone library is not advisable or has to be interpreted with caution. The second molecular method, ARISA, reflected community structure shifts that were indeed recovered by 454-pyrosequencing. The indicative limitation of ARISA on sequence length variances however, makes the method more suitable for a preceded sample selection than for a robust 454-pyrosequencing support. The assessment with traditional methods as light microscopy and HPLC presented good analogies. Since light microscopy is biased against small cells and HPLC against heterotrophic protists, the comparison was adapted to the respective limitations. A quantitative comparison of the diatom assemblage showed similar percentages within light microscopy and 454-pyrosequencing (≥10 µm filter), and approved the use of diatoms for the evaluation of 454-pyrosequencing accuracy. The comparison with HPLC was further in accordance with 454-pyrosequencing and agreed in the portions of autotrophic protists. In summary, three out of four evaluation methods presented good analogies with 454-pyrosequencing data and approved the suitability of the molecular method for assessing natural protist diversity.
ii) To address the hypothesis if water masses host specific protist communities, 454-pyrosequencing was tested in different hydrographic environments for different size classes. In the process, the picoplankton community structure was investigated at four stations in the Eastern Fram Strait during the expedition ARK XXIV/2. The community structure of all protists was analyzed at five stations in the Western Fram Strait (ARK XXV/2) and at eight stations in the Central Arctic Ocean (ARK XXVI/3). A relation of water mass, distinguished by abiotic factors such as temperature, salinity and/or nutrients, and protist community structure was observed in all three studies. However, while the water mass regimes in the Fram Strait promoted distinct community structures in protist assemblages of the pico size spectrum and the entire size spectrum, populations of the Central Arctic Ocean showed a less definite association. In the Eastern Fram Strait picoplankton community (0.2-3 µm) in the Atlantic Water was mostly dominated by Phaeocystis cells and in the ice-covered station by small dinophytes (e.g. Dinophyte 1). The influence of cold, coastal water at one station however, shifted the community structure from a Phaeocystis-based to a Micromonas-based protist assemblage. In the Western Fram Strait, the protist community showed a distribution pattern that also corresponded to the different water properties. While Polar Water (EGC) was dominated by diatoms in recently light exposed stations and by dinoflagellates in a station longer exposed to light, the Atlantic Water (WSC) presented a high dominance of Micromonas that was not associated with the light availability. The two different
protist communities observed in the Polar Water, however, suggest a high influence of light availability on the protist assemblage by promoting a protist succession. In the Central Arctic Ocean, in contrast, protist communities showed a less pronounced relation to the four water masses (Atlantic Water, Pacific Water, Mixed Water I and II) with no switch of dominant protist community members. All water masses were mainly dominated by dinoflagellates (e.g. Syndiniales 2), and at two stations by Micromonas. In principle, different water masses were reflected more significantly by whole community structure changes (ARISA) than by the appearance or disappearance of single protists (454-pyrosequencing) in the abundant biosphere. In particular, protist communities of the so-called Mixed Water I and II were difficult to separate from Atlantic Water and Pacific Water communities. The formation of mixed water, e.g. the mixing of two separate water masses, resulted in a combination of the abiotic characteristics (temperature, salinity, and nutrients) and of the protist communities. Moreover, the high sea-ice concentration and thus low light availability in the Central Arctic Ocean represented a strong selective force that eventually led to a unification of the protist communities.
iii) 454-pyrosequencing revealed an adequate tool not just for investigating the protist diversity but also for reflecting hydrographical situations, as the recirculation of AW in the Fram Strait by protist community structure shifts. The hydrographic system of the Central Arctic Ocean, including Atlantic, Pacific, and mixed water masses, was not that strongly reflected by community structure shifts. The consistent presence of various ice concentrations strongly controlled the community composition and promoted heterotrophic and/or mixotrophic cells. In this regard, the Arctic protist assemblage presented high contributions of dinoflagellates under higher sea-ice concentrations (low light areas) and a higher contribution of diatoms under low sea-ice concentrations (high light areas). The strong response of the Arctic protist assemblage to the changing light conditions finally hampered the identification of water mass associated protist communities and hence, the reflection of the hydrographical situations.
This thesis showed the suitability of 454-pyrosequencing for molecular studies of protist diversity and biogeography, independent of organisms cell size, or organisms nutritional strategy (hetero-, mixo-, or autotrophy). The application of 454-pyrosequencing, to study Arctic protist distribution facilitated to reveal even complex hydrographical situations and indicated the presence of different microbial habitats in polar regions, determined by water
mass properties (T, S, and nutrients) and altered by sea ice concentration. This hypothesizes an improved differentiation of the microbial habitats in the Central Arctic Ocean under continuously sea ice decrease. Moreover, the study of the rare biosphere revealed a constant distribution of taxonomic groups. In contrast to the abundant biosphere, the rare biosphere did not respond to changing nutrient or sea ice concentrations
Fast time scale modulation of pattern generators realized by Echo State Networks
No full textThere are many attempts in the field of robotics and artificial intelligence towards achieving a well-functioning architecture that can control an agent in a stable and elegant manner, while allowing for mixing of behaviors. However, state of the arts robots are far away from the coordinated, complex and graceful behavior that we can observe in animals. A popular approach towards tackling complex motor control problems is the employment of pattern generators. Several architectures have shown good performance, modulating and switching between different patterns. However, those methods perform gradual changes rather than the fast and yet smooth modulations of a pattern for a short duration of time, characteristic for reflexive actions. In this report I present a practical study of a technique that achieves changes in amplitude, frequency and shift of a periodic signal in the time frame of less than a period
Measuring Stress: Assessment of cortisol as psychobiological marker for chronic work stress and burnout
No full textChronic stress has since long been identified as risk factor for physical and mental health. Evidence from epidemiological research has been accumulated showing that chronic stress at work is a relevant risk factor for the development of disease. In order to design appropriate stress intervention and prevention strategies, a deeper understanding of how stress affects human physiology is essential. The biological mechanisms underlying the adverse effects of stress on health are not yet well understood. Recent studies suggest that an altered regulation of the hypothalamus-pituitary-adrenal (HPA) axis with its end-product cortisol might be a biological pathway underlying the link between stress and disease. Cortisol thus might serve as a pre-clinical marker for work stress and burnout and could be used for diagnostic and therapeutic purposes. However, the direction of HPA axis dysregulation remains unclear with reported HPA hyper- or hypoactivity. One potential source for the divergence in the literature might be of methodological origin. Differences in study design, assessment techniques of cortisol and HPA axis regulation as well as assessments of confounding variables might have contributed to the inconclusive pattern of results. Also, different levels of HPA axis functioning might be differentially affected by distinct work stress conditions. The present work thus aimed to identify factors in assessment of cortisol that could help to reduce this inconsistency. Three empirical studies were conducted. The studies contribute to our understanding of how stress can be "measured" by using cortisol as pre-clinical marker. Several methodological issues are addressed concerning the assessment of different dimensions of HPA axis functioning and recommendations for future research in the area of psychoneuroendocrinological stress research are given. Furthermore, some conclusions regarding altered HPA axis activity under conditions of chronic stress exposure are drawn as well
Protist diversity and biogeography in the Pacific sector of the Southern Ocean
No full textThe objectives of this thesis were the establishment of molecular approaches in the diversity investigation of eukaryotic protists in the Southern Ocean and the delivery of a comprehensive and taxon detailed overview of protist assemblages in the Pacific sector of the Southern Ocean, especially in the Amundsen Sea. The molecular approaches used to achieve these goals were automated ribosomal intergenic spacer analysis (ARISA) and 454-pyrosequencing. First, the hypothesis that distinct protist community assemblages characterize large-scale water masses was tested. The composition and biogeography of late summer eukaryotic protist assemblages along a transect from the coast of New Zealand to the eastern Ross Sea was determined. Distinct biogeographic patterns defined by the different oceanic regions were revealed. Different water masses harboured different microbial communities, and environmental gradients limited their dispersal. Picoeukaryotes were of minor importance throughout the investigated transect and were nearly absent south of the Polar Front. Dinoflagellates, Syndiniales, and small stramenopiles dominated the Subantarctic Zone, whereas the importance of diatoms increased southwards, in the Polar Frontal Zone, the Antarctic Zone and the Subpolar Region. South of the Polar Front, haptophytes were the dominating group. Second, the investigation focused on the Amundsen Sea to see if the protist community assemblages vary in different areas of a single large-scale water mass. The composition and structure of late summer eukaryotic protist assemblages along a west-east transect in the Amundsen Sea were analysed. Characteristic communities offshore and inshore were revealed. In general, total chlorophyll a and microeukaryotic contribution were higher in inshore samples. Diatoms were the dominating group across the entire area, at which Eucampia sp. and Pseudo-nitzschia sp. were dominating inshore and Chaetoceros sp. was dominating offshore. At the eastern most station, the assemblage was dominated by Phaeocystis sp. Under the ice, ciliates showed their highest and haptophytes their lowest abundance. This thesis constitutes as groundwork for future investigations of protist assemblage changes in this area
Expression and regulation of levansucrase in Pseudomonas syringae
No full textPseudomonas syringae pv. glycinea PG4180 is an opportunistic plant pathogen which causes bacterial blight disease of soybean (Glycine max). This organism can utilize sucrose, the most abundant plant sugar, with the help of enzyme levansucrase (Lsc). Sucrose is eventually cleaved into glucose, which is further metabolised by the cell, and fructosyl residues, which are polymerized by the same enzyme to form the exopolysaccharide levan. PG4180 has three genes for Lsc, of which two, lscB and lscC are expressed while lscA is not expressed. The first part of this study dealt with determining the presence of multiple lsc genes in P. syringae. Nucleotide sequence alignments between lscB/C and lscA helped identify a prophage associated promoter element present in the upstream of lscB/C ORF but not associated with lscA. We could, for the first time, express lscA in PG4180 under the lscB promoter. However, lscB could not be expressed under the lscA promoter indicating that lscA upstream region does not contain a promoter region. We speculate that lscA might be an ancestral Lsc variant in front of which an active promoter, possibly derived from a bacteriophage, got inserted leading to expression of lscB/C. The second part of this study dealt with identifying transcription factors involved in regulation of Lsc expression in P. syringae. The binding site for a hexose metabolism regulator, HexR, was identified in the promoter region of lscB. As compared to the wild-type, the hexR mutant had severely hampered growth characteristics in medium containing glucose or sucrose as sole source of carbon as well as in planta. DNA affinity chromatography was done to identify two H-NS-like regulatory proteins MvaT-MvaU binding to the promoter region of lscB. Protein-DNA binding assay and overexpression of MvaU in PG4180 suggested that these two proteins could act as repressors of lsc expression. The data suggest a co-regulation of genes involved in extra-cellular sugar acquisition with those involved in intra-cellular energy-providing metabolic pathways as well as virulence and biofilm expression in P. syringae
First Impressions of Trustworthiness: The Roles of the Impression Target, the Impression Holder, and the Physical Environment
No full textFirst impressions of trustworthiness are formed quickly and can have long-term consequences. The literature proposes a large number of factors that could influence first impressions of trustworthiness. However, there exists no single integrative line of research that studies the relative importance and the combined effects of these factors on the first impression of trustworthiness. Therefore, it was the purpose of this dissertation to provide a first systematic investigation in this direction. The PhD project focused on three main groups of factors – those relating to the impression holder (e.g., personality), those relating to the impression target (e.g., structural facial qualities), and those relating to the physical environment (e.g., temperature). Five experiments were conducted that addressed the ways in which these factors affected the trustworthiness impressions – both in isolation and in combination with one another. The results clearly indicate a considerable role of the physical appearance of the impression target in the formations of the trustworthiness impression, whereas the relative importance of the personality of the impression holder and the physical environment appear less straightforward and pose interesting questions for future research. The implications of the findings for theory, research, and everyday life are discussed, limitations of the project are pointed out, and suggestions are put forward regarding the next steps in the study of the factors influencing the first impression of trustworthiness
Identification of bacterial genes required for diatom-bacteria interactions : Gene expression analysis in Marinobacter adhaerens HP15 interacting with Thalassosira weissflogii
No full textAggregate 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
Robotic Mapping in the Real World : Performance Evaluation and System Integration
No full textMapping is an important task for mobile robots. The assessment of the quality of maps in a simple, efficient and automated way is not trivial and an ongoing research topic. In this thesis, evaluation methods for the maps produced by robotic systems are developed. The algorithm has to analyze and evaluate the maps in a systematic, repeatable and reproducible way. The problem is approached systematically: First the different terms and concepts are introduced and the state of the art in map evaluation is presented. Then a special type of mapping using video data is introduced and a path-based evaluation of the performance of this mapping approach is made. Afterwards a number of algorithms to process those maps are presented. Then the first novel map evaluation method, the Fiducial algorithm, is developed. In this place-based method, artificial markers that are distributed in the environment are detected in the map. The errors of the positions of those markers with respect to the known ground truth positions are used to calculate a number of attributes of the map. The main contribution of this thesis is the second novel map evaluation algorithm, that uses a graph that is representing the environment topologically. This structure-based approach abstracts from all other information in the map and just uses the topological information about which areas are directly connected to asses the quality of the map. Different ways to compare the similarity of two vertices from two graphs are presented and compared. This is needed to match two graphs to each other - the graph from the map to be evaluated and the graph of a known ground truth map. Using this match, a number of map attributes can be computed, including the interesting the map brokenness. Experiments made on many maps from different environments are then performed for both map metrics