Broader background of the proposed research project

The extraordinary molecular complexity of DOM combined with its almost infinite dilution of each single species suggests that this material could be chemical waste resulting from erroneous metabolic transformations (Wortmann et al. 2015). The synthesis of fatty acids from acetyl-CoA via malonyl-CoA (Bae 2015) is a metabolic pathway commonly used by all marine organisms (Finzel et al. 2015, Dibrova et al. 2014, Li et al. 2014). We propose the hypothesis that synthetic mistakes in the five step elongation by two carbon atoms (Claisen condensation, decarboxylation, carbonyl reduction, water elimination, C-C double bond reduction) could lead to such complex mixtures of useless "waste" byproducts without biological function, which are secreted into the marine environment. Actually, further postbiosynthetic intra- or extracellular processing by aldol condensation could be assumed leading to polyketide type constitutions. Furthermore, by incorporation of propionyl instead of acetyl-CoA building blocks, also species with odd carbon numbers could be generated.

Outline for the proposed PhD research project

Aim of the PhD thesis is to simulate the molecular complexity of DOM by chemical synthesis. The molecular complexity of DOM should be synthetically modelled by the product distribution obtained by Claisen condensations from beta-dicarbonyl compounds (as a mimic of malonyl-CoA). Products of these transformations are prone for subsequent aldol (in particular Knoevenagel) processes under suitable basic or acidic reaction conditions, thus, leading to polyketide type products including hydroxylated and annulated aromatic rings. Of course, not the physiological reaction conditions shall be mimicked, but the products distribution patterns. Thus, reaction conditions will preferably be elevated temperatures, high concentrations and strongly acidic or basic heterogeneous catalysts, such as aluminum silicates ("zeolites"). Product distributions will be analyzed by GC, GC-MS-coupling techniques (for higher molecular weights LC, LC-MS) and ultrahigh-resolution MS (FT-ICR-MS) and compared with data obtained from natural DOM. Furthermore, selected unique species with be isolated by preparative LC, and their constitution elucidated by NMR-spectroscopic methods. If required, independent synthetic routes to selected species will be developed. Molecular analyses will be performed in the central analytical facility of IfC, and in cooperation with WPs 3 and 4. The synthetic diverse mixtures will used as substrates for microbiological incubations in WP 2.

Broader background of the proposed research project

Sulfate-reducing prokaryotes (SRP) are major drivers of the carbon cycle in marine sediments. At highly active sites (e.g. Wadden Sea), terminal oxidation of organic matter (OM) is channeled to a large part through microbial sulfate reduction. This rests upon the nutritional versatility combined with complete substrate oxidation (to CO2) essentially confined to the deltaproteobacterial family of Desulfobacteraceae (Rabus et al. 2000). Indeed, its members were repeatedly demonstrated to dominate the SRP community in diverse marine sediments (e.g. Llobet-Brossa et al. 2002). Recent (proteo)genomic studies, e.g. of chemolithoautotrophic Desulfobacterium autotrophicum (Strittmatter et al. 2009) and aromatic compound-degrading Desulfobacula toluolica (Wöhlbrand et al. 2013), have revealed complex metabolic networks and thereby underpinned the necessity and benefit of comparative metabolic reconstruction of other Desulfobacteraceae members (Rabus et al. 2015). Combined proteogenomic and metabolomic studies of SRP promise to provide a valuable knowledge base on how these microbes shape the to date mostly unexplored “geo-metabolome” in marine sediments.

Outline for the proposed PhD research project

Metabolic and protein networks of Desulfobacteraceae are in the focus of this PhD project. The proposed project is based on the hypothesis that Desulfobacteraceae members by virtue of their broad metabolic capacities significantly imprint on OM composition in marine sediments. Comparative proteogenomics of metabolic networks and transporter complements will assess the strain-specific vs. cumulative metabolic potential of selected Desulfobacteraceae members with respect to (i) which organic substances can be depleted from sedimentary OM and (ii) which metabolites and cellular components are released from the cells and thereby add to OM complexity. This project will be based on (proteo)genomic data already existing in AG Rabus for D. autotrophicum, D. toluolica (both published), Desulfococcus multivorans (unpublished) and on genomes of further SRP (incl. filamentous and oil-degrading species). Thus, the main part of this project will be concerned with comparative analysis. Differential (subcellular) proteomics will be conducted for the newly genome-sequenced strains (full cycle proteomics is well established in AG Rabus). In addition, all studied SRPs will be batch-cultured with selected substrates to provide samples for targeted (WP3) and global (WP4) analysis of metabolites. To integrate the proteogenomic and metabolomic data and to unravel unnoticed causal connections, mathematical modelling will be performed by WP10. The data integration/modelling will also allow a comparison of OM dynamics between pelagic and benthic environments, generating a unique added value for EcoMol.

In her PhD-project Development and Application of a Method for the Identification and Quantification of Organic Acids in Microbial Exometabolomes Simone Heyen developed new methods for a more detailed and specific analysis of metabolits, whith a wide range of application not limited to microbial samples.

Anaerobic bacteria capable of thriving in the absence of oxygen transform hydrocarbons employing well-understood degradation pathways. During this process, a variety of intracellular metabolites are released into the extracellular environment – the exometabolome – and are ultimately integrated into the dissolved organic matter pool when encountered in aquatic environments. However, the mechanisms behind the release of exometabolites have not been extensively studied so far.

As organic acids are the main contributors to the metabolite diversity in bacterial cells, a selective and sensitive method for the extraction and analysis of these target compounds in microbial samples was developed and validated. Applying anionic exchange solid-phase extraction coupled to a subsequent gas chromatographic-mass spectrometric analysis, the new method was established for reference standards of 24 aromatic and aliphatic carboxylic acids with and without further functional groups. In comparison with existing methods, improved analytical performance like higher recovery as well as the lower limits of detection and quantification were achieved. This method was applied to exometabolomes of the n-alkane-degrading, denitrifying betaproteobacterium Aromatoleum sp. HxN1 supplied with various carboxylic acids, n-hexane or crude oil as substrates, respectively. An enhanced release of transformation products linked to the co-metabolic degradation of non growth-promoting compounds was observed during growth with crude oil. Additionally, the dissolved organic matter composition was investigated via FT-ICR-MS, revealing major variances in the presence and concentration of metabolites released by the cells during growth, especially when crude oil was supplied as substrate.

The method developed in this project is already in use for further studies and is not limited to microbial samples. Various follow-up experiments involving other substrates, organisms and an increasing complexity of the bacterial community can potentially be useful to gain further insights into the systematics behind the release of exometabolites, finally approaching compositions encountered in natural environments.

Broader background of the proposed research project

Dissolved organic matter (DOM) is the main nutrient and energy source for marine bacterioplankton (Azam et al 1983, Fuhrman et al. 2015). To understand the interactions between DOM and the bacterial community, it is important to identify the key players on both sides in detail, i.e. chemically distinct moieties in DOM and the various bacterial taxa (Osterholz et al. 2015b). Technical advances in molecular microbial ecology (Edwards & Dinsdale 2007) and organic geochem-istry (Dittmar & Paeng 2009) have enabled the ultrahigh-resolution analysis of both, the microbial community and DOM. Pyrosequencing-based analysis facilitates the classification of millions of reads of environmental DNA and RNA amplicons and ultrahigh-resolution mass spectrometry (via the Fourier-transform ion cyclotron resonance technique, FT-ICR-MS) yields up to 10,000 DOM molecular formulae in a marine water sample. Linking this detailed biological and chemical information is a crucial first step towards a mechanistic understanding of the role of microorganisms in the marine carbon cycle (Romano et al. 2014, Osterholz et al. 2015b).

Outline for the proposed PhD research project

Over the past few years, we have collaboratively collected complex data sets on several hundred samples on experimental exometabolomes, natural geometabolomes, and associated microbial communities. For each sample, (semi-)quantitative information of >10,000 individual variables are available. These data have been interpreted in the context of the respective individual study, but not yet in the framework of EcoMol. Aim of this PhD thesis is to analyze the available data on correlative patterns between microbial community and molecular DOM composition. The emerging patterns will be interpreted in a biogeochemical context. We hypothesize that the bacterial community composition, especially of the active heterotrophic community, is closely related to the composition of the main energy and carbon source, DOM. We will interpret the complex microbiological and molecular information via a novel combination of multivariate statistics, as pioneered by Osterholz et al. (2015b). The same data will be analyzed by associated WP 8 (microbial communities), WP 10 (ecological modelling), and WP 11 (ecological network and time series modelling). Each of these WPs has their own focus, with different research questions and different data analysis tools. Close interactions within this sub-cluster will yield a holistic picture of molecule-organism interactions in an ecological context. In addition, we will closely interact with all WPs that require support with non-targeted molecular analysis. The PhD student of WP 4 will be involved in all interactions, but actual analytical work would be beyond the scope of the proposed PhD thesis. Practical analytical support will be provided by the technical personal in Dittmar’s research group.

Broader background of the proposed research project

There is accumulating evidence that marine DOM is an extraordinarily diverse blend of indi-vidual organic compounds and consists of thousands of different molecular formulae (Flerus et al. 2012, Lechtenfeld et al. 2015, Osterholz et al. 2015a). The DOM diversity patterns appear to be very constant over space and time in marine pelagic systems, assuming that sources and sinks act rather similarly. Phytoplankton primary production and microbial DOM processing, are the primary biological sources and sinks but still little is known about the impact of individual phytoplankton algae and heterotrophic bacteria on the composition of marine DOM. It has been shown that the molecular diversity of the exometabolome of a marine pelagic alphaproteobacterium, Pseudovibrio sp., growing in rich organic media is very different from that of marine DOM (Romano et al. 2014) but, when growing at natural DOM concentrations, has a distinct impact on the diversity of marine DOM (Schwedt et al. 2015). Also natural marine bacterial communities clearly influence the diversity and composition of marine DOM (Lechtenfeld et al. 2015, Osterholz et al. 2015a).

Outline for the proposed PhD research project

We hypothesize that the concerted action of individual marine microbes are instrumental in shaping the marine geometabolome. To address the above mentioned hypothesis we will carry out a comprehensive comparative data analysis of the exometabolomes of individual and mixed bacterial species, phytoplankton communities, geometabolomic DOM patterns and the composition und functional properties of bacterioplankton communities. The aim is to elucidate how the marine geometabolome is shaped by the metabolic activity of microbes and to identify the significance and function of individual microbial species and communities for these processes. Data will be provided by ongoing projects on the exometabolome of marine Roseobacter species within TRR 51 Roseobacter and approved research cruises of RV Sonne in the Pacific in 2016 (transect at ~180°E from 30°S to 60°N) and 2017 (transect at ~180°E from 30°S to 60°S) for marine DOM and bacterioplankton community data. Further, if granted, additional data will be provided by large scale mesocosm experiments to investigate phytoplankton blooms under different nutrient stoichiometry which are planned for 2016 and 2017 (PlanktoDyn). If necessary, these data will be complemented by few specific experiments to address distinct questions on the role of individual microbes, bacteria and/or phytoplankton algae, in the context of DOM processing and their role in geometabolomics.

Broader background of the proposed research project

Water samples that are collected over a time series from a mesocosm experiment render a sequence of snapshots that reflect dynamical changes of the microbial and molecular community (waxing and waning of concentrations). The concentration dynamics can be resolved down to microbial operational taxonomic units (OTUs) and molecular species level by advanced analytic techniques (next generation sequencing of the 16S rRNA gene for the microbial community, ultrahigh-resolution mass spectrometry for DOM). When applied to the sequence of water samples these analyses yield a wealth of time series reflecting the microbial and molecular concentration dynamics. An explanation of the observed concentration changes from the microbial/molecular interaction and a reconstruction of the association network (Fuhrman 2009, Fuhrman et al. 2015) from the mentioned time series poses a methodological challenge (in view of the multitude of species and the necessarily limited length of the time series). The empirical network reconstruction may be based on a class of generic models (VAR) or on paradigmatic models that are widely used in the context of ecological dynamics. An early ecological model description (Billen et al. 1980) of a single species bacteria/substrate dynamics shall serve as the starting point for many-species generalizations.

Outline for the proposed PhD research project

Time series data of the microbial and the DOM compartment are available for Helgoland Roads and for mesocosm experiments. These data will be provided by WPs 4, 8 and 13 and will be analyzed in collaboration with these associated WPs. The resulting high-dimensional time series will be aggregated to describe bacterial consortia and molecular clusters using recently advanced cluster analysis methods (Röder et al. in preparation). An assessment of resulting clusters will be done in collaboration with WPs 4 and 8 (Teeling et al. 2012). The consensus time series of each cluster will serve as an input to the network reconstruction. Besides measures of undirected pair-correlation we aim also at applying measures detecting directed interactions. The concept of Granger causality is well established for time-discrete linear stochastic processes (VAR) and a model-free counterpart based on transfer entropy exists; unfortunately, the last-mentioned approach works only for sufficiently long time series. Instead we will investigate in how far ecological model descriptions of the interaction between microbial OTUs and DOM clusters can be used to faithfully reconstruct the interaction network. In the setup and development of paradigmatic network models we will collaborate with WP10.

Monitoring the succession of a phytoplankton spring bloom within the North Sea is challenging due to environmental conditions and water movements. To monitor changes within a distinct and controlled water body, Master student Nils Hintz established a North Sea spring bloom in eight large-scale indoor mesocosms (Planktotrons).

Over the experimental duration of 38 days, two distinct blooms occurred and were high frequently sampled. These blooms were investigated under aspects of quantitative phytoplankton properties (biovolume and -mass) as well as qualitative phytoplankton properties (accessory pigment composition and ecological stoichiometry). Phytoplankton community succession turned from a Bacillariophyceae (Thalassiosira spp.) dominated first bloom towards a Prymnesiophyceae (P. globosa) dominated second bloom. The molar ratios of C:N and C:P increased, indicating a decrease in food quality for heterotrophic grazers. The phytoplankton diversity and pigment diversity decreased. In conclusion, the quantitative properties were significantly stronger affected by the bloom event as the qualitative properties. Further work will synthesize these and other results of biotic and abiotic investigations within this mesocosm experiment.

The work of doctoral student Miriam Gerhard Phytoplankton community responses to nutrient availability: interactions with temperature and diversity was associated to WP 14 of the EcoMol project. She investigated the responses of freshwater phytoplankton communities to different nutrient conditions considering interactions of temperature regimes (i.e., temporal variability in temperature) and diversity under controlled laboratory conditions. These aspects were evaluated conducting three experiments where nutrient conditions, temperature regimes and diversity were differently combined. With her work she gained important insights into the complex interactions among environmental factors and community`s characteristics in freshwater ecosystems. The significance and quality of her work was consequently acknowledged by being granted the OLB Wissenschaftspreis in 2020.

Freshwater ecosystems provide critical ecosystem services to human societies, but are strongly affected by anthropogenic activities. Nutrient enrichment is one of the most prevalent disturbance in lakes, producing changes in primary producers functional and structural aspects continuing at higher trophic levels and whole ecosystems. Temperature is another crucial factor driving changes in phytoplankton communities influencing metabolic and physiological processes. Investigating temperature increases on biological communities have become relevant to predict responses to global warming, but also temperature dynamics have been shown to affect ectotherms performance. Single effects of nutrient availability and temperature changes on phytoplankton communities are modulated

by interactive effects of these factors as well as with community features. The ability of phytoplankton communities to respond to environmental changes is critical for the maintenance of ecosystem processes, and has been related to high species diversity and the associated functional diversity.

However, many of these complex interactions among environmental factors and community`s characteristics have been poorly tested under controlled experimental conditions, and have been mostly approached using single species or artificial assemblages.

Miriam found that

1. fluctuating temperatures decreased phytoplankton community growth rate in comparison to constant temperature as has been shown for single species. However, the phytoplankton carrying capacity increased under temperature fluctuations, which has not been investigated before. The effect size (i.e. magnitude of the effect) between thermal regimes were mediated by the nutrient context, highlighting the importance of the interactive effects between the evaluated factors. Thus, multiple-nutrient balance and thermal regimes mediate phytoplankton responses to environmental change.
2. that more realistic biodiversity loss scenarios in experiments can yield different results from those found using classical assemblage`s approaches.
3. heterogeneous environments combined with high diversity promote complementarity effects according to theoretical and modelling predictions. Hence, the effect of rare species losses on ecosystem functioning can be compensated by the persistent species in the community when nutrient ratios are similar, but might have important consequences for ecosystems under changing nutrient context.

Overall, the results highlight the relevance of investigations at the phytoplankton community level and the importance of including multiple factors evaluations when analyzing responses to change.

As well in the framework of WP 13, Nilz Hintz conducted his Master thesis, entitled:

Community composition and stoichiometry of a phytoplankton spring bloom: an indoor mesocosm (Planktotron) experiment with high temporal resolution

Monitoring the succession of a phytoplankton spring bloom within the North Sea is challenging due to environmental conditions and water movements. To monitor changes within a distinct and controlled water body, a North Sea spring bloom was established in eight large-scale indoor mesocosms (Planktotrons). Over the experimental duration of 38 days, two distinct blooms occurred and were high frequently sampled. These blooms were investigated under aspects of quantitative phytoplankton properties (biovolume and -mass) as well as qualitative phytoplankton properties (accessory pigment composition and ecological stoichiometry). Phytoplankton community succession turned from a Bacillariophyceae (Thalassiosira spp.) dominated first bloom towards a Prymnesiophyceae (P. globosa) dominated second bloom. The molar ratios of C:N and C:P increased, indicating a decrease in food quality for heterotrophic grazers. The phytoplankton diversity and pigment diversity decreased. In conclusion, the quantitative properties were significantly stronger affected by the bloom event as the qualitative properties. Further work will synthesize these and other results of biotic and abiotic investigations within this mesocosm experiment.

Based on their open ocean concentrations pattern, trace metal (TM) behavior is operationally subdivided into nutrient-, scavenging and conservative-type elements. Regardless of this classification, they can be actively and/or passively involved in biological processes. They can serve as potentially primary production limiting bio-essential as well as non-bio-essential or even potentially harmful agents. TM cycling in often dynamic and highly productive coastal environments is more complex compared to open ocean observations, which results in behavioral deviations from this open ocean classification system. Along the coastal transect from the riverine freshwater endmember to the edge of the continental margin, TM are subject to intensive physico-biogeochemical element cycling, all of which shape and potentially leave their imprint on their concentration patterns. The coastal areas are an important interface between land and open ocean and act as gateways to the atmosphere and sediment reservoirs. They function as filter-, alteration-, if not even catalyst-systems and significantly shape the export of allochthonous and autochthonous, naturally as well as anthropogenically derived organic and inorganic materials to the open ocean environment.

Corinna’s goals were to identify key processes which shape coastal TM concentration along the land-ocean continuum, to assess possible reasons for TM behavior deviating from the open ocean framework, and to estimate the impact of coastal processes on open ocean export fluxes. Her work focused on the TM manganese (Mn), iron (Fe), molybdenum (Mo) and thallium (Tl).

Using interdisciplinary approaches and by combining field and mesocosm-based studies of varying complexity, Corinna could show that the abundance and composition of the OM and the inorganic elemental pool is significantly altered along the coastal continuum.

The field-based case study of the mangrove-framed estuarine system in Australia revealed that fluvial and estuarine derived aromatic, sulfur- and nitrogen-containing dissolved organic matter (DOM) compounds were removed and altered by the estuarine filter before reaching the coastal ocean, leading to a discharge of aliphatic DOM. Crucial for the pool budgets and composition was the tidally driven exchange of estuarine surface waters with the mangrove-porewaters of the adjacent redox-stratified sediment reservoir. Thereby, the estuarine system served as a net source for Mn via discharged of Mn-enriched reducing porewaters, and as a sink for riverine derived Fe, which was captured in the sulfidic areas of the sediment reservoir.

Two mesocosm-based case studies both focused on the response of TM cycling to phytoplankton blooms and therewith associated processes. These studies focused mainly on the supposedly conservative type elements Mo and Tl, whose concentration patterns should not be influenced by active or passive coupling with OM cycling. A circumstance, however, which has already been challenged by previous studies. Since Mo as well as Tl cycling are often associated with the cycling of bio-essential and redox-sensitive metals Fe and Mn, those were investigated as well. The first mesocosm setup was based on the tidal influenced area in the southern North Sea at the time of a phytoplankton summer bloom. This approach, however, made it impossible to clearly identify whether the temporal pattern was induced by biologically derived or lithogenic processes, in particular in the case of Tl. This could  be better examined in the second mesocosm approach, where a spring bloom was incubated in artificial seawater (in order to minimize the terrigenous DOM background) without an adjacent sediment reservoir.

In both case studies Fe was found to be predominantly present as organometallic complex and thus shielded from potential oxidation-induced flocculation. The temporal pattern of Mn was in both approaches highly correlated to the bloom itself. The temporal concentration pattern was mainly determined by its assimilation as micro-nutrient and/or by scavenging by OM.

The first mesocosm approach revealed a non-conservative behavior of Mo as well as Tl during the incubation of a phytoplankton summer bloom. While the positive concentration anomalies of Mo in the water column were attributed to the oxidation of reduced bottom sediments, negative Tl-anomalies were suggested to be induced by its immobilization by algae-derived OM prior to a potential fixation in the sulfidic areas of the adjacent sediment reservoir. Both anomaly patterns seemed to depend on both the bloom intensity and related processes as well as on the properties of the adjacent sediment reservoir.

Non-conservative Tl depletion was also observed in the second mesocosm approach reflecting a phytoplankton spring bloom in the North Sea. Here, the concentration patterns were neither disturbed by lithogenic background nor by a terrigenous OM background pool. The results suggest that the negative Tl anomalies resulted from its immobilization and fixation by Phaeocystis sp. derived OM and associated processes. The extent of the Tl-anomalies seemed to be dependent on the bloom intensity and the related excretion of DOM (e.g. carbohydrates, amino acids) by phytoplankton cells themselves, as well as the formation of redox-stratified hydrogels.

In general, the findings of this thesis reveal that OM (living and non-living) and TM cycling are tightly coupled and influence each other. Thereby the response of the studied TM depends on the abundance as well as composition of the present OM (living and non-living) pool. It could be shown, that the processes in terms of filter capacity and alteration potential for the studied TM are spatially and temporarily highly variable.

Climate change and the resulting increase of ocean surface temperatures are threatening coral reefs at unprecedented levels, often leading to severe mass bleaching events on a global scale. The survival of these biodiverse ecosystems largely depends on the recruitment of new generations of corals, and we urgently need to extend our knowledge on the responsible molecular processes and crucial environmental drivers involved in this process. PhD candidate Lars-Erik Petersen investigated the role of bacteria as well as the responsible signaling molecules in coral settlement, a process in which planktonic larvae attach to a surface and metamorphose into sessile polyps. Testing the settlement-inducing effect of algae-associated bacterial mono- and multispecies biofilms on coral larvae revealed that specific bacterial species are potent inducers (> 70%) and that multispecies biofilms consisting of highly-inductive strains can synergistically increase settlement levels (> 90%). Subsequent analyses of their secondary metabolites demonstrated the impact of chemical signaling in coral settlement not only through the discovery of a previously unknown chemical settlement inducer and the importance of light (and its absence) for its activity, but also by uncovering its molecular mechanism of action as a redox-reactive energy storage molecule and proposing a model system for photochemical degradation and signaling between bacteria and corals. The discoveries of Lars’ PhD provided novel insights into the settlement process of reef-building corals that will advance a wide range of functional studies on coral settlement biology and thus will aid scientists in conducting reef restoration. 

Broader background of the proposed research project

DOM can act as olfactory cues potentially influencing migration behavior of marine species. While laboratory tests have shown olfactory preferences for specific cues, very little is known whether and how species are responding to DOM in their natural habitat and ecosystem. To test this, we will establish a new detection method of animals in the marine environment and correlate the presence of species with presence or absence of specific DOM. For detecting species we will develop an eDNA method. Free DNA molecules are ubiquitous, released from skin, mucous, urine eggs etc. and are collectively referred to as eDNA (Bohmann et al. 2014). Extraction and identification of waterborne potentially degraded environmental DNA (eDNA) originating from many different organisms, has proven noteworthy in detecting the presence of species. While eDNA can persist in sediments for centuries or millennia, waterborne eDNA may degrade much faster within short time spans (2 weeks) (Thomsen et al. 2011). Studies have shown eDNA concentration to be directly related to number of individuals in mesocosms and natural ponds, but several issues still need to be addressed to validate this method in the marine environment. The implementation of so-called ecosystem-based approaches (Foote et al. 2012) will take a more holistic view than single species studies.

Outline for the proposed PhD research project

The project aims to determine waterborne environmental DNA (eDNA) of migrating species in the North Sea to test for any correlation of their appearance and presence of specific DOM compounds which can serve as chemical attractants. In our project we aim to establish a genetically based detection method to monitor the occurrence of species close to a source of chemical attractants. We will first focus on crustaceans species such as the invading crab Hemigrapsus takanoi and the native crab Carcinus maenas; develop species specific primers that amplify a fragment of the CO1 gene. Chemical attractants which will be identified under laboratory conditions beforehand will be released by chemical source devices. By taking water samples and by eDNA analysis we will determine the presence and attraction of responding species even when we do not see – or do not catch them.

At the beginning, the Ph.D. student will test for the specificity of primers to detect mtDNA CO1 fragments of single individuals and of groups consisting of single and mixed species. He/she will verify qualitatively and quantitatively the detection of species eDNA. Once established, we will take and analyze water samples for eDNA in different distances to the chemical source device in the North Sea to determine the density of species. This project will be conducted in close collaboration with Dittmar (WP 4) to assess the attraction of species responding to chemical signals in the wild. We will also closely collaborate with WP 6 where a different model system is investigated in a similar context.

Broader background of the proposed research project

Assessing the dynamics of the DOM pool and its interactions with the marine ecosystem re-quires rapid in situ sensing techniques that can complement sophisticated laboratory-based analytical methodologies like FT-ICR-MS. Namely these rapid sensing approaches target inherent optical properties including the absorption of chromophoric dissolved organic matter (CDOM) and the fluorescent fraction (FDOM) the latter typically analyzed by excitation-emission-matrix-spectroscopy (EEMS) (Moore et al. 2009; Zielinski et al. 2009). While EEMS are an established laboratory method and extensive field data is (e.g. Garaba et al. 2014) and will be available from seagoing expeditions, recent developments achieved in the EU project NeXOS (www.nexosproject.eu) make in situ quasi-EEMS sensing possible (Delory et al. 2014). Additionally combining these methodologies with a microfluidic sample treatment system (Gaßmann et al. 2015) will result in a highly specific FDOM sensor system setup that can be operated underway, that means continuously during the vessels movement, thus providing unchallenged high resolution optical (F)DOM characteristics that can be linked to the “ecology of molecules”.

Outline for the proposed PhD research project

In the proposed project, we will test the hypothesis that FDOM excitation-emission-matrices show a high variability and complexity that can be linked to ecosystem dynamics. To address this topic we will correlate multidimensional FDOM data collected and processed during the approved research cruises on RV Sonne in 2016 and 2017, as well as data obtained from supplemental studies, with the EcoMol observables, namely from microbial communities (WP8) the pelagic chemical diversity (WP4), molecular interactions (WPs 6 and 7) and the sea surface processes (WP15). Underway and in situ sensing of complex FDOM spectral characteristics will be achieved by excitation-emission-matrix spectroscopy (EEMS) a) from discrete water samples with available state-of-the-art laboratory instrumentation and b) from an online quasi-EEMS sensing (a novel technological innovation) that is optionally c) coupled with a microfluidic sample treatment system (WP5) exposing the sample to high UV-radiation and/or adsorbing processing. All three approaches will use statistical chemometric analysis and establish a database of the molecular FDOM complexity of the non-living environment and the diversity of interactions with organisms thus contributing to the overall EcoMol concept.

Broader background of the proposed research project

For the investigation of DOM numerous sophisticated laboratory analysis methodologies are available, still further methodologies need to be developed, especially with respect to the field applicability. With the help of a microfluidic system well defined abiotic conditions (e.g. temperature, UV radiation, etc.) can be achieved in a uniform, precise and rapid way. Thus DOM samples can be exposed to these conditions and subsequent changes of the sample can be studied. This will help to understand the complex behavior of the DOM.

In previous experiments, the microfluidic treatment of DOM samples with temperature was already carried out (Gassmann et al. 2015, Miranda et al. 2015). The advantage of the microfluidic setup was demonstrated and a significant change in the fluorescence excitation-emission-matrix spectrum of the DOM sample was observed. The response of DOM to UV is well documented and occurs especially near the sea surface (Wurl et al. 2009, Blough 1997). Specific conditions of the surface layer can be mimicked using the microfluidic approach for a better understanding. Further investigations will include separating columns or electrical fields added to the system. The in-depth investigation of the temperature influenced structural change of DOM as well as the treatment with high ultraviolet (UV) radiation will be done in the framework of EcoMol. Once the response of DOM under the treatment is understood this methodology can be used as an improvement of existing sensing methodologies. So the sample is treated first with the microfluidic system potentially helping to remove cross sensitivities and leading to more precise sensing result.

Outline for the proposed PhD research project

During this research project a microfluidic system will be developed which is able to apply different treatments to DOM samples. First, a system for laboratory use will be developed. Different sample treatments should be implemented (e.g. temperature, UV, electrical field, separation columns). Once the system is operable different treatments with known samples will be performed. The results need to be validated with sophisticated laboratory analysis methodologies available in EcoMol (e.g. FT-ICR-MS, WPs 3 and 4). Main questions concerning UV treatment could be: Can the DOM break up at the sea surface (WP 15) as modeled in the microfluidic system? How will DOM behave when parameters like wavelength or intensity are changed? This aims towards a better understanding of the complex behavior of the DOM and should be used to improve in situ sensors. The PhD research will be combining engineering work with aspects of the biology and chemistry of the ocean. The EcoMol training group will be the ideal framework for this cooperation.