Metabolite profiling of the chemosphere of the macroalga ulva (ulvales, chlorophyta) and its associated bacteria

The eukaryotic green marine algae Ulva spp. (Chlorophyta), are usually associated with marine bacteria and exhibit therefore microbe-dependent growth and morphotypes. Ulva spp. might actively affect their microbiome by releasing specific compounds in its chemosphere. For instance, algal oxylipins including polyunsaturated aldehydes (PUAs) derived from polyunsaturated acids (PUFAs) might play an important structuring role for the microbiome. In the present study, Ulva spp. collected at various sampling sites in the lagoon of the Ria Formosa (Portugal) have been studied with respect to (1) their ability to produce polyunsaturated aldehydes and (2) their ability to communicate with their surrounding bacteria via infochemicals. Lipoxygenase/hydroperoxidelyase mediated transformations convert polyunsaturated fatty acids into various oxylipins. These fatty acid transformations are highly diverse in marine algae and play a crucial role in e.g., signaling, chemical defense, and stress response often mediated through polyunsaturated aldehydes (PUAs). In this study, Ulva spp. were surveyed for PUAs. Ulva species with sea-lettuce like morphotype were demonstrated to produce elevated amounts of volatile C10-polyunsaturated aldehydes (2,4,7-decatrienal and 2,4-decadienal) upon tissue damage in contrast to Ulva species with tube-like morphotype. Moreover, morphogenetic and phylogenetic analyses of the collected Ulva species revealed chemotaxonomic significance of the perspective biosynthetic pathways. The aldehydes are derived from omega-3 and omega-6 polyunsaturated fatty acids (PUFA) with 20 or 18 carbon atoms including eicosapentaenoic acid (C20:5 n-3), arachidonic acid (C20:4 n-6), stearidonic acid (C18:4 n-3), and gamma-linolenic acid (C18:3 n-6). As first evidences in this study, it was found that lipoxygenase-mediated (11-LOX and 9-LOX) eicosanoid and octadecanoid pathways catalyze the transformation of C20- and C18-polyunsaturated fatty acids into PUAs and concomitantly into short chain hydroxylated fatty acids. Ulva mutabilis Foyn (sl) with tube-like morphotype was used as an objective to investigate the chemical mediated interaction (infochemicals) within the chemosphere of tripartite community consisting of U. mutabilis and its associated marine bacteria i.e., Roseobacter and Cytophaga species. In the absence of these bacteria (axenic conditions), U. mutabilis forms callus-like colonies. However, the combination of the two bacterial strains, Roseobacter sp. and Cytophaga sp. can completely restore the morphogenesis of U. mutabilis forming a symbiotic tripartite community. The exo-metabolome of the chemosphere of this tripartite community was surveyed along with the biological metadata. Two different approaches and cultivation conditions i.e., sterile 25 L bioreactor cultures and non-sterile 200 L outdoor aquacultures were conducted which cultures were inoculated with axenic seven days old germlings. Indeed, it was feasible to observe the whole life cycle of the gametophyte under these conditions when the appropriate bacteria were inoculated as well. Hereby, the medium did not need to be changed. Bioassays revealed that U. mutabilis passed through three statuses of gametogenesis inducibility which can be distinguished whether Ulva is able to onset the gametogenesis: (1) gametogenesis is not inducible, (2) gametogenesis can be induced or (3) it starts even spontaneously. The nutrient depletion over the reproductive cycle shows that the utilization rate of nitrate as a limiting growth factor was significantly high during the inducible status, when the macroalgae was growing. The waterborne metabolites were extracted by solid phase extraction. The samples were directly analyzed by ultra-high performance liquid chromatography (UHPLC) and by gas chromatography (after derivatization) coupled with a time-of-flight mass spectrometer (TOF-MS). Interestingly, discriminant analysis proofed that all waterborne metabolites obtained either from GC-MS or LC-MS were corresponding to the inducibility status of gametogenesis of U. mutabilis in both cultivation conditions. Even more interesting, many unknown biomarkers were found to be common in both bioreactor cultures and aquaculture, insuring the high probability of using these biomarkers as indicators to determine the growth phases corresponding to the status of gametogenesis inducibility in U. mutabilis under any cultivation condition in future land based aquacultures. Moreover, the present study revealed remarkable metabolic fingerprints which might due to the adaptation of U. mutabilis to changes in its surrounding environment. For instance, the algal biomarker 2,4,6-tribromophenol was detected in the chemosphere of the tripartite community under sterile cultivation (bioreactor) but not in the well-defined bacterial community under non-sterile cultivation (aquaculture). In summary, the changes of the metabolite profile between the growth phases were significant. Therefore, various statues in algal growth and life cycle can be predicted based on the dynamics of waterborne metabolites. This knowledge will be essential in order to maintain land based aquacultures providing economical relevant amounts of biomasses.