Biofilm cultures to understand Ostreopsis benthic HABs

BIOFILM CULTURES TO UNDERSTAND OSTREOPSIS BENTHIC HABS Roberta Congestri1 (roberta.congestri@uniroma2.it), Francesca Di Pippo2 (francesca.di.pippo@uniroma2.it), Patrizia Albertano1 (albertano@uniroma2.it) 1University of Rome “Tor Vergata”, Department of Biology, LBA- Laboratory of Biology of Algae, Via della Ricerca scientifica, 00133 Rome, 2CNR–IAMC, National Research Council, Institute for Coastal Marine Environment, Località Sa Mardini, Torregrande, 09072 Oristano, Italy. An incubator prototype specially designed for culturing aquatic phototrophic biofilms on substrata at controlled environmental conditions was used to perform semi-continuous flow microcosm experiments with microphytobenthos sampled during toxic Ostreopsis ovata outbreaks in summer along the middle Tyrrhenian Sea coast. The system provided the possibility of simultaneously controlling environmental conditions and co-culturing biofilm organisms on a set of artificial substrata in a closed photobioreactor, providing a realistic assessment of community processes and maintaining biofilm integrity during sampling. The design allowed to study the effect of time, site, substratum of in situ growth and the role of key species in community development starting from natural inocula, cultured on polycarbonate slides, in a moving film of K/2 medium (50 L h-1 flow velocity) under controlled temperature (25°C) and light conditions (110 mol photons m-2s-1). Benthic dinoflagellates, diatoms and filamentous cyanobacteria constituted significant fractions, in taxon richness and biomass, of the natural biofilms formed on macroalgae and bryozoans used as inocula. Biofilm cells were all embedded in a common mucilage matrix (EPS, exopolymeric substances). Non-destructive, real time monitoring of biomass accumulation was obtained by recordings of transmittance values by light sensors positioned under the slides. Growth curves indicated that biofilms reached a mature stage in 40 to 50 days. Species accrual, growth and compositional shifts over time were monitored using a variety of microscopical technique, including confocal laser scanning microscopy. Initial adhesion and substratum colonisation appeared as stochastic processes, then community structure and physiognomy, especially of the photosynthetic fraction, visibly changed with time. Prevalence of cyanobacteria at late growth stages was detected in all cultures, which showed complex layering, inner voids and channels. HPLC on matrix sugars revealed variations in total amounts and composition. No toxic compounds were detected in the final communities tested by LC-MS and MALDI-TOF MS techniques.