Modelling the Stoichiometric Regulation of C-Rich Toxins in Marine Dinoflagellates

Toxin production in marine microalgae was previously shown to be tightly coupled with cellular stoichiometry. The highest values of cellular toxin are in fact mainly associated with a high carbon to nutrient cellular ratio. In particular, the cellular accumulation of C-rich toxins (i.e., with C:N > 6.6) can be stimulated by both N and P deficiency. Dinoflagellates are the main producers of C-rich toxins and may represent a serious threat for human health and the marine ecosystem. As such, the development of a numerical model able to predict how toxin production is stimulated by nutrient supply/deficiency is of primary utility for both scientific and management purposes. In this work we have developed a mechanistic model describing the stoichiometric regulation of C-rich toxins in marine dinoflagellates. To this purpose, a new formulation describing toxin production and fate was embedded in the European Regional Seas Ecosystem Model (ERSEM), here simplified to describe a monospecific batch culture. Toxin production was assumed to be composed by two distinct additive terms; the first is a constant fraction of algal production and is assumed to take place at any physiological conditions. The second term is assumed to be dependent on algal biomass and to be stimulated by internal nutrient deficiency. By using these assumptions, the model reproduced the concentrations and temporal evolution of toxins observed in cultures of Ostreopsis cf. ovata, a benthic/epiphytic dinoflagellate producing C-rich toxins named ovatoxins. The analysis of simulations and their comparison with experimental data provided a conceptual model linking toxin production and nutritional status in this species. The model was also qualitatively validated by using independent literature data, and the results indicate that our formulation can be also used to simulate toxin dynamics in other dinoflagellates. Our model represents an important step towards the simulation and prediction of marine algal toxicity

Dynamics and sources of organic carbon in suspended particulate matter and sediments in Pialassa Baiona lagoon (NW Adriatic Sea, Italy)

Organic carbon (OC), total nitrogen (TN) and stable carbon isotopic signature (d13C) of suspended particulate matter and of surface sediments were investigated to assess temporal dynamics and relative contribution of autochthonous and allochthonous OC sources in the coastal eutrophic Pialassa Baiona lagoon. Water sampling for particulate organic carbon (POC), total nitrogen (PTN), nutrients and chlorophyll a (Chl a) concentrations, and bacterial abundance was carried out over a year. Seasonal changes of d13C POC signature and C/N ratio discriminated two main areas within the lagoon: the partially preserved northern-central area and the southernmost impacted area. Except for the southernmost impacted area, d13C POC (mean value: -22.11 per mil) showed the highest depleted values in winter and the most enriched in summer following phytoplankton seasonal development as also suggested by changes in C/N ratios, and by the significant correlations found between Chl a and POC, and d13C POC. Moreover, the two areas showed different timing and isotopic signature of phytoplankton blooms (e.g. Chl a 23.22 and 29.27 mg L-1, and d13C POC -19.15 and -30.70 per mil, June and March, in the northern-central and southern area, respectively). The summer bloom in the northern-central area was also associated with high bacterial abundances (up to 7.30 x 109 cells L-1), suggesting the establishment of a strong structured microbial food loop and organic matter recycling. Surface sediments from the southern impacted area showed significant higher values in OC and TN contents (3.05 and 1.44 %), and significant depleted d13C signal (-23.03 per mil) when compared to the control area (1.09 and 0.16 %, -19.40 per mil); whereas no differences were found in C/N ratios (8.1 and 8.2, in northern-central and southern impacted area, respectively). Elemental and isotopic composition data showed a strong coupling between POC and surface sediments. The relative contribution of three different sources (marine, estuarine, terrestrial) to POC and surface sediments were estimated using a mixing model, which predicted a predominant fraction of marine phytoplankton in POC during spring-summer and in surface sediments from the northern-central area. Conversely, dominant allochthonous sources were predicted for POC in winter months and in impacted area sediments. d13C values of surface sediment reflected an isotopic overprint of refractory terrestrial-derived (allochthonous) organic carbon agreeing with urban/industrial wastewaters origin

Assimilation of inorganic nitrogen for scaling up Desmodesmus communis (Scenedesmaceae) biomass production.

The feasibility of the green alga Desmodesmus communis for biomass production was investigated, firstly testing different nitrogen forms in the growth medium and the effect of CO2-enriched air supply, secondarily scaling up the cultivation system in 70 L photobioreactors (PBRs). Maximum nitrogen uptake rate obtained in the performed kinetic experiment was higher for ammonium than for nitrate (188.0 vs 11.7 \u3bcmol g 121 h 121); however, D. communis cultured in PBRs with only aeration grew faster with nitrate reaching a biomass yield (1.23 g L 121) and a productivity (0.036 g L 121 day 121) about twofold higher than with ammonium, which caused a pH decrease in the medium affecting the algal growth. CO2 supply allowed algal growth optimization, maintaining a high productivity with both nitrogen sources, slightly higher with nitrate (0.050 vs 0.038 g L 121 ay 121). Additionally, nitrate-supplied cells showed higher lipids (19.0 vs 9.4%) and proteins (33.0 vs 27.2%) values than those grown with ammonium. The semi-continuous scaled-up cultivation performed for 5 months attests the potential utilization of this species for valuable algal biomass production exploitable in various industrial applications

Preliminary results on phylogenetic structure of the bacterial community associated with Ostreopsis cf. ovata in batch cultures.

Extensive blooms of the toxic epiphytic/benthic dinoflagellate Ostreopsis cf. ovata are being reported with increase frequency and spatial distribution in temperate coastal regions including the Mediterranean Sea. O. cf. ovata outbreaks are of human and environmental health concern due to the production of isobaric palytoxin and a wide range of ovatoxins. In the last years, among the ecological factors that trigger or regulate the algal bloom dynamics, bacteria-microalgae interactions have received increasingly attention. This study investigated the microbial dynamics and the phylogenetic structure of the bacterial community co-occurring with Ostreopsis cf. ovata in batch cultures during different algal growth phases. Cultures of a O. cf. ovata strain isolated along the NW Adriatic Sea were maintained under controlled conditions for a period of 42 days. O. cf. ovata, bacterial cell abundances were monitored along with removal of major nutrients from the medium. Phylogenetic composition of bacterial community was assessed by next generation sequencing of bacterial 16S rDNA hypervariable regions. Bacterial growth showed two different exponential steps, the first step occurring in parallel with the algal exponential phase and the second one in concomitance with the algal mid-stationary phase. Ion torrent data revealed the presence of 12 bacterial phyla, 17 classes and 150 genera all along the experiment. A self-sufficient consortium for vitamin synthesis composed by only few genera belonging to Alphaproteobacteria (65-96%) and Sphingobacteria (2-34%) dominated the community. Our results allow to postulate on specific mutualistic and antagonistic interactions between O. cf. ovata and the associated microbial community in batch cultures, which ultimately will affect algal cellular physiology and potentially toxin dynamics. Moreover, the data pave the way for further investigations on relationships between bacteria-O. cf. ovata interactions and vitamins availability in the environment

Phylogenetic structure of bacterial assemblages co-occurring with Ostreopsis cf. ovata bloom

Extensive blooms of the toxic epiphytic/benthic dinoflagellate Ostreopsis cf. ovata are being reported with increasing frequency and spatial distribution in temperate coastal regions including the Mediterranean. These blooms are of human and environmental health concern due to the production of isobaric palytoxin and a wide range of ovatoxins by Ostreopsis cf. ovata. Bacterial-microalgal interactions are important regulators in algal bloom dynamics and potentially toxin dynamics. This study investigated the bacterial assemblages co-occurring with O. cf. ovata (OA) and from ambient seawaters (SW) during the early and peak phases of bloom development in NW Adriatic Sea. Fractions of the bacterial assemblages co-occurring with O. cf. ovata (OA) and more closely associated to the mucilage layer (LA) embedding O. cf. ovata cells were also reported. In total, 14 bacterial phyla were detected by targeted 454 pyrosequencing of the 16S rRNA gene. The dominant bacterial phyla in the OA assemblages were Proteobacteria and Bacteroidetes; while at the class level, Alphaproteobacteria were the most abundant (83 and 66%, relative abundance, early and peak bloom phases), followed by Flavobacteria (7 and 19%, early and peak phases). Actinobacteria and Cyanobacteria were of minor importance (<5% of the relative bacterial abundance each). Gammaproteobacteria showed a notably presence in OA assemblage only at the early phase of the bloom (genus Haliea, 13%). The Alphaproteobacteria were predominately composed by the genera Ruegeria, Jannaschia and Erythrobacter which represented about half of the total phylotypes contribution of OA at both early and peak phases of the O. cf. ovata bloom, suggesting interactions between this consortium and the microalga. Moreover, the highest contribution of Ruegeria (30% of the total phylotypes) was observed at the early phase of the bloom in LA assemblage. Microbial assemblages associated with the ambient seawaters while being also dominated by Alphaproteobacteria and Flavobacteria were partially distinct from those associated with O. cf. ovata due to the presence of genera almost not retrieved in the latter assemblages

Osservazioni preliminari di produzione di dimetilsolfoniopropionato (DMSP) nella dinoflagellata tossica Ostreopsis cf. ovata.

An increase in the frequency of extensive blooms of the toxic benthic dinoflagellate Ostreopsis cf. ovata has been reported in temperate coastal regions including the Mediterranean. Dimethylsulfoniopropionate (DMSP) can mediate a mutualist interaction between dinoflagellates and their associated bacteria that could have an effect on the development of blooms. In this study the production of DMSP by O. cf. ovata is reported for the first time

Effects of different levels of N- and P-deficiency on cell yield, okadaic acid, DTX-1, protein and carbohydrate dynamics in the benthic dinoflagellate Prorocentrum lima.

Prorocentrum lima (Ehrenberg) Dodge is a cosmopolitan epiphytic dinoflagellate that produces biotoxins which are causative of diarrhetic shellfish poisoning (DPS). Here we report on effects of several nitrogen (N) and phosphorous (P) limited conditions on cell yield, okadaic acid (OA) and dinophysistoxin-1 (DTX-1) contents synoptically with cell carbohydrate, exopolysaccharide (EPS) and cell protein concentrations in a P. lima strain isolated from the Sacca di Goro lagoon (Northern Adriatic Sea). Batch culture experiments were set to assess changes induced by four nitrogen-limited levels (1/3-N, 1/10-N, 1/20-N, and 1/50-N) and four phosphorus-limited levels (1/3-P, 1/10-P, 1/20-P, and 1/50-P) with respect to control nutrient conditions (f/2 medium; NO3- and PO4 3- concentrations: 883 and 36.3 mM, respectively; N/P ratio: 24). Low nutrients availability determined lower cell yields starting from 1/10-N and 1/3-P levels and the pattern observed was dependent on nutrient dynamics, as shown byNand P analyses performed in culture media during growth. Final cell yield decreased significantly up to 4.7- and 5.6-fold under 1/50-N and 1/50 P-limited levels with respect to control values, while cell volume increased with respect to control (up to 30% and 35% for N- and P-experiment, respectively). On overall, OA concentration ranged from 6.69 to 15.80 pg cell-1, while DTX-1 ranged from 0.12 and 0.39 pg cell-1 resulting in unusual high OA/DTX-1 ratios. The study indicates that protein, carbohydrate, EPS, and toxin concentrations displayed remarkable different patterns under the two kinds of nutrient deficiencies. The main differences can be summarised as: (i) significant decrease of cell protein concentration (up to 2-fold) under N-limitation, conversely no significant changes in protein concentration under P-limitation; (ii) significant increase of cell carbohydrate (up to 2.8-fold and 3.4-fold for N- and P-limitation, respectively) and cell OA amount (up to 1.9-fold and 2.3-fold, N- and P-limitation, respectively) under both N- and P-limitations, however different level-deficiency dependent patterns were displayed under the two nutrient conditions; (iii) significant increase of EPS concentration (up to 6.50-fold) under P-limitation, conversely no significant changes in EPS concentration under N-limitation. Data presented here indicate that P. lima adopts different eco-physiological strategies to face N-limitation or P-limitation. This study provides the first evidence for an increase in EPS production by benthic dinoflagellates under P-limited conditions; the ecological significance of this increase is discussed