Improvement of In Vivo Fluorescence Tools for Fast Monitoring of Freshwater Phytoplankton and Potentially Harmful Cyanobacteria.

The use of multi-wavelength spectrofluorometers for the fast detection of algal taxa, based on chlorophyll a (Chl-a) emission spectra, has become a common practice in freshwater water management, although concerns about their accuracy have been raised. Here, inter-laboratory comparisons using monoalgal cultures have been performed to assess the reliability of different spectrofluorometer models, alongside Chl-a extraction methods. Higher Chl-a concentrations were obtained when using the spectrofluorometers than extraction methods, likely due to the poor extraction efficiencies of solvents, highlighting that traditional extraction methods could underestimate algal or cyanobacterial biomass. Spectrofluorometers correctly assigned species to the respective taxonomic group, with low and constant percent attribution errors (Chlorophyta and Euglenophyceae 6-8%, Cyanobacteria 0-3%, and Bacillariophyta 10-16%), suggesting that functioning limitations can be overcome by spectrofluorometer re-calibration with fresh cultures. The monitoring of a natural phytoplankton assemblage dominated by Chlorophyta and Cyanobacteria gave consistent results among spectrofluorometers and with microscopic observations, especially when cell biovolume rather than cell density was considered. In conclusion, multi-wavelength spectrofluorometers were confirmed as valid tools for freshwater monitoring, whereas a major focus on intercalibration procedures is encouraged to improve their reliability and broaden their use as fast monitoring tools to prevent environmental and public health issues related to the presence of harmful cyanobacteria.

Allelopathic interactions between phytobenthos and meiofaunal community in an Adriatic benthic ecosystem: Understanding the role of aldehydes and macroalgal structural complexity.

Macroalgae produce several allelopathic substances, including polyunsaturated aldehydes (PUAs), which may inhibit photosynthesis and growth rates of other algal species, and grazing. Additionally, macroalgal structural complexity is an important factor in determining abundance patterns and size structure of epiphytic organisms. In this study the PUAs production of two Mediterranean macroalgae, Dictyopteris polypodioides, (DP, Phaeophyceae, Dictyotales) and Cystoseira compressa (CC, Phaeophyceae, Fucales), was characterized to clarify the relationships between the meiobenthic and microphytobenthic communities. Results showed a higher PUAs production and a diverse qualitative profile for DP, which reported long-chain compounds (i.e. C14-C16) as main aldehydes, than CC, with the short-chain C6:2 as the main compound, as well as variability among sampling times. A clear separation of the meiofauna and microphytobenthos assemblages was found for the macroalgae, but with different temporal trends. Dissimilarities were due to five microalgal orders, namely Naviculales, Lyrellales, Gonyaulacales (i.e. Ostreopsis), Bacillariales, and Licmophorales, and to the meiofaunal groups nematodes, copepods, and copepod nauplii, which were more abundant on DP than on CC. Results indicate that macroalgal complexity is a major determinant of the meiofaunal community structure (accounting for 26% of the variation), rather than PUAs production itself (17%). PUAs effects seem species-specific, thus affecting some grazers instead of the entire community. Conversely, microphytobenthos affected the meiofauna assemblages, particularly harpacticoids, confirming the role of these organisms as the primary food source of all marine food chain producers. Since PUAs are produced also by several epiphytic diatoms, the understanding of their effects on the community structure and on the relationships among taxa in the field is complicated and requires further in-depth investigations in simplified systems (i.e. microcosms).

Production of polyhydroxybutyrate by the cyanobacterium cf. Anabaena sp.

Polyhydroxybutyrate (PHB) production by the cyanobacterium cf. Anabaena sp. was here studied by varying the medium composition and the carbon source used to induce mixotrophic growth conditions. The highest PHB productivity (0.06 gPHB gbiomass-1 d-1) was observed when cultivating cf. Anabaena sp. in phosphorus-free medium and in the presence of sodium acetate (5.0 g L-1 concentration), after an incubation period of 7 days. A content of 40% of PHB on biomass, a dry weight of 0.1 g L-1, and a photosynthetic efficiency equal to the control were obtained. The cyanobacterium was then grown on a larger scale (10 L) to evaluate the characteristics of the produced PHB in relation to the main composition of the biomass (the content of proteins, polysaccharides, and lipids): after an incubation period of 7 days, a content of 6% of lipids (52% of which as unsaturated fatty acids with 18 carbon atoms), 12% of polysaccharides, 28% of proteins, and 46% of PHB was reached. The extracted PHB had a molecular weight of 3 MDa and a PDI of 1.7. These promising results demonstrated that cf. Anabaena sp. can be included among the Cyanobacteria species able to produce polyhydroxyalkanoates (PHAs) either in photoautotrophic or mixotrophic conditions, especially when it is grown under phosphorus-free conditions.

Survey of the allelopathic potential of Mediterranean macroalgae: production of long-chain polyunsaturated aldehydes (PUAs).

Chemical interactions between macroalgae and other organisms play an important role in determining species compositions and dominance patterns, and can explain the widespread success of some species in establishing their predominant populations in a specific coastal area. Allelopathy could act as a self-regulatory strategy of the algal community, being not only a succession regulator but also an active mechanism maintaining the species diversity especially in a delimited environment, such as the benthic ecosystem. Polyunsaturated aldehydes (PUAs) are among the most studied allelopathic compounds and are commonly released into the aquatic environment by different phytoplankton species in response to environmental stressors (e.g. wounding, grazing, or competition for nutrients). Diatom-released PUAs were observed to affect phytoplankton community dynamics and structure, and showed inhibitory effects on the reproduction and development of marine invertebrates. As for macroalgae, there are only a few reports that attest to the production of PUAs, and mostly refer to Ulva spp. In this study, the production of PUAs by several Mediterranean macroalgae was investigated at different sampling times, aiming at providing the first evidence of potential allelochemical activity. Results highlighted the potential production by macroalgae of a variety of aldehydes, among which some have never reported so far. Some species (i.e. D. polypodioides and U. cf. rigida) were found to produce higher PUAs amounts than others, and even a wider variety of structures (e.g. length of the carbon chain); these species might exert strong effects on epiphytic species or other organisms of the benthic community, especially considering the differential sensitivities of the various taxa. A high dPUA concentration (order of µM) potentially due to the release of PUAs by algal species was found, and might affect the population dynamics of the epiphytic organisms (e.g. microalgae, meiofauna), of grazers, as well as of the microbial community.

Use of waste carbon dioxide and pre-treated liquid digestate from biogas process for Phaeodactylum tricornutum cultivation in photobioreactors and open ponds.

Phaeodactylum tricornutum is considered a promising source of polyunsaturated fatty acids (PUFAs), in particular eicosapentaenoic acid (EPA). In this study, P. tricornutum cultivation using waste products from anaerobic digestion (i.e. liquid digestate and CO2) was tested and scaled-up in closed and open prototype systems. The chemical composition of algal biomass was evaluated to optimize the lipid content. Algal productivity and composition, especially in terms of PUFAs, were not modified by the use of waste CO2. Digestate led to a lower protein (24%) content than medium (36-37%), without affecting lipid amount (about 37%). Algal and EPA productivity were nearly two-fold higher by using photobioreactors (0.075 g biomass L-1 day-1 and 1.62 mg EPA g-1 day-1) than open ponds, which are more influenced by environmental conditions. This study highlights that economic and environmental benefits could be achieved by using waste CO2 and liquid digestate from anaerobic digestion for microalgae cultivation.

Allelopathic effects of diatom filtrates on the toxic benthic dinoflagellate Ostreopsis cf. ovata.

Ostreopsis blooms regularly occur in many Mediterranean coastal areas in late summer-autumn. In the northern Adriatic Sea, Ostreopsis blooms affect diatom-dominated microphytobenthic communities. In this study, the effects of the filtrates of some diatom species, both benthic (Tabularia affinis, Proschkinia complanatoides and Navicula sp.) and planktonic (Thalassiosira sp. and Skeletonema marinoi) on cell morphology, cytological features and growth of O. cf. ovata were investigated. Our results showed a marked decrease of O. cf. ovata growth when cells were exposed to all diatom filtrates tested. The highest inhibitions were observed for exposures to P. complanatoides and Navicula sp. filtrates (92.5% and 80%, respectively) and increased with the age of diatom culture. Moreover, a clear DNA degradation and abnormal forms of O. cf. ovata cells (83.8% of the total) were found at the highest concentrations using Navicula sp. filtrate after 10 days of the inoculum.

PUFAs and PUAs production in three benthic diatoms from the northern Adriatic Sea.

The production of polyunsaturated aldehydes (PUAs) has been reported by many planktonic diatoms, where they have been implicated in deleterious effects on copepod reproduction and growth of closeby microbes or suggested as infochemicals in shaping plankton interactions. This study investigates the production of PUAs by diatoms commonly occurring in the microphytobenthic communities in temperate regions: Tabularia affinis, Proschkinia complanatoides and Navicula sp. Results highlight the production of PUAs by the three benthic diatoms during stationary and decline phases, with intracellular concentrations from 1.8 to 154.4 fmol cell-1, which are within the range observed for planktonic species. The existence of a large family of PUAs, including some with four unsaturations, such as decatetraenal, undecatetraenal and tridecatetraenal, was observed. Since particulate and dissolved PUAs were positively correlated, together with cell lysis, equivalent concentrations may be released during late growth stages, which may affect benthic invertebrates grazing on them and other microalgae.

The dawn of dentistry in the late upper Paleolithic: An early case of pathological intervention at Riparo Fredian.

OBJECTIVES: Early evidence for the treatment of dental pathology is found primarily among food-producing societies associated with high levels of oral pathology. However, some Late Pleistocene hunter-gatherers show extensive oral pathology, suggesting that experimentation with therapeutic dental interventions may have greater antiquity. Here, we report the second earliest probable evidence for dentistry in a Late Upper Paleolithic hunter-gatherer recovered from Riparo Fredian (Tuscany, Italy). MATERIALS AND METHODS: The Fredian 5 human consists of an associated maxillary anterior dentition with antemortem exposure of both upper first incisor (I1 ) pulp chambers. The pulp chambers present probable antemortem modifications that warrant in-depth analyses and direct dating. Scanning electron microscopy, microCT and residue analyses were used to investigate the purported modifications of external and internal surfaces of each I1 . RESULTS: The direct date places Fredian 5 between 13,000 and 12,740 calendar years ago. Both pulp chambers were circumferentially enlarged prior to the death of this individual. Occlusal dentine flaking on the margin of the cavities and striations on their internal aspects suggest anthropic manipulation. Residue analyses revealed a conglomerate of bitumen, vegetal fibers, and probable hairs adherent to the internal walls of the cavities. DISCUSSION: The results are consistent with tool-assisted manipulation to remove necrotic or infected pulp in vivo and the subsequent use of a composite, organic filling. Fredian 5 confirms the practice of dentistry-specifically, a pathology-induced intervention-among Late Pleistocene hunter-gatherers. As such, it appears that fundamental perceptions of biomedical knowledge and practice were in place long before the socioeconomic changes associated with the transition to food production in the Neolithic.

Photobioreactor cultivation and catalytic pyrolysis of the microalga Desmodesmus communis (Chlorophyceae) for hydrocarbons production by HZSM-5 zeolite cracking.

The study evaluated the growth of Desmodesmus communis on column photobioreactor and its thermochemical treatment by catalytic pyrolysis using HZSM-5 zeolite. D. communis showed good results in terms of growth (0.05gL-1d-1). Analytical pyrolysis of original algae and derived bio-oil mixed with zeolite was used as a screening method in order to gather information on the cracking process. Preparative pyrolysis on bench scale reactor was performed on algae biomass over a zeolite bed at 1:10 ratio (wt/wt). Py-GC-MS of biomass/catalyst mixture showed that the denitrogenation/deoxygenation increased with increasing zeolite load from 1:5 to 1:20 ratio and became significant at 1:10 ratio. The composition observed by analytical pyrolysis was featured by the predominance of alkylated monoaromatic hydrocarbons. The scaling-up to bench scale confirmed the results obtained with analytical pyrolysis in terms of monoaromatic hydrocarbons. However, low yield of catalytic oil (8% by weight) was observed.

Inhibitory effect of polyunsaturated aldehydes (PUAs) on the growth of the toxic benthic dinoflagellate Ostreopsis cf. ovata.

Diatoms have been shown to produce and release a wide range of secondary metabolites that mediate interactions between individuals of different species. Among these compounds, different types of fatty acid derived long-chained polyunsaturated aldehydes (PUAs) have been related to multiple functions such as intra- or interspecific signals and adverse effect on the reproduction of marine organisms. Several studies have reported changes on growth, cell membrane permeability, flow cytometric properties and cell morphology in phytoplankton organisms exposed to PUAs, but little information is available on the effect of these compounds on benthic microalgae. Ostreopsis cf. ovata is a toxic benthic dinoflagellate which causes massive blooms along the Mediterranean coasts typically during the late summer period. In this study the effects of three toxic PUAs known to be produced by several algae (2E,4E-decadienal, 2E,4E-octadienal and 2E,4E-heptadienal) on the growth, cytological features and cell morphology of O. cf. ovata were investigated. Our results show a clear decrease of O. cf. ovata growth with longer-chain molecules than with shorter-chain ones, confirmed also by EC50 values calculated at 48h for 2E,4E-decadienal and 2E,4E-octadienal (6.6±1.5, 17.9±2.6µmolL(-1) respectively) and at 72h for 2E,4E-heptadienal (18.4±0.7µmolL(-1)). Moreover, morphological analysis highlighted up to 79% of abnormal forms of O. cf. ovata at the highest concentrations of 2E,4E-decadienal tested (9, 18 and 36µmolL(-1)), a gradual DNA degradation and an increase of lipid droplets with all tested PUAs. Further studies are needed to better clarify the interactions between diatoms and O. cf. ovata, especially on bloom-forming dynamics.

Modeling of photosynthesis and respiration rate for Isochrysis galbana (T-Iso) and its influence on the production of this strain.

Isochrysis galbana is a widely-used strain in aquaculture in spite of its low productivity. To maximize the productivity of processes based on this microalgae strain, we have developed a model considering the influence of irradiance, temperature, pH and dissolved oxygen concentration on the photosynthesis and respiration rate. Results demonstrate that this strain tolerates temperatures up to 35 °C but it is highly sensitive to irradiances higher than 500 µE m(-2)s(-1) and dissolved oxygen concentrations higher than 11 mgl(-1). We have validated the developed model using data from an industrial-scale outdoor tubular photobioreactor demonstrating that inadequate temperature and dissolved oxygen concentrations reduce productivity to half that which is maximal, according to light availability under real outdoor conditions. The developed model is a useful tool for managing working processes, but especially in the development of new processes based on this strain and to take decisions regarding optimal control strategies.

Effects of N and P availability on carbon allocation in the toxic dinoflagellate Ostreopsis cf. ovata.

Blooms of the toxic dinoflagellate Ostreopsis cf. ovata are usually associated with shallow and calm coastal waters, characterized by low nutrient concentrations. The algal cells typically cover the benthic substrates, such as the macroalgal and invertebrate communities and rocks, forming a mucilaginous film. Data reported on O. cf. ovata toxin production observed under both field and culture conditions show high variability in terms of toxic profile and cellular content; little is known about the environmental and physiological aspects which regulate the toxin dynamics. In this study, O. cf. ovata physiology was investigated using batch cultures supplied with nutrient concentrations similar to those found in the Adriatic Sea during the recurrent blooms and the observed cellular dynamics were compared with those found in a culture grown under optimal conditions, used as a reference. Data on the cellular C, N and P content during the growth highlighted a possible important role of the cellular nutritional status in regulating the toxin production that resulted to be promoted under specific intervals of the C:N and C:P ratios. The variable toxicity found for O. cf. ovata in various geographic areas could be related to the different in situ prevalent environmental conditions (e.g., nutrient concentrations) which affect the cellular elemental composition and carbon allocation. The obtained results strongly suggest that in the environment toxin production is steadily sustained by a low and constant nutrient supply, able to maintain appropriate cellular C:N (>12) or C:P (>170) ratios for a long period. These results explain to some extent the variability in toxicity and growth dynamics observed in blooms occurring in the different coastal areas.

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.

Ovatoxin-a, A Palytoxin Analogue Isolated from Ostreopsis cf. ovata Fukuyo: Cytotoxic Activity and ELISA Detection.

This study provides the first evaluation of the cytotoxic effects of the recently identified palytoxin (PLTX) analog, ovatoxin-a (OVTX-a), the major toxin produced by Ostreopsis cf. ovata in the Mediterranean Sea. Its increasing detection during Ostreopsis blooms and in seafood highlights the need to characterize its toxic effects and to set up appropriate detection methods. OVTX-a is about 100 fold less potent than PLTX in reducing HaCaT cells viability (EC50 = 1.1 × 10(-9) M vs 1.8 × 10(-11) M, MTT test) in agreement with a reduced binding affinity (Kd = 1.2 × 10(-9) vs 2.7 × 10(-11) M, saturation experiments on intact cells). Similarly, OVTX-a hemolytic effect is lower than that of the reference PLTX compound. Ost-D shows the lowest cytotoxicity toward HaCaT keratinocytes, suggesting the lack of a hydroxyl group at C44 as a critical feature for PLTXs cytotoxic effects. A sandwich ELISA developed for PLTX detects also OVTX-a in a sensitive (LOD = 4.2 and LOQ = 5.6 ng/mL) and accurate manner (Bias = 0.3%), also in O. cf. ovata extracts and contaminated mussels. Although in vitro OVTX-a appears less toxic than PLTX, its cytotoxicity at nanomolar concentrations after short exposure time rises some concern for human health. The sandwich ELISA can be a viable screening method for OVTXs detection in monitoring program.

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.

Nutrient removal efficiency and physiological responses of Desmodesmus communis at different HRTs and nutrient stress condition using different sources of urban wastewater effluents.

The objective of the present study was to evaluate the nutrient removal efficiency and the physiological responses in terms of growth, biochemical composition and photosynthetic activity of the autochthonous freshwater algal strain Desmodesmus communis. Microalgae were grown in a primary municipal effluent under different hydraulic retention times (HRTs) and in a two-phases process using both primary and secondary wastewater effluents. Semi-continuous cultures were operated for 7 day at 5-, 3- and 1.5-day HRT and the different dilution rate showed a greater influence on the biomass composition and nutrient removal efficiency. Removal of N-NH3 and P-PO4 was over 99 % and the highest accumulation of polysaccharides (57.2 wt.%) was obtained at high HRT (5 day); the maximum content of proteins (26.9 wt.%) was achieved at 1.5-day HRT, even if, under this condition, a clear inefficiency in terms of ammonia removal was observed. Moreover the accumulation of N-NH3 occurring at 1.5-day HRT caused the decrease of the photosynthetic response in terms of efficiency of light capture (α) and relative electron transport rate (rETR), both parameters extracted from the rapid light curves (RLC) measurements. No significant differences were observed for the total fatty acids (TFAs), with a content of 2-3.5 wt.% for each HRT condition. On the other hand, in the two-phases process, when a nutrient deprivation condition was induced by diluting the culture with the secondary wastewater effluent, the algal cells accumulated TFAs, achieving a maximum content of 9.7 wt.% and a great increment in terms of biomass (1.64 ± 0.02 g L(-1)) due to the ability of this algal strain to accumulate intracellular N. The wide and accurate investigation of the different aspects related to the whole process represents a relevant point of novelty in this research field and suggests the operational conditions for the start-up of an open pond system for wastewater treatment and biomass production for further applications.

Skeletonema marinoi (Bacillariophyceae) sensitivity to herbicides and effects of temperature increase on cellular responses to terbuthylazine exposure.

The North East area of Italy is an intensively farmed area, where the use of herbicides has increased dramatically during the last years. Some of the most detected herbicides are triazine compounds, such as: simazine (SIM), terbuthylazine (TBA), its degradation product desethyl-terbuthylazine (D-TBA) and other herbicides, such as metolachlor (MET). In this paper, the sensitivity of the diatom Skeletonema marinoi to the most detected herbicides (TBA, D-TBA, SIM and MET) was preliminarily studied. All the pollutants tested significantly inhibited the diatom growth and photosynthetic efficiency (from the concentration of 15 µg L(-1)) with the exception of TBA which had the strongest effects on S. marinoi starting from the concentration of 5 µg L(-1). Consequently, cellular physiological responses to TBA exposure (1, 5, 10, 20 and 30 µg L(-1)) were further studied at increasing temperature conditions (15, 20 and 25°C). Inhibition of growth rate and photosynthetic efficiency was observed earlier and determined by lower TBA levels than those affecting cell growth. These responses were significantly enhanced at increasing temperature conditions when growth rates were higher than those measured at 15°C. Carbon cell content increased in the cultures exposed to high concentrations of TBA (from 20 µg L(-1)) compared to the controls, especially at high temperatures. Cell chlorophyll significantly increased from the added concentration of 10 µg L(-1) of TBA at all the temperatures and, as a consequence, also the Chl:C ratio significantly increased. The C:N ratio followed the pattern of nitrate uptake and was characterized, at all the temperatures, by low values during the lag phase in cultures with 20 and 30 µg L(-1) of TBA; in these conditions, in fact, the nutrient in the medium was exhausted later then in the controls. Only cultures exposed to 30 µg L(-1) of TBA at 25°C, which stopped to take up nutrients earlier and could not increase chlorophyll levels, did not display any growth capacity. This study shows that S. marinoi is affected by TBA concentrations lower than those affecting some harmful flagellate species frequently observed in the Adriatic Sea. Thus, it raises the question of the combined effects of herbicides pollution and high temperature pressures on phytoplankton composition.

Combined effects of the herbicide terbuthylazine and temperature on different flagellates from the Northern Adriatic Sea.

The triazinic herbicide terbuthylazine (TBA) is becoming an emergent contaminant in Italian rivers and in coastal and groundwater. A preliminary analysis of the sensitivity of marine flagellates to TBA was performed by monitoring the photosynthetic efficiency of nine species (belonging to the Dinophyceae or Raphidophyceae class) isolated from the Adriatic Sea. Different sensitivity levels for each flagellate were observed and the most sensitive microalgae, based on PSII inhibition, were: Gonyaulax spinifera>Fibrocapsa japonica>Lingulodinium polyedrum while the most resistant were two species belonging to the Prorocentrum genus. Then the response of two microalgae to drivers, such as temperature and terbuthylazine, applied in combination was also investigated. Two potentially toxic flagellates, Prorocentrum minimum and G. spinifera, were exposed, under different temperature conditions (15, 20 and 25°C), to TBA concentrations that did not completely affect PSII. For both flagellates, effects of TBA on algal growth, measured through cell density and carbon analysis, as well as on the photosynthetic activity are reported. All parameters analyzed showed a negative effect of TBA from the exponential phase. TBA effect on algal growth was significantly enhanced at the optimal temperature conditions (20 and 25°C), while no difference between control and herbicide treatments were detected for G. spinifera grown at 15°C, which represented a stress condition for this species. The maximum inhibition of photosynthetic efficiency was found at 20°C for both organisms. Both flagellates increased cell carbon and nitrogen content in herbicide treatments compared to the control, except G. spinifera grown at 15°C. Chlorophyll-a production was increased only in G. spinifera exposed to 5 µg L(-1) of TBA and the effect was enhanced with the increase of temperature. Herbicide-induced variations in cellular components determined changes in cellular carbon:nitrogen (C:N) and chlorophyll:carbon (Chl:C) ratios. The C:N ratio decreased in both species, while only G. spinifera showed an increase in the Chl:C ratio at all temperature conditions. In response to TBA exposure G. spinifera increased extracellular polysaccharides release at 20 and 25°C, while no difference was reported for P. minimum. Changes in nutrient uptake rates were also observed for P. minimum. Nitrate and phosphate uptake significantly increased in the presence of TBA and this response was enhanced at 25°C, while nitrate uptake increased in G. spinifera only when grown at 25°C. As for growth rates, the observed changes in intracellular component contents increased at optimal temperature conditions. In this work it is shown that temperature conditions can have an important role on the effect of terbuthylazine on algal growth and on the physiological responses of different species. Furthermore, the algal resistance and recovery can be dependent on nutrient availability.

Growth and nitrogen removal capacity of Desmodesmus communis and of a natural microalgae consortium in a batch culture system in view of urban wastewater treatment: part I.

The microalgal biomass applications strongly depend on cell composition and the production of low cost products such as biofuels appears to be economically convenient only in conjunction with wastewater treatment. As a preliminary study, in view of the development of a wastewater treatment pilot plant for nutrient removal and algal biomass production, a biological wastewater system was carried out on a laboratory scale growing a newly isolated freshwater algal strain, Desmodesmus communis, and a natural consortium of microalgae in effluents generated by a local wastewater reclamation facility. Batch cultures were operated by using D. communis under different growth conditions to better understand the effects of CO2, nutrient concentration and light intensity on the biomass productivity and biochemical composition. The results were compared with those obtained using a natural algal consortium. D. communis showed a great vitality in the wastewater effluents with a biomass productivity of 0.138-0.227 g L⁻¹ d⁻¹ in the primary effluent enriched with CO2, higher biomass productivity compared with the one achieved by the algal consortium (0.078 g L⁻¹ d⁻¹). D. communis cultures reached also a better nutrient removal efficiency compared with the algal consortium culture, with almost 100% for ammonia and phosphorous at any N/P ratio characterizing the wastewater nutrient composition. Biomass composition was richer in polysaccharides and total fatty acids as the ammonia concentration in the water decreased. In view of a future application of this algal biomass, due to the low total fatty acids content of 1.4-9.3 wt% and the high C/N ratio of 7.6-39.3, anaerobic digestion appeared to be the most appropriate biofuel conversion process.