Harmful algae and pathogens on plastics in three mediterranean coastal lagoons.

Plastic is now a pervasive pollutant in all marine ecosystems. The microplastics and macroplastic debris were studied in three French Mediterranean coastal lagoons (Prevost, Biguglia and Diana lagoons), displaying different environmental characteristics. In addition, biofilm samples were analyzed over the seasons to quantify and identify microalgae communities colonizing macroplastics, and determine potentially harmful microorganisms. Results indicate low but highly variable concentrations of microplastics, in relation to the period and location of sampling. Micro-Raman spectroscopy analyses revealed that the majority of macroplastic debris corresponded to polyethylene (PE) and low-density polyethylene (LDPE), and to a far lesser extent to polypropylene (PP). The observations by Scanning Electron Microscopy of microalgae communities colonizing macroplastic debris demonstrated differences depending on the seasons, with higher amounts in spring and summer, but without any variation between lagoons and polymers. Among the Diatomophyceae, the most dominant genera were Amphora spp., Cocconeis spp., and Navicula spp.. Cyanobacteria and Dinophyceae such as Prorocentrum cordatum, a potentially toxic species, were also found sporadically. The use of Primer specific DNA amplification tools enabled us to detect potentially harmful microorganisms colonizing plastics, such as Alexandrium minutum or Vibrio spp. An additional in situ experiment performed over one year revealed an increase in the diversity of colonizing microalgae in relation to the duration of immersion for the three tested polymers PE, LDPE and polyethylene terephthalates (PET). Vibrio settled durably after two weeks of immersion, whatever the polymer. This study confirms that Mediterranean coastal lagoons are vulnerable to the presence of macroplastic debris that may passively host and transport various species, including some potentially harmful algal and bacterial microorganisms.

Functional diversity of microboring Ostreobium algae isolated from corals.

The filamentous chlorophyte Ostreobium sp. dominates shallow marine carbonate microboring communities, and is one of the major agents of reef bioerosion. While its large genetic diversity has emerged, its physiology remains little known, with unexplored relationship between genotypes and phenotypes (endolithic versus free-living growth forms). Here, we isolated nine strains affiliated to two lineages of Ostreobium (>8% sequence divergence of the plastid gene rbcL), one of which was assigned to the family Odoaceae, from the fast-growing coral host Pocillopora acuta Lamarck 1816. Free-living isolates maintained their bioerosive potential, colonizing pre-bleached coral carbonate skeletons. We compared phenotypes, highlighting shifts in pigment and fatty acid compositions, carbon to nitrogen ratios and stable isotope compositions (δ13 C and δ15 N). Our data show a pattern of higher chlorophyll b and lower arachidonic acid (20:4ω6) content in endolithic versus free-living Ostreobium. Photosynthetic carbon fixation and nitrate uptake, quantified via 8 h pulse-labeling with 13 C-bicarbonate and 15 N-nitrate, showed lower isotopic enrichment in endolithic compared to free-living filaments. Our results highlight the functional plasticity of Ostreobium phenotypes. The isotope tracer approach opens the way to further study the biogeochemical cycling and trophic ecology of these cryptic algae at coral holobiont and reef scales.

Ecotoxicological Studies of ZnO and CdS Nanoparticles on Chlorella vulgaris Photosynthetic Microorganism in Seine River Water.

: Seine river water was used as natural environmental medium to study the ecotoxicological impact of ZnO and CdS nanoparticles and Zn2+ and Cd2+ free ions using Chlorella vulgaris as a biological target. It was demonstrated by viability tests and photosynthetic activity measurements that free Zn2+ (IC50 = 2.7 × 10-4 M) is less toxic than free Cd2+ and ZnO nanoparticles (IC50 = 1.4 × 10-4 M). In the case of cadmium species, free Cd2+ (IC50 = 3.5 × 10-5 M) was similar to CdS nanoparticles (CdS-1: IC50 = 1.9 × 10-5 M and CdS-2: IC50 = 1.9 × 10-5 M), as follows: CdS > Cd2+ > ZnO > Zn2+. Adenosine-5'-triphosphate (ATP) assay and superoxide dismutase (SOD) enzymatic activity confirmed these results. Transmission electron microscopy (TEM), coupled with energy-dispersive X-ray spectroscopy (EDS), confirmed the internalization of CdS-1 nanoparticles after 48 h of contact with Chlorella vulgaris at 10-3 M. With a higher concentration of nanoparticles (10-2 M), ZnO and CdS-2 were also localized inside cells.

Effect of Multi-walled Carbon Nanotubes on Metabolism and Morphology of Filamentous Green Microalgae.

Multi-walled carbon nanotubes (MWCNTs) have potential applications in the industrial, agricultural, pharmaceutical, medical, and environmental remediation fields. However, many uncertainties exist regarding the environmental implications of engineered nanomaterials. This study examined the effect of the MWCNTs on metabolic status and morphology of filamentous green microalgae Klebsormidium flaccidum. Appropriate concentrations of MWCNT (1, 50, and 100 µg mL-1) were added to a microalgal culture in the exponential growth phase and incubated for 24, 48, 72, and 96 h. Exposure to MWCNT led to reductions in algal growth after 48 h and decreased on cell viability for all experimental endpoints except for 1 µg mL-1 at 24 h and 100 µg mL-1 after 72 h. At 100 µg mL-1, MWCNTs induced reactive oxygen species (ROS) production and had an effect on intracellular adenosine triphosphate (ATP) content depending on concentration and time. No photosynthetic activity variation was observed. Observations by scanning transmission electron microscopy showed cell damage. In conclusion, we have demonstrated that exposure to MWCNTs affects cell metabolism and microalgal cell morphology. To our best knowledge, this is the first case in which MWCNTs exhibit adverse effects on filamentous green microalgae K. flaccidum. These results contribute to elucidate the mechanism of MWCNT nanotoxicity in the bioindicator organism of terrestrial and freshwater habitats.

Coccomyxa actinabiotis sp. nov. (Trebouxiophyceae, Chlorophyta), a new green microalga living in the spent fuel cooling pool of a nuclear reactor.

Life can thrive in extreme environments where inhospitable conditions prevail. Organisms which resist, for example, acidity, pressure, low or high temperature, have been found in harsh environments. Most of them are bacteria and archaea. The bacterium Deinococcus radiodurans is considered to be a champion among all living organisms, surviving extreme ionizing radiation levels. We have discovered a new extremophile eukaryotic organism that possesses a resistance to ionizing radiations similar to that of D. radiodurans. This microorganism, an autotrophic freshwater green microalga, lives in a peculiar environment, namely the cooling pool of a nuclear reactor containing spent nuclear fuels, where it is continuously submitted to nutritive, metallic, and radiative stress. We investigated its morphology and its ultrastructure by light, fluorescence and electron microscopy as well as its biochemical properties. Its resistance to UV and gamma radiation was assessed. When submitted to different dose rates of the order of some tens of mGy · h-1 to several thousands of Gy · h-1 , the microalga revealed to be able to survive intense gamma-rays irradiation, up to 2,000 times the dose lethal to human. The nuclear genome region spanning the genes for small subunit ribosomal RNA-Internal Transcribed Spacer (ITS) 1-5.8S rRNA-ITS2-28S rRNA (beginning) was sequenced (4,065 bp). The phylogenetic position of the microalga was inferred from the 18S rRNA gene. All the revealed characteristics make the alga a new species of the genus Coccomyxa in the class Trebouxiophyceae, which we name Coccomyxa actinabiotis sp. nov.

Application of a spectrofluorimetric tool (bbe BenthoTorch) for monitoring potentially toxic benthic cyanobacteria in rivers.

Over the last decade reports of animal poisoning following accidental consumption of neurotoxin-producing benthic cyanobacteria (mainly Phormidium spp.) have increased. There is a need for rapid and cost-effective tools to survey benthic cyanobacteria. In this study we assessed the performance of the BenthoTorch, a fluorometric probe that provides in situ estimations of cyanobacteria, diatoms and green algae biomass in biofilms. Biofilms (n = 288) were analysed from two rivers in France and eight in New Zealand. Correlations between chlorophyll-a measured using the BenthoTorch and spectrophotometry were higher for thin (<2 mm) compared to thick (>2 mm) biofilms (r(2) = 0.58 and 0.27 respectively; p < 0.001). When cyanobacteria represented less than 50% of the total biomass (based on biovolumes), microscopic and BenthoTorch compositional estimations were significantly correlated (r(2) = 0.53, p < 0.001). Conversely, there was no correlation when cyanobacteria exceeded 50% of the total biomass. Under this scenario diatoms were overestimated. Our results suggest that the observed biases occur because the BenthoTorch only measures the upper biofilm layer and it underestimates the biomass of phycoerythrin-containing cyanobacteria. To improve the performance of this sensor and render it a useful tool for a rapid evaluation of benthic cyanobacterial biomass in rivers, we propose that: (i) the algorithms based on the LEDs responses currently available on this tool need revision, (ii) new excitation wavelengths should be included that allow the fingerprints of phycoerythrin-containing cyanobacteria to be discriminated, and (iii) a sensor that penetrates the biofilms is needed to obtain more accurate estimates of cyanobacterial biomass.

Direct and indirect toxic effects of cotton-derived cellulose nanofibres on filamentous green algae.

Recently, cellulose nanofibers (CNFs) have attracted considerable attention as natural, abundant polymers with excellent mechanical properties and biodegradability. CNFs provide a new materials platform for the sustainable production of high-performance nano-enable products for various applications. Given the increasing rates of CNF production, the potential for their release to the environment and the subsequent impact on ecosystem is becoming an increasing concern that needs to be addressed. Here, we used the Klebsormidium flaccidum as a bioindicator organism of terrestrial and freshwater habitats pollution using a battery of biomarkers. Our results show that cotton CNFs inhibit the proliferation of algae and induce morphological changes in them. The two main toxicity mechanisms induced by cotton CNFs are: (i) a direct contact of CNFs with the cell wall and cellular membrane and (ii) an indirect effect through the generation of reactive oxygen species (ROS).

Size-dependent ecotoxicity of barium titanate particles: the case of Chlorella vulgaris green algae.

Studies have been demonstrating that smaller particles can lead to unexpected and diverse ecotoxicological effects when compared to those caused by the bulk material. In this study, the chemical composition, size and shape, state of dispersion, and surface's charge, area and physicochemistry of micro (BT MP) and nano barium titanate (BT NP) were determined. Green algae Chlorella vulgaris grown in Bold's Basal (BB) medium or Seine River water (SRW) was used as biological indicator to assess their aquatic toxicology. Responses such as growth inhibition, cell viability, superoxide dismutase (SOD) activity, adenosine-5-triphosphate (ATP) content and photosynthetic activity were evaluated. Tetragonal BT (~170 nm, 3.24 m(2) g(-1) surface area) and cubic BT (~60 nm, 16.60 m(2) g(-1)) particles were negative, poorly dispersed, and readily aggregated. BT has a statistically significant effect on C. vulgaris growth since the lower concentration tested (1 ppm), what seems to be mediated by induced oxidative stress caused by the particles (increased SOD activity and decreased photosynthetic efficiency and intracellular ATP content). The toxic effects were more pronounced when the algae was grown in SRW. Size does not seem to be an issue influencing the toxicity in BT particles toxicity since micro- and nano-particles produced significant effects on algae growth.

Ecotoxicological studies of micro- and nanosized barium titanate on aquatic photosynthetic microorganisms.

The interaction between live organisms and micro- or nanosized materials has become a current focus in toxicology. As nanosized barium titanate has gained momentum lately in the medical field, the aims of the present work are: (i) to assess BT toxicity and its mechanisms on the aquatic environment, using two photosynthetic organisms (Anabaena flos-aquae, a colonial cyanobacteria, and Euglena gracilis, a flagellated euglenoid); (ii) to study and correlate the physicochemical properties of BT with its toxic profile; (iii) to compare the BT behavior (and Ba(2+) released ions) and the toxic profile in synthetic (Bold's Basal, BB, or Mineral Medium, MM) and natural culture media (Seine River Water, SRW); and (iv) to address whether size (micro, BT MP, or nano, BT NP) is an issue in BT particles toxicity. Responses such as growth inhibition, cell viability, superoxide dismutase (SOD) activity, adenosine-5-triphosphate (ATP) content and photosynthetic efficiency were evaluated. The main conclusions are: (i) BT have statistically significant toxic effects on E. gracilis growth and viability even in small concentrations (1µgmL(-1)), for both media and since the first 24 h; on the contrary of on A. flos-aquae, to whom the effects were noticeable only for the higher concentrations (after 96 h: ≥75 µg mL(-1) for BT NP and =100 µg mL(-1) for BT MP, in BB; and ≥75 µg mL(-1) for both materials in SRW), in spite of the viability being affected in all concentrations; (ii) the BT behaviors in synthetic and natural culture media were slightly different, being the toxic effects more pronounced when grown in SRW - in this case, a worse physiological state of the organisms in SRW can occur and account for the lower resistance, probably linked to a paucity of nutrients or even a synergistic effect with a contaminant from the river; and (iii) the effects seem to be mediated by induced stress without a direct contact in A. flos-aquae and by direct endocytosis in E. gracilis, but in both organisms the contact with both BT MP and BT NP increased SOD activity and decreased photosynthetic efficiency and intracellular ATP content; and (iv) size does not seem to be an issue in BT particles toxicity since micro- and nano-particles produced significant toxic for the model-organisms.

Ecotoxicological effects of carbon nanotubes and cellulose nanofibers in Chlorella vulgaris.

BACKGROUND: MWCNT and CNF are interesting NPs that possess great potential for applications in various fields such as water treatment, reinforcement materials and medical devices. However, the rapid dissemination of NPs can impact the environment and in the human health. Thus, the aim of this study was to evaluate the MWCNT and cotton CNF toxicological effects on freshwater green microalgae Chlorella vulgaris. RESULTS: Exposure to MWCNT and cotton CNF led to reductions on algal growth and cell viability. NP exposure induced reactive oxygen species (ROS) production and a decreased of intracellular ATP levels. Addition of NPs further induced ultrastructural cell damage. MWCNTs penetrate the cell membrane and individual MWCNTs are seen in the cytoplasm while no evidence of cotton CNFs was found inside the cells. Cellular uptake of MWCNT was observed in algae cells cultured in BB medium, but cells cultured in Seine river water did not internalize MWCNTs. CONCLUSIONS: Under the conditions tested, such results confirmed that exposure to MWCNTs and to cotton CNFs affects cell viability and algal growth.

Intracellular biosynthesis of superparamagnetic 2-lines ferri-hydrite nanoparticles using Euglena gracilis microalgae.

The intracellular biosynthesis of superparamagnetic (blocking temperature 5.6K) 2-lines ferrihydrite (Fh2L) nanoparticles was observed within living Euglena gracilis microalgae.

Recycling and adaptation of Klebsormidium flaccidum microalgae for the sustained production of gold nanoparticles.

Targeting the development of cell-based bioreactors for the production of metal nanoparticles, the possibility to perform the sustained synthesis of colloidal gold using Klebsormidium flaccidum green algae was studied. A first strategy relying on successive growth/reduction/reseeding recycling steps demonstrated maintained biosynthesis capability of the microalgae but limitation in metal content due to toxic effects. An alternative approach consisting of progressive gold salt addition revealed to be suitable to favor cell adaptation to larger metal concentrations and supported particle release over month periods.

Micro-algal biosensors.

Fighting against water pollution requires the ability to detect pollutants for example herbicides or heavy metals. Micro-algae that live in marine and fresh water offer a versatile solution for the construction of novel biosensors. These photosynthetic microorganisms are very sensitive to changes in their environment, enabling the detection of traces of pollutants. Three groups of micro-algae are described in this paper: chlorophyta, cyanobacteria, and diatoms.

Ecotoxicological studies of CdS nanoparticles on photosynthetic microorganisms.

The potential ecotoxicity of nanosized cadmium sulfide (CdS), synthesized by the polyol process, was investigated using common Anabaena flos-aquae cyanobacteria and Euglena gracilis euglenoid microalgae. The photosynthetic activities of these microorganisms, after addition of free Cd2+ ions and CdS nanoparticles, varied with the presence of tri-n-octylphosphine oxide (TOPO) used to protect surface particle to avoid toxicity and also to control particle size and shape during the synthesis. The nanoparticle concentration was varied from 10(-3) to 5 x 10(-4) M. It was observed that the cadmium concentration, the addition of TOPO protective agent and the particle dissolution process in the culture medium play an important role during the ecotoxicological tests. Viability tests were followed by PAM fluorimetry. Cd2+ ions were very toxic for Anabaena flos aquae. The same behavior was observed after contact with CdS and CdS-TOPO nanoparticles. However, for Euglena gracilis, the photosynthetic activity was stable for more than 1 month in the presence of Cd2+ ions. Moreover, it was observed that the toxicity varies with the concentration of CdS and CdS-TOPO nanoparticles, both kind of nanoparticles are toxic for this microorganism. Transmission electron microscopy (TEM) analyses of microorganisms ultrathin sections showed that polysaccharides produced by Anabaena flos-aquae, after contact with CdS and CdS-TOPO nanoparticles, protect the microalgae against particle internalization. Only some particles were observed inside the cells. Moreover, the nanoparticle internalization was observed after contact with all nanoparticles in the presence of Euglena gracilis by endocytosis. All nanoparticles are inside vesicles formed by the cells.

ZnO nanoparticles: synthesis, characterization, and ecotoxicological studies.

The potential ecotoxicity of nanosized zinc oxide (ZnO), synthesized by the polyol process, was investigated using common Anabaena flos-aquae cyanobacteria and Euglena gracilis euglenoid microalgae. The photosynthetic activities of these microorganisms, after addition of ZnO nanoparticles, varied with the presence of protective agents such as tri-n-octylphosphine oxide (TOPO) and polyoxyethylene stearyl ether (Brij-76) used to control particle size and shape during the synthesis. In the case of Anabaena flos-aquae , the photosynthetic activity, after addition of ZnO, ZnO-TOPO, and ZnO-Brij-76, decreased progressively due to stress induced by the presence of the nanoparticles in the culture medium. After contact with ZnO-TOPO nanoparticles, this decrease was followed by cell death. On the other hand, after 10 days, a progressive increase of the photosynthetic activity was observed after contact with ZnO and ZnO-Brij-76 nanoparticles. In the case of Euglena gracilis , cell death was observed after contact with all nanoparticles. Transmission electron microscopy (TEM) analyses of ultrathin sections of microorganisms showed that polysaccharides produced by Anabaena flos-aquae avoid particle internalization after contact with ZnO and ZnO-Brij-76 nanoparticles. On the other hand, nanoparticle internalization was observed after contact with all nanoparticles in the presence of Euglena gracilis and also with ZnO-TOPO nanoparticles after contact with Anabaena flos-aquae .

Photosynthetic microorganism-mediated synthesis of akaganeite (beta-FeOOH) nanorods.

Common Anabaena and Calothrix cyanobacteria and Klebsormidium green algae are shown to form intracellularly akaganeite beta-FeOOH nanorods of well-controlled size and unusual morphology at room temperature. X-ray diffraction (XRD), transmission electron microscopy (TEM), and scanning electron microscopy X-ray energy dispersive spectrometry (SEM-EDS) analyses are used to investigate particle structure, size, and morphology. A mechanism involving iron-siderophore complex formation is proposed and compared with iron biomineralization in magnetotactic bacteria.

Cyanobacteria as bioreactors for the synthesis of Au, Ag, Pd, and Pt nanoparticles via an enzyme-mediated route.

Common Anabaena, Calothrix, and Leptolyngbya cyanobacteria are shown to form Au, Ag, Pd, and Pt nanoparticles of well-controlled size. These nanoparticles are synthesized intra-cellularly, and naturally released in the culture medium, where they are stabilized by algal polysaccharides, allowing their easy recovery. The size of the recovered particles as well as the reaction yield is shown to depend on the cyanobacteria genus. Investigations of nanoparticle formation indicate that the intracellular nitrogenase enzyme is responsible for the metal reduction but that the cellular environment is involved in the colloid growth process.

[Harmful and red-tide dinoflagellates in the Annaba bay (Algeria)]. / Dinoflagellés toxiques et/ou responsables de blooms dans la baie d'Annaba (Algérie).

We present here the first description of harmful and non-toxic red-tide dinoflagellates of the Annaba bay (Algeria), during 2002. The qualitative and quantitative phytoplankton analyses reveal the presence of eleven dinoflagellates; two species, Alexandrium catenella and Gymnodinium catenatum, are responsible for efflorescences and known to be harmful. Seven species, Dinophysis caudata, D. fortii, D. rapa, D. rotundata, D. tripos, Lingulodinium polyedrum, and Protoperidinium crassipes, are considered to produce a toxin. Two others, P. triestinum, and Scrippsiella trochoidea, have sometimes been associated with blooms. Analyses of physical and chemical parameters show that stations 1 and 2 are eutrophic, whereas station 3 is oligotrophic.

Seasonal dynamics and toxicity of Cylindrospermopsis raciborskii in Lake Guiers (Senegal, West Africa).

Cylindrospermopsis raciborskii is a toxic bloom-forming cyanobacterium that occurs at tropical and temperate latitudes. Despite several reports from Africa, no data were previously available about its dynamics or toxic potential there. We therefore carried out a 1-year survey of the dynamics of C. raciborskii in the main water reservoir in Senegal, Lake Guiers. Cylindrospermopsis raciborskii never formed a bloom in this lake during the period studied, but was dominant during the dry season. The only observed bloom-forming species was a diatom, Fragilaria sp., which displayed a seasonal pattern contrary to that exhibited by C. raciborskii. Principal component analysis applied to environmental and phytoplankton data showed that high C. raciborskii biomasses were mainly related to high temperature and water column stability. Tests for C. raciborskii species-related toxicity and/or toxin synthesis were performed on 21 isolated clones. All the strains isolated tested negative in mouse toxicity bioassays, toxin analysis (MS/MS) and tests for known cylindrospermopsin genes (ps, pks). The limited number of isolates studied, and the occurrence of toxic and nontoxic clones in natural cyanobacterial populations, mean that we cannot conclude that there is no C. raciborskii-associated health risk in this drinking water reservoir.

Factors involved in the colonisation of building façades by algae and cyanobacteria in France.

Algae and cyanobacteria are colonisers of building façades. A multivariate analysis of data gathered during a sampling campaign around France proved that precipitation, hygrometry, thermal amplitude, distance from the sea and proximity to vegetation were environmental parameters influencing this colonisation. Other influencing factors could be attributed to the nature of the façade coating, mineral substrata being more frequently colonised, and to the architecture, favouring in some cases the formation of damp conditions and thus the colonisation of the building envelope.