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- Toxicological effects induced by the biocide triclosan on Pseudokirchneriella subcapitataPublication . Machado, Manuela D.; Soares, Eduardo V.Triclosan, a widely used biocide broadly found in aquatic environments, is cause of concern due to its unknown effects on non-targets organisms. In this study, a multi biomarker approach was used in order to evaluate the 72 h-effect of triclosan on the freshwater alga Pseudokirchneriella subcapitata (Raphidocelis subcapitata). Triclosan, at environmental relevant concentrations (27 and 37 μg L−1), caused a decrease of proliferative capacity, which was accompanied by an increase of cell size and a profound alteration of algae shape. It was found that triclosan promoted the intracellular accumulation of reactive oxygen species, the depletion of non-enzymatic antioxidant defenses (reduced glutathione and carotenoids) and a decrease of cell metabolic activity. A reduction of photosynthetic pigments (chlorophyll a and b) was also observed. For the highest concentration tested (37 μg L−1), a decrease of photosynthetic efficiency was detected along with a diminution of the relative transport rate of electrons on the photosynthetic chain. In conclusion, triclosan presents a deep impact on the microalga P. subcapitata morphology and physiology translated by multiple target sites instead of a specific point (cellular membrane) observed in the target organism (bacteria). Additionally, this study contributes to clarify the toxicity mechanisms of triclosan, in green algae, showing the existence of distinct modes of action of the biocide depending on the microalga.
- Harmful effects of metal(loid) oxide nanoparticlesPublication . Soares, Eduardo V.; Soares, HMVMThe incorporation of nanomaterials (NMs), including metal(loid) oxide (MOx) nanoparticles (NPs), in the most diversified consumer products, has grown enormously in recent decades. Consequently, the contact between humans and these materials increased, as well as their presence in the environment. This fact has raised concerns and uncertainties about the possible risks of NMs to human health and the adverse effects on the environment. These concerns underline the need and importance of assessing its nanosecurity. The present review focuses on the main mechanisms underlying the MOx NPs toxicity, illustrated with different biological models: release of toxic ions, cellular uptake of NPs, oxidative stress, shading effect on photosynthetic microorganisms, physical restrain and damage of cell wall. Additionally, the biological models used to evaluate the potential hazardous of nanomaterials are briefly presented, with particular emphasis on the yeast Saccharomyces cerevisiae, as an alternative model in nanotoxicology. An overview containing recent scientific advances on cellular responses (toxic symptoms exhibited by yeasts) resulting from the interaction with MOx NPs (inhibition of cell proliferation, cell wall damage, alteration of function and morphology of organelles, presence of oxidative stress bio-indicators, gene expression changes, genotoxicity and cell dead) is critically presented. The elucidation of the toxic modes of action of MOx NPs in yeast cells can be very useful in providing additional clues about the impact of NPs on the physiology and metabolism of the eukaryotic cell. Current and future trends of MOx NPs toxicity, regarding their possible impacts on the environment and human health, are discussed.
- Palmelloid-like phenotype in the alga Raphidocelis subcapitata exposed to pollutants: A generalized adaptive strategy to stress or a specific cellular response?Publication . Machado, Manuela D.; Soares, Eduardo V.This work focuses on the formation of palmelloid-like phenotype in the freshwater alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum), when exposed to adverse conditions generated by the presence of organic [the antibiotic erythromycin (ERY) and the herbicide metolachlor (MET)] or inorganic [the heavy metals, cadmium (Cd) and zinc (Zn)] pollutants, at environmentally relevant concentrations. This alga in absence of stress or when exposed to ERY or Zn, up to 200 µg/L, essentially showed a single-nucleus state, although algal growth was reduced or stopped. R. subcapitata “switched” to a multinucleated state (palmelloid-like morphology) and accumulated energy-reserve compounds (neutral lipids) when stressed by 100–200 µg/L MET or 200 µg/L Cd; at these concentrations of pollutants, growth was arrested, however, the majority of the algal population (≥83 %) was alive. The formation of palmelloid-like phenotype, at sub-lethal concentrations of pollutants, was dependent on the pollutant, its concentration and exposure time. The multinucleated structure is a transitory phenotype since R. subcapitata population was able to revert to a single-nucleus state, with normal cell size, within 24–96 h (depending on the impact of the toxic in the alga), after being transferred to fresh OECD medium, without pollutants. The obtained results indicate that the formation of a palmelloid-like phenotype in R. subcapitata is dependent on the mode of action of toxics and their concentration, not constituting a generalized defense mechanism against stress. The observations here shown contribute to understanding the different strategies used by the unicellular alga R. subcapitata to cope with severe stress imposed by organic and inorganic pollutants.
- Reproductive cycle progression arrest and modification of cell morphology (shape and biovolume) in the alga Pseudokirchneriella subcapitata exposed to metolachlorPublication . Machado, Manuela Dias; Soares, Eduardo V.Metolachlor (MET) is an herbicide widely used and frequently found (at μg L-1) in aquatic systems. This work aimed to study the modes of action of MET on the green microalga Pseudokirchneriella subcapitata. Algae exposed to 115 or 235 μg L-1 MET, for 48 or 72 h, presented a reduction of metabolic activity, chlorophyll a and b content and photosynthetic efficiency. The exposure to 115 or 235 μg L-1 MET also induced growth yield reduction, mean cell biovolume increase and alteration of the typical algae shape (cells lunate or helically twisted) to "French croissant"-type; at these MET concentrations, algal population was mainly composed by multinucleated cells (≥ 4 nuclei), which suggest that MET impairs the normal progression of the reproductive cycle but did not hinder nuclear division. The accumulation of multinucleated cells seems to be the consequence of the incapacity of the parent cell to release the autospores. In conclusion, MET disrupts the physiology of P. subcapitata cells; the disturbance of the progression of the reproductive cycle should be in the origin of growth slowdown (or even its arrest), increase of mean cell biovolume and modification of algal shape. This work contributed to elucidate, in a systematically and integrated way, the toxic mechanism of MET on the non-target organism, the alga P. subcapitata.
- Life and death of Pseudokirchneriella subcapitata: physiological changes during chronological agingPublication . Machado, Manuela Dias; Soares, Eduardo V.The green alga Pseudokirchneriella subcapitata is widely used in ecotoxicity assays and has great biotechnological potential as feedstock. This work aims to characterize the physiology of this alga associated with the aging resulting from the incubation of cells for 21 days, in the OECD medium, with continuous agitation and light exposure, in a batch mode. After inoculation, cells grow exponentially during 3 days, and the culture presents a typical green color. In this phase, “young” algal cells present, predominantly, a lunate morphology with the chloroplast occupying a large part of the cell, maximum photosynthetic activity and pigments concentration, and produce starch as a reserve material. Between the 5th and the 12th days of incubation, cells are in the stationary phase. The culture becomes less green, and the cells stop dividing (≥ 99% have one nucleus) and start to age. “Old” algal cells present chloroplast shrinkage, an abrupt decline of chlorophylls content, and photosynthetic capacity (Fv/Fm and ɸPSII), accompanied by a degradation of starch and an increase of neutral lipids content. The onset of the death phase occurs after the 12th day and is characterized by the loss of cell membrane integrity of some algae (cell death). The culture stays, progressively, yellow, and the majority of the population (~93%) is composed of live cells, chronologically “old,” with a significant drop in photosynthetic activity (decay > 75% of Fv/Fm and ɸPSII) and starch content. The information here achieved can be helpful when exploring the potential of this alga in toxicity studies or in biotechnological applications.
- Perspective on the biotechnological production of bacterial siderophores and their usePublication . Soares, Eduardo V.Iron (Fe) is an essential element in several fundamental cellular processes. Although present in high amounts in the Earth’s crust, Fe can be a scarce element due to its low bioavailability. To mitigate Fe limitation, microorganism (bacteria and fungi) and grass plant biosynthesis and secret secondary metabolites, called siderophores, with capacity to chelate Fe(III) with high affinity and selectivity. This review focuses on the current state of knowledge concerning the production of siderophores by bacteria. The main siderophore types and corresponding siderophore-producing bacteria are summarized. A concise outline of siderophore biosynthesis, secretion and regulation is given. Important aspects to be taken into account in the selection of a siderophore-producing bacterium, such as biological safety, complexing properties of the siderophores and amount of siderophores produced are summarized and discussed. An overview containing recent scientific advances on culture medium formulation and cultural conditions that influence the production of siderophores by bacteria is critically presented. The recovery, purification and processing of siderophores are outlined. Potential applications of siderophores in different sectors including agriculture, environment, biosensors and the medical field are sketched. Finally, future trends regarding the production and use of siderophores are discussed.
- Modulation of Siderophore Production by Pseudomonas fluorescens Through the Manipulation of the Culture Medium CompositionPublication . Vindeirinho, João M.; Soares, Helena M.V.M.; Soares, Eduardo V.Pseudomonas fluorescens has the ability to produce the siderophore pyoverdine, a biotechnologically significant iron chelator, which has a wide range of potential applications, such as in agriculture (iron fertilizers) and medicine (development of antibiotics). The present work aimed to evaluate the influence of culture medium composition on the production of siderophores by P. fluorescens DSM 50090, an industrial relevant strain. It was found that the bacterium grown in minimal medium succinate (MMS) had a higher siderophore production than in King B medium. The replacement of succinate by glycerol or dextrose, in minimal medium, originated lower siderophore production. The increase of succinate concentration, the addition of amino acids or the reduction of phosphate in the culture medium did not improve siderophore production by P. fluorescens. The results obtained strongly suggest that (i) MMS is more appropriate than King B for large-scale production of siderophores; (ii) the modification of the culture medium composition, particularly the type of carbon source, influences the level of siderophore secreted; (iii) the production of siderophore by P. fluorescens seems to be a tightly regulated process; once a maximum siderophore concentration has been reached in the culture medium, the bacterium seems to be unable to produce more compound
- Exposure of the alga Pseudokirchneriella subcapitata to environmentally relevant concentrations of the herbicide metolachlor: Impact on the redox homeostasisPublication . Machado, Manuela D.; Soares, Eduardo V.This study investigated the effect of the herbicide metolachlor (MET) on the redox homeostasis of the freshwater green alga Pseudokirchneriella subcapitata. At low MET concentrations (≤40 μg L−1), no effects on algal cells were detected. The exposure of P. subcapitata to 45–235 μg L−1 MET induced a significant increase of reactive oxygen species (ROS). The intracellular levels of ROS were particularly increased at high (115 and 235 μg L−1) but environmentally relevant MET concentrations. The exposure of algal cells to 115 and 235 μg L−1 MET originated a decrease in the levels of antioxidants molecules (reduced glutathione and carotenoids) as well as a reduction of the activity of scavenging enzymes (superoxide dismutase and catalase). These results suggest that antioxidant (non-enzymatic and enzymatic) defenses were affected by the excess of MET. As consequence of this imbalance (ROS overproduction and decline of the antioxidant system), ROS inflicted oxidative injury with lipid peroxidation and damage of cell membrane integrity. The results provide further insights about the toxic modes of action of MET on a non-target organism and emphasize the relevance of toxicological studies in the assessment of the impact of herbicides in freshwater environments.