Crecimiento y composición bioquímica de la microalga marina Tetraselmis suecica en cultivos mixotróficos con distintos azúcares y aminoácidos

[Résumé] Croissance et composition biochimique de la microalgue TetrasellIIis sllecica dans des cultures mixotrophiques avec différents sucres et aminoacides. Trois sucres, fructose, saccharose et manose et trois acides aminés (alanyne, lysine et méthyonine) ont été testés comme stimulants potentiels de la croissance chez la microalgue Tetraselmis suecica. Celle-ci est cultivée en absence de bactéries et a une intensité de lumière de 850 lux. La croissance et la composition biochimique chez T. suecica sont affectées par les substrats organiques testés. Le fructose stimule la croissance mais les autres sucres et l'alanyne sont sans effet. La lysine et la méthyonine réduisent la croissance par rapport aux cultures servant de contrôle. La composition biochimique cellulaire dépend de la nature du substrat organique testé mais elle varie à peine en fonction de la concentration. Les cultures enrichies en sucres présentent les protéines cellulaires les plus abondantes, tandis que pour les cultures enrichies en acides aminés, les lipides sont plus abondants.[Abstract] Growth and biochemical composition of the marine microalga TetrasellIIis sllecica in mixotrophic cultures witf7 different sugars and amino acids. Three sugars (fructose, sucrose and mannose) and three amino acids (alanine, lysine, and methionine) have been tested as potential growth factors for the marine microalgae Tetraselmis suecica, in axenic cultures, at 850 lux light intensity. The organic compounds assayed affected the growth and biochemical composition of T. suecica. Growth stimulation was achieved only in cultures added with fru ctose, whereas growth was not affected in the cultures with the remaining sugars and alanine. Growth was inhibited in the cultures added with lysine and methionine. Biochemical cellular composition was affected by the nature of the organic compound added, but not by the concentration used. Proteins were the main cellular constituent in the cultures carried out with sugars, while in those with amino acids the main cellular fraction was lipids.Ministerio de Educación y Ciencia; PB 87-049

High yield mixotrophic cultures of the marine microalga Tetraselmis suecica (Kylin) Butcher (Prasinophyceae)

The effects of three organic compounds were tested on one of the most used marine micro-algae in the aquaculture of molluscs and crustaceans, Tetraselmis suecica. Studies were made in axenic conditions with yeast extract, peptone and glucose added to the culture medium, each alone, in combinations of two or all together. Medium without any organic compound was used for the control. Cultures containing yeast extract grew best, reaching maximum cell density of 3.79 × 106 and 3.84 × 106 cells ml−1. The organic carbon source affected the biochemical composition. The components most affected were the carbohydrates, with values between 6.5 pg cell−1 in control cultures and 48.5 pg cell−1 in glucose cultures. Protein content ranged between 27.5 pg cell−1 in control cultures and 88.6 pg cell−1in yeast + glucose + peptone cultures. The lipid content changed little. Maximum protein yields were reached in cultures with yeast + glucose and with yeast - glucose - peptone, with values of 24.6 and 28.2 mg 1−1 d−1, respectively. These values are 22 and 25 times those in control cultures. A maximum carbohydrate yield of 7.9 mg carbohydrate per litre per day was obtained in yeast + glucose + peptone cultures, 27 times that in the control cultures. The maximum lipid yield was obtained with yeast + glucose + peptone and yeast + glucose. Maximum energy values were 308 kcal 1− in yeast extract - glucose - peptone cultures and 279 kcal 1−1 in yeast extract + glucose cultures. Gross energy values in control cultures were 24.5 kcal 1−1, but peptone cultures presented the minimum energy value, 22 kcal 1−1. The yeast extract: glucose ratio in the culture medium was optimized. A ratio 2:1 produced the best yields in cells, protein, carbohydrate and gross energy.Dirección General de Investigación Científica y Técnica; PB 87-049

Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: Screening cytotoxicity and genotoxicity endpoints

Since recent evidence has demonstrated that many types of chemicals exhibit oxidative and/or genotoxic potential on living organisms, reactive oxygen species (ROS) formation and DNA damage are currently the best accepted paradigms to assess the potential hazardous biological effects of a wide range of contaminants. The goal of this study was to evaluate the sensitivity of different cytotoxicity and genotoxicity responses on the model microalga Chlamydomonas reinhardtii exposed to the prooxidant herbicide paraquat. In addition to the growth endpoint, cell viability, mitochondrial membrane potential and presence of reactive oxygen species (ROS) were assayed as potential markers of cytotoxicity using flow cytometry (FCM). To study the effects of paraquat on C. reinhardtii DNA, several genotoxicity approaches were implemented for the first time in an ecotoxicological study on microalgae. Oxidative DNA base damage was analysed by measuring the oxidative DNA lesion 8-OHdG by FCM. DNA fragmentation was analysed by different methods: comet assay, and cell cycle analysis by FCM, with a particular focus on the presence of subG1-nuclei. Finally, effects on morphology of nuclei were monitored through DAPI staining. The evaluation of these endpoints showed that several physiological and biochemical parameters reacted to oxidative stress disturbances with greater sensitivity than integrative parameters such as growth rates or cell viability. The experiments revealed concentration-dependent cytotoxicity (ROS formation, depolarization of mitochondrial membrane), genotoxicity (oxidative DNA damage, DNA strand breakage, alterations in nuclear morphology), and cell cycle disturbances (subG1-nuclei, decrease of 4N population) in paraquat-treated cells. Overall, the genotoxicity results indicate that the production of ROS caused by exposure to paraquat induces oxidative DNA damage followed by DNA single- and double-strand breaks and cell cycle alterations, possibly leading to apoptosis in C. reinhardtii cells. This is supported by the observation of typical hallmarks of apoptosis, such as mitochondrial membrane depolarization, alterations in nuclear morphology and subG1 nuclei in cells exposed to the highest assayed concentrations. To our knowledge, this is the first study that provides a comprehensive analysis of oxidative DNA base damage in unicellular algal cells exposed to a prooxidant pollutant, as well as of its possible relation with other physiological effects. These results reinforce the need for additional studies on the genotoxicity of environmental pollutants on ecologically relevant organisms such as microalgae that can provide a promising basis for the characterization of potential pollutant hazards in the aquatic environment

Screening acute cytotoxicity biomarkers using a microalga as test organism

The present study checked the suitability of the integration of flow cytometry (FCM) as technique and a freshwater microalga (Chlamydomonas moewusii) as cell system model for ecotoxicological studies, looking for sensitive biomarkers of acute cytotoxicity of potential contaminants in aquatic systems. The detection of the potential acute toxicity of a pollutant is of interest because pulse discharges of contaminants to natural waters could lead to high concentrations of these substances that are only present for short periods of time but can affect aquatic organisms such as microalgae. Physiological alterations in C. moewusii cells were analysed after 1 h of exposure to different concentrations of the herbicide paraquat. Cell viability was not affected, but the acute toxicity of paraquat was evident at other levels of cell physiology. Herbicide-treated cells showed lower autofluorescence and higher size and internal complexity, lower esterase activity and lower mitochondrial membrane potential. Paraquat induced the depolarisation of the plasma membrane and the increase of intracellular free calcium level and cytosolic pH in a concentration-dependent percentage of cells. All these effects can be related to the oxidative stress induced by the herbicide, as revealed the significantly increased intracellular levels of reactive oxygen species in cultures exposed to paraquat concentrations which induced the physiological alterations mentioned above. Excluding cell viability and mitochondrial membrane potential, these cytotoxicity endpoints could be considered sensitive biomarkers for the short-term exposure to pollutants such as herbicides. Highlights: • This study examine the acute cytotoxicity of paraquat on microalgal physiology. • Flow cytometric protocols assayed allow the screening of different toxicity cellular endpoints. ► Cell viability is not a sensitive biomarker. • Short-term paraquat exposure induces alteration in the overall cellular ionic homeostasis. • Most alterations observed could be related with the overproduction of ROS.Xunta de Galicia; 08MDS020103P

Population growth study of the rotifer Brachionus sp. fed with triazine-exposed microalgae

[Abstract]Few data exist on potential toxic effects that pollutants may have on zooplankton fed microalgae exposed to pesticides. For that reason, microalgal cultures were exposed to different concentrations of the triazine herbicide terbutryn, and used as exclusive food source to Brachionus sp. females, with the aim to evaluate potential deleterious effects upon population growth, survival, reproduction and feeding of the rotifer.Chlorella vulgaris cells were able to accumulate terbutryn, removing more than 90% of the total amount of herbicide in all the exposed cultures. Growth curves of Brachionus sp. showed that population density decreased as terbutryn concentration increased in the microalgal cells. In fact, this species of rotifer did not survive beyond four days when fed with microalgae exposed to 500 nM terbutryn. Percentage of reproductive females in rotifer populations fed with terbutryn-exposed microalgae decreased significantly as herbicide concentration increased. In control cultures, reproductive females laid a maximum of three eggs per individual; whereas in 100 nM cultures, reproductive females laid only one egg per individual throughout the treatment period. Terbutryn accumulated in C. vulgaris provoked a decrease in the feeding rate ofBrachionus sp. cultures fed with these microalgae with respect to control cultures. After this treatment period, all the rotifer populations, except those fed with 500 nM terbutryn-exposed microalgae, showed recovery patterns when they were returned to fresh medium containing herbicide-free microalga. Taking into account the results obtained, uptake of pesticides by phytoplankton can directly affect higher trophic levels.Xunta de Galicia; PGIDIT04RFO103946P

Toxicity induced by three antibiotics commonly used in aquaculture on the marine microalga Tetraselmis suecica (Kylin) Butch

Aquaculture facilities are a potential source of antibiotics to the aquatic ecosystems. The presence of these compounds in the environment may have deleterious effects on non-target aquatic organisms such as microalgae, which are often used as biological indicators of pollution. Therefore, the toxicity induced by chloramphenicol (CHL), florphenicol (FLO) and oxytetracycline (OTC), three antibiotics widely used in aquaculture, on the marine microalga Tetraselmis suecica was evaluated. Growth inhibition and physiological and biochemical parameters were analysed. All three antibiotics inhibited growth of T. suecica with 96 h IC50 values of 11.16, 9.03 and 17.25 mg L−1 for CHL, FLO and OTC, respectively. After 24 h of exposure no effects on growth were observed and cell viability was also unaffected, whereas a decrease in esterase activity, related with cell vitality, was observed at the higher concentrations assayed. Photosynthesis related parameters such as chlorophyll a cellular content and autofluorescence were also altered after 24 h of antibiotics addition. It can be concluded that T. suecica was sensitive to the three antibiotics tested

Cytotoxic effects of pesticides on microalgae determined by flow cytometry

As much as ca. 99.7% of the applied load of pesticides is dispersed in the environment, not reaching the target pests. In this way, pesticides enter into aquatic ecosystems from agricultural runoff or leaching and, as a consequence, have become some of the most frequently ocurring organic pollutants in aquatic ecosystems. Most phytotoxicological research with herbicides has been conducted on target plants. The sensitivity of algae to many herbicides is very high, and a better understanding of their environmental effects is probably acquired by using test species representing non-target groups. Because of their short generation times, microalgae respond rapidly to environmental changes. Furthermore, microalgal tests are generally sensitive, rapid and low-cost effective. For these reasons, the use of microalgal toxicity tests is increasing, and today these tests are frequently required by authorities for notifications of chemicals and are also increasingly being used to manage chemical discharges. Cytotoxic effects of aquatic pollutants on microalgae are very heterogeneous, and they are influenced by the environmental conditions and the test species. Growth, photosynthesis, chlorophyll fluorescence and others parameters reflect the toxic effects of pollutants on microalgae; however, other relevant endpoints are less known because experimental difficulties, especially under in vivo conditions. Flow cytometry offers multiparametric analysis of cells on a cell-by-cell basis in near in vivo conditions. Applied in the study of the aquatic environments since the 80's, this technique has achieved extensive use for the study of microalgae and has been introduced as an alternative to more traditional techniques of analyzing cells in culture and from natural populations. Several parameters determined by flow cytometry, such as cell volume and granularity, chlorophyll a fluorescence, cell viability, cell proliferation, oxidative stress, membrane potential or intracellular calcium level, were employed to assess changes in the physiological status of different microalgae as a consequence of the toxic action of herbicides. The variety of results obtained in the present study reveals that flow cytometry is a useful tool in the toxicity tests with microalgae.Xunta de Galicia; PGIDIT04RFO103946PRMinisterio de Educación y Ciencia CGL2004-02037BO

Disorders provoked by copper in the marine diatom Phaeodactylum tricornutum in short-time exposure asays

[Abstract] Copper toxicity on the marine diatom Phaeodactylwn tricomutum was assessed after 24.liours of exposure, using different parameters: growth, 14C-bicarbonate assimilation, 3H-thymidine incorporation, photosynthetic pigment content, and others assayed by flow cytometry, such as cellular volume, chlorophyll a autofluorescence and cell viability. Different degrees of sensitivity were observed for the different analysis. Cell viability assayed by flow cytometry was the less sensitive parameter.[Resume] La toxicite du cuivre sur la diatomee marine Phaeodactylum tricomutum. Cette toxicite a ete etudiee apres 24 heures d'exposition, par la mesure de differents parametres: croissance, assimilation de 14C-bicarbonate, incorporation de 3H-thymidine, teneur en pigment photosynthetique, et d'autres parametres evalues par cytometrie de flux, tels que Ie volume cellulaire, l' autofluorescence de la chlorophylle a et la viabilite cellulaire. Differents degres de sensibilite ont ete observes pour les differentes analyses. La viabilite cellulaire, etudiee par cytometrie de flux, s'est averee comme Ie parametre Ie moins sensible.Xunta de Galicia; XUGA1030IB9

Removal of cadmium ions by the marine diatom Phaeodactylum tricornutum Bohlin accumulation and long-term kinetics of uptake

The marine diatom Phaeodactylum tricornutum Bohlin was exposed to different cadmium concentrations (1–100 mg l−1) for 4 days. The amount of cadmium removed was recorded, with particular attention paid to long-term uptake kinetics, and to the cellular location of cadmium. Cadmium accumulation occurred at all concentrations assayed. The EC50 of cadmium to P. tricornutum was 22·39 mg l−1 after 4 days of exposure. Cadmium uptake followed a saturation kinetic at cadmium concentrations ≥25 mg l−1. However, at lower cadmium concentrations, the uptake of this metal followed a linear trend for all days of culture. At cadmium concentrations in the medium lower than 25 mg l−1, P. tricornutum removed cadmium mainly within the cell. At higher cadmium concentrations, the amount of cadmium removed by adsorption to the cell surface was higher than intracellular cadmium, because of the toxic effects of cadmium on P. tricornutum cells. This toxicity reduced the cadmium accumulation within the cells.Xunta de Galicia; XUGA 10301B9

The herbicide paraquat induces alterations in the elemental and biochemical composition of non-target microalgal species

[Abstract]Huge quantities of pesticides are dispersed in the environment, affecting non-target organisms. Since paraquat affects the photosynthetic process, the biochemical composition of affected species should be altered. The effect of paraquat on Chlamydomonas moewusii, a freshwater non-target species, was studied. After 48 h of herbicide exposure, growth rate, dry weight, and chlorophyll a and protein content were affected by paraquat concentrations above 0.05 μM. C/N ratio was also affected due to a decrease in nitrogen content in the dry biomass, while the carbon content remained constant for all paraquat concentrations assayed. Enzymes involved in nitrogen assimilation were affected by paraquat, being nitrate reductase activity more sensitive to paraquat than nitrite reductase. Based on the results obtained in the present study, paraquat exerts adverse effects upon a common freshwater green microalga, thus the application of this herbicide for weed control must be carried out very carefully, so that any disturbance affecting algae will have severe repercussions on higher trophic levels and on the elemental biogeochemical cycles.Ministerio de Educación y Ciencia; CGL 2004/02037 BO