Freshwater Green Microalgae Research Articles

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

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.

Rapid determination of arsenic species in freshwater organisms from the arsenic-rich Hayakawa River in Japan using HPLC-ICP-MS

Speciation analyses of water-soluble arsenicals from freshwater and biological samples collected from the Hayakawa River (Kanagawa, Japan), which contains a high concentration of arsenic, were performed using high performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC-ICP-MS). River water contained only arsenate, which is a pentavalent inorganic arsenical. The water bug Stenopsyche marmorata contained inorganic arsenicals accounting for 77% of the water-soluble arsenicals, followed by oxo-arsenosugar-glycerol, which is a type of dimethylarsinoylriboside (arsenosugar). The freshwater green macroalga Cladophora glomerata contained oxo-arsenosugar-glycerol and oxo-arsenosugar-phosphate as 64% of the water-soluble arsenicals. Production of the same types of arsenosugars was confirmed in the freshwater green microalga Chlamydomonas reinhardtii CC125 experimentally exposed to arsenate. The muscle tissues of all freshwater fish and crustaceans analyzed contained arsenobetaine, oxo-arsenosugar-glycerol, and/or oxo-arsenosugar-phosphate in various concentrations. Seven freshwater fish ( Cobitis biwae, Leuciscus hakonensis, Phoxinus lagowski steindachneri, Plecoglossus altivelis, Rhinogobius sp. CB, Rhinogobius sp. CO, Sicyopterus japonicus) and the crustacean Macrobracbium nipponenese contained arsenobetaine in their muscle tissues as the predominant form, contributing up to 80% of the water-soluble arsenicals, while the freshwater fish Anguilla japonica muscle tissues primarily contained dimethylarsinic acid as 77% of the water-soluble arsenicals, followed by arsenobetaine. The freshwater fish Zacco platypus muscle tissues predominantly contained oxo-arsenosugar-phosphate, accounting for 51% of the water-soluble arsenicals, followed by dimethylarsinic acid and arsenobetaine. These biological samples possessed non-extractable arsenical(s) accounting for more than 50% of the total arsenic concentration.

Chemical fingerprinting of algaenans using RuO 4 degradation

The freshwater green microalgae, Oocystus solitaria Wittrock f. maior Wille, Pediastrum braunii Wartmann, Pediastrum kawraiskyi Schmidle, Sorastrum spinulosum Nägeli and Coelastrum reticulatum (Dangeard) Senn were investigated with respect to the presence of algaenan and the corresponding RuO 4 chemical fingerprint. All species, with the exception of O. solitaria, were shown to possess a cell wall resistant toward strong acid and base treatment. By analysis of the RuO 4 chemical degradation products from these materials a molecular fingerprint was obtained having similar features to the RuO 4 fingerprints of Tetraedron minimum, Scenedesmus communis and Pediastrum boryanum algaenans reported in earlier studies. Previously it had been shown that these RuO 4 fingerprints could also be obtained from ancient sediments, indicating the presence of algaenan as part of the organic matter. The results from this study show that the chemical composition of algaenans is possibly a chemotaxonomic feature and can be used to chemically explore the algal contribution to sedimentary organic matter on a family level. Furthermore, it illustrates that the use of RuO 4 degradation combined with gas chromatography is a suitable analytical tool for the purpose of chemical fingerprinting of algaenans.

Bioavailability Models for Predicting Copper Toxicity to Freshwater Green Microalgae as a Function of Water Chemistry

We investigated whether an earlier-developed bioavailability model for predicting copper toxicity to growth rate of the freshwater alga Pseudokirchneriella subcapitata could be extrapolated to other species and toxicological effects (endpoints). Hardness and dissolved organic carbon did not significantly affect the toxicity of the free Cu2+ ion to P. subcapitata (earlier study) and Chlorella vulgaris(this study), but a higher pH resulted in an increased toxicity for both species. Regression analysis showed significant linear relationships between ECxpCu (= "effect concentration" that produces x% adverse effect, expressed as pCu = -log of the Cu2+ activity) and pH. By linking these regression models with a geochemical metal speciation model, dissolved copper concentrations that elicit a given adverse effect (EC(X)dissolved) can be predicted. Within the pH range investigated (5.5-8.7), slopes of the linear EC(X)pCu vs pH regression models varied between 1.301 and 1.472 depending on the species and the effect level (10% or 50%) considered. In a statistical sense these slopes were all significantly different from one another (p < 0.05), suggesting that this empirical regression model does not yet capture the full complexity of toxicological copper bioavailability to algae. However, we demonstrated that regression models with an "average" slope of 1.354 had predictive power very similar to those of regression models with species and effect-specific slopes. Additionally, the "average" regression model was further successfully validated for other species (Chlamydomonas reinhardtii and Scenedesmus quadricauda) and for different toxicological effects/endpoints (growth rate, biomass yield, and phosphorus uptake rate). For all these toxicity datasets effect concentrations of copper could be predicted with this "average" model by errors of less than a factor of 2 in 94-100% of the cases. The success of this "average" model suggests the possibility that the pH-based linear regression model may form a sound conceptual basis for modeling the toxicological bioavailability of copper to green algae in regulatory assessments, although a full mechanistic understanding is lacking and should be the focus of future studies.

Toward a Biotic Ligand Model for Freshwater Green Algae: Surface-Bound and Internal Copper Are Better Predictors of Toxicity than Free Cu2+-Ion Activity When pH Is Varied

The freshwater green microalgae Chlorella sp. and Pseudokirchneriella subcapitata (P. subcapitata) were chronically (48 and 72 h, respectively) exposed to copper at various pH levels, i.e., pH 6-7.5 and pH 5.9-8.5, respectively. Concentrations resulting in 50% inhibition of exponential growth rate (EC50) were determined as dissolved Cu, estimated chemical activity of the free Cu2+ ion (as pCu = - log{Cu2+ activity as molarity}), and as external (surface-bound) Cu and internal Cu in the algal cells. With increasing pH, EC50dissolved decreased from 30 to 1.1 microg of Cu L(-1) for Chlorella sp. and from 46 to 18 microg of Cu L(-1) for P. subcapitata. The pH effect on copper toxicity was even more obvious when expressed as Cu2+ activity. The EC50pCu increased on average 1.4 pCu unit per pH unit for Chlorella sp. and 1.1 pCu unit per pH unit for P. subcapitata, thus indicating a marked increase of Cu2+ toxicity at higher pH (more than 1 order of magnitude per pH unit). In contrast, it was found that EC50 values expressed as surface bound or external copper (EC50external) and as internal copper (EC50internal) did not vary substantially when pH was increased. External Cu was operationally defined as the Cu fraction removable from the algal cell by short-term contact with ethylenediaminetetraacetic acid; internal copper was defined as the nonremovable fraction. For Chlorella sp. the EC50external varied between 5 and 10 fg of Cu/ cell (factor of 2 difference) and the EC50internal between 25 and 40 fg of Cu/cell (factor of 1.6 difference). For P. subcapitata the EC50external varied between 10 and 28 fg of Cu/cell (factor of 2.8 difference) and the EC50internal between 42 and 71 fg of Cu/cell (factor of 1.7 difference). Because the observed variation in EC50external and EC50internal is much less than the variation in EC50Cu2+, it is concluded that both external and internal copper are better predictors of copper toxicity than Cu2+ when pH is varied. From the perspective of toxicity modeling, this observation is the first step toward considering the use of the cell surface as the algal biotic ligand for Cu in a similar way as fish gills fulfill this role in the biotic ligand model for predicting metal toxicity to fish species.

The Effect of Salt Stress on the Production of Canthaxanthin and Astaxanthin by Chlorella zofingiensis Grown Under Limited Light Intensity

The fresh water green microalga Chlorella zofingiensisis known to accumulate ketocarotenoids – primarily astaxanthin but also canthaxanthin – when grown under stress conditions of high light irradiance and low nitrogen. We found that salt stress can replace light stress with respect to inducing carotenoid production: cells of C. zofingiensis grown under low light irradiance and subjected to salt and low nitrogen stress accumulated higher amounts of total secondary carotenoids than those growing under high light and low nitrogen stress. Furthermore, C. zofingiensis growing under conditions of salt stress and low light accumulated higher amounts of canthaxanthin than astaxanthin. It is suggested that for canthaxanthin accumulation under salt stress, light is not a limiting factor, but for astaxanthin accumulation high light irradiance is mandatory. These results may be applied in the future for the commercial production of canthaxanthin by C. zofingiensis in systems in which light availability is poor.

Zoospore adhesion and germination upon non-toxic substances in Pseudulvella sp. (Chlorophyta)

One of the most significant processes in the life history of an alga is the colonization of a new substratum. In the present study, we evaluate whether different organic compounds, such as agar, gelatine, chicken albumin, glycerine and polylysine, promote zoospore recruitment and germination in a periphytic, fresh-water green microalga of the genus Pseudulvella (Chlorophyta). Given the low adhesion capacity of its zoospores a series of experiments were conducted in order to find a substance and its optimal concentration that increases zoospore recruitment and allows us to follow the processes of settlement, attachment and germination of zoospores. Polylysine significantly increased the number of zoospores attached with no significant effect on the germination rate. The minimum effective concentration of polylysine for improving zoospore settlement was 0.1%.

Accumulation of arachidonic acid-rich triacylglycerols in the microalga Parietochloris incisa (Trebuxiophyceae, Chlorophyta)

The freshwater green microalga Parietochloris incisa is the richest known plant source of the polyunsaturated fatty acid (PUFA), arachidonic acid (20:4ω6, AA). While many microalgae accumulate triacylglycerols (TAG) in the stationary phase or under certain stress conditions, these TAG are generally made of saturated and monounsaturated fatty acids. In contrast, most cellular AA of P. incisa resides in TAG. Using various inhibitors, we have attempted to find out if the induction of the biosynthesis of AA and the accumulation of TAG are codependent. Salicylhydroxamic acid (SHAM) affected a growth reduction that was accompanied with an increase in the content of TAG from 3.0 to 6.2% of dry weight. The proportion of 18:1 increased sharply in all lipids while that of 18:2 and its down stream products, 18:3ω6, 20:3ω6 and AA, decreased, indicating an inhibition of the Δ12 desaturation of 18:1. Treatment with the herbicide SAN 9785 significantly reduced the proportion of TAG. However, the proportion of AA in TAG, as well as in the polar lipids, increased. These findings indicate that while there is a preference for AA as a building block of TAG, the latter can be produced using other fatty acids, when the production of AA is inhibited. On the other hand, inhibiting TAG construction did not affect the production of AA. In order to elucidate the possible role of AA in TAG we have labeled exponential cultures of P. incisa kept at 25 °C with [1- 14C]arachidonic acid and cultivated the cultures for another 12 h at 25, 12 or 4 °C. At the lower temperatures, labeled AA was transferred from TAG to polar lipids, indicating that TAG of P. incisa may have a role as a depot of AA that can be incorporated into the membranes, enabling the organism to quickly respond to low temperature-induced stress.

The adsorption kinetics of metal ions onto different microalgae and siliceous earth

In the present work the adsorption kinetics of the six metal ions aluminum, zinc, mercury, lead, copper, and cadmium onto living microalgae were measured. The freshwater green microalga Scenedesmus subspicatus, the brackish water diatom Cyclotella cryptica, the seawater diatom Phaeodactylum tricornutum, and the seawater red alga Porphyridium purpureum were the subject of investigation. In most cases the adsorption rate of the metals could be well described by using the equation of the Langmuir adsorption rate expression. Inverse parameter estimation allowed the determination of the rate constants of the adsorption process and the maximum metal content of the algae. The highest values for the rate constant were obtained for Porphyridium purpureum followed by Phaeodactylum tricornutum. High values for the maximum content were obtained for Cyclotella cryptica and Scenedesmus subspicatus. The maximum rate constant was 24.2 l h −1 for the adsorption of Hg to Porphyridium purpureum whereas the maximum metal content (0.243 g g −1) was obtained for Zn on Cyclotella cryptica. A comparison of these values with those obtained for the mineral siliceous earth exhibiting low maximum content and high adsorption rates reveals that the mechanism of adsorption onto the algae is a mixture of adsorption and accumulation.

Phytotoxicity of the New Antifouling Compound Irgarol 1051 and a Major Degradation Product

The phytotoxicity of the new antifouling compound Irgarol 1051 (2-methylthio-4- tert-butylamino-6-cyclopropylamino- s-triazine) and its degradation product M1 (2-methylthio-4- tert-butylamino-6-amino- s-triazine) was assessed by a battery of bioassays with marine and freshwater species. Both compounds at low concentrations affected aquatic plant species such as the red macroalga Porphyra yezoensis conchospores, the brown macroalga Eisenia bicyclis gametophytes, the freshwater green microalga Closterium ehrenbergii, the floating macrophytes Lemna gibba G3 and Lemna minor 1769. The toxicity of Irgarol 1051 was higher than that of M1 for all the aquatic plant species tested, but M1 showed a higher toxicity to root elongation of a terrestrial plant. The NOEC (0.3 μg/ l) of Irgarol to the brown seaweed was identical to the maximum concentration (0.296 μg/ l) detected in Japanese coastal waters, and the maximum concentration of M1 (1.87 μg/ l) detected amounted to only 20% of the NOEC value. The bioassay results suggest the possibility that both Irgarol 1051 and M1 may influence the primary producer community in the aquatic environment.

Arsenic compounds in the freshwater green microalga Chlorella vulgaris after exposure to arsenite

Arsenic compounds in the freshwater green microalga Chlorella vulgaris after exposure to arsenite

Chemical structure of algaenans from the fresh water algae Tetraedron minimum, Scenedesmus communis and Pediastrum boryanum

The cell walls of the fresh water green microalgae Tetraedron minimum, Scenedesmus communis and Pediastrum boryanum are composed of highly resistant, non-hydrolyzable aliphatic biopolymers as revealed by 13C-NMR, FTIR and thermal and chemical degradations. The biopolymers are composed of long-chain even-carbon-numbered ω 9-unsaturated ω-hydroxy fatty acid monomers varying in chain length from 30 to 34 carbon atoms. These monomers are intermolecularly ester linked to form linear chains in which the unsaturations act as the starting position of ether cross-linking. S. communis biosynthesises a more densely cross-linked algaenan than T. minimum and P. boryanum. The monomers of T. minimum have, on average, larger chain lengths than those of P. boryanum and S. communis. The polyether nature of these algaenans makes them highly resistant against degradation, so that they are selectively preserved in the sedimentary record. Therefore, these algaenans probably are important precursors for Type I kerogens.

Cell wall-specific ω-hydroxy fatty acids in some freshwater green microalgae

Ester-bound lipids from the cell walls of the green algae, Tetraedron minimum, Scenedesmus communis and Pediastrum boryanum, were analysed by gas chromatography-mass spectrometry. The double bond positions of the C 30, C 32 and C 34 mono- and C 30 and C 32 diunsaturated ω-hydroxy fatty acids obtained were determined by derivatisation with dimethyl disulphide (DMDS) prior to mass spectrometric identification. All unsaturated ω-hydroxy fatty acids have a double bond located at the ω9-position, whilst the C 30 and C 32 diunsaturated acids have a second double bond at the ω19 and ω18 positions, respectively. Unsaturated ω-hydroxy fatty acids are released upon saponification of the isolated cell walls in all three algae, but the distribution patterns of these compounds differ between species. ω-Hydroxy fatty acids are the main building blocks of the highly cross-linked constituent of the cell walls, herein termed algaenan, in which linear chains of esterified monomers are ether cross-linked at the position of the double bonds. Because none of these monomeric compounds were observed in the cytosolic fraction of the cells, it is believed that they are rapidly secreted and incorporated in the aliphatic cell walls of these algae.

Resistant biomacromolecules in marine microalgae of the classes Eustigmatophyceae and Chlorophyceae: Geochemical implications

Non-hydrolysable macromolecular constituents (i.e. algaenans) were isolated from two out of seven marine microalgae investigated. Nannochloropsis salina and Nannochloropsis sp. from the class of Eustigmatophyceae produce highly aliphatic algaenans. Flash pyrolysis and chemical degradations with HI and RuO 4 allowed for the identification of their chemical structure, which is mainly composed of polyether-linked long-chain (up to C 36) n-alkyl units. The building blocks of this polymer were also recognized in lipid fractions. The green microalgae (Chlorophyceae) Chlorella spaerckii, Chlorococcum sp. and Nannochloris sp. were earlier thought to biosynthesize algaenans comprising aliphatic and/or aromatic moieties. However, a new isolation method utilizing trifluoroacetic acid (TFA) prior to the other hydrolyses revealed that the macromolecular material isolated from these three chlorophytes was either hydrolysable with TFA or artefacts from the former method. Similar to algaenans from fresh water green microalgae, the aliphatic eustigmatophyte algaenans are likely to be selectively preserved in depositional environments and might ultimately serve as source rock organic matter of marine crude oils. Furthermore, they may play an important role in the cycling of carbon.

Toxıcity of anionic surfactants to green microalgae “Scenedesmus Subspicatus” and “Selenastrum Capricornutum”

This study was undertaken to compare the chronic toxicity results (EC-50-72 hours) of common anionic surfactants - LAS (Linear Alkylbenzene Sulphonate), AS (Alcohol Sulphate) and AES (Alcohol Ether Sulphate) - to selected fresh water green microalgae. For LAS, not only commercial products have been tested but also its single homologues C 10 -C 14 were assessed. As a general rule for LAS, the higher the molecular weight, the greater the toxicity. Other anionic surfactants appear to be slightly more toxic than LAS when using standard toxicity tests.

Uptake of technetium by freshwater green microalgae

Accumulation of [99Tc]pertechnetate ions (99TcO4−) by the freshwater microalgae Chlorella emersonii, Chlamydomonas reinhardtii and Scenedesmus obliquus has been characterized. In all three species, accumulation consisted of a single rapid energy-independent phase (biosorption), and no energy-dependent accumulation was observed. Biosorption of 99TcOinf4sup−by all three species was concentration dependent, followed a Freundlich adsorption isotherm, and was dependent on pH with increased accumulation by cells with decreasing external pH. Elevated external NaCl concentrations also caused increased accumulation of 99TcOinf4sup−by the cells, as did increased external osmotic potential. Concentrations of K+, Mg2+ and Ca2+ increased accumulation of 99TcOinf4sup−, but concentrations of HCOinf3sup−, SOinf4sup2−and COinf3sup2−decreased 99TcOinf4sup−accumulation by the cells. Most of the 99TcOinf4sup−accumulated by all three species was easily desorbed by 10 mm buffers at various pH values, 0.5 m NaCl, 10 mm Na2CO3 or 10 mm Na2SO4. No differences in the amount of desorption were observed between the various desorption agents used.

Inorganic Carbon Limitation and Chemical Composition of Two Freshwater Green Microalgae

Two freshwater chlorophytes, Chlorella vulgaris and Scenedesmus obliquus, were grown in inorganic carbon-limited continuous cultures in which HCO(3) was the sole source of inorganic carbon. The response of the steady-state growth rate to the external total inorganic carbon concentration was reasonably well described by the Monod equation; however, the response to the internal nutrient concentration was only moderately well represented by the Droop equation when the internal carbon concentration was defined on a cellular basis. The Droop equation was totally inapplicable when total biomass (dry weight) was used to define internal carbon because the ratio of carbon to dry weight did not vary over the entire growth rate spectrum. In batch cultures, maximum growth rates were achieved at the CO(2) levels present in atmospheric air and at HCO(3) concentrations of 3 mM. No growth was observed at 100% CO(2). Both nitrogen uptake and chlorophyll synthesis were tightly coupled to carbon assimilation, as indicated by the constant C/N and C/chlorophyll ratios found at all growth rates. The main influence of inorganic carbon limitation appears to be not on the chemical structure of the biomass, but rather on cell size; higher steady-state growth rates lead to bigger cells.