Biomass, photosynthetic activity, and biomolecule composition in Chlorella fusca (Chlorophyta) cultured in a raceway pond operated under greenhouse conditions.

Raceways are widely used as microalgae culture systems due to their low cost, but they are not the best option for biomass yield. Understanding in situ photosynthetic performance can be a first step to increase their biomass productivity. This study aimed at comparing the real time photosynthetic activity in a greenhouse raceway culture (250 L) with discrete measurements under laboratory conditions. We evaluated the photophysiology and biochemical composition of Chlorella fusca culture up to 120 h. In situ photosynthetic activity was continuously monitored and compared to discrete ex situ measurements; biochemical compounds were measured daily. The results showed a final biomass density of 0.45 g L-1 (5 days - 120 h) and an increase of the electron transport rate (ETR) up to 48 h but decreased thereafter. When the relative ETR was estimated considering the absorption coefficient (a) positive correlations of this parameter with photosynthetic capacity, cell density, biomass, biocompounds and antioxidant activity were obtained, whereas no correlation was detected without considering a. In situ photosynthesis monitoring showed higher absolute maximal ETR (10 - 160 µmol m-3s-1) than discrete ex situ measurements. We demonstrated the importance of considering the light absorption coefficient for expressing photosynthetic capacity and showed that C. fusca can produce, in the short-term, bioactive compounds that are correlated to photosynthetic conditions.

Study of the growth and biochemical composition of 20 species of cyanobacteria cultured in cylindrical photobioreactors.

BACKGROUND: Cyanobacteria are prokaryotic organisms with wide morphological and metabolic diversity. By means of photosynthesis, they convert inorganic compounds into biomolecules, which may have commercial interest. In this work, we evaluated 20 cyanobacterial strains regarding their physiological aspects such as growth, photosynthesis and biochemical composition, some of which are revealed here for the first time. The organisms were cultivated in cylindrical photobioreactors (CPBR) for 144 h and the biomass was obtained. The light inside cultures was constant throughout experimental time and maintained at the saturation irradiance (Ik) of each species. Culture pH was maintained within 7.8 and 8.4 by automatic CO2 bubbling. Growth rate, dry biomass, chlorophyll a, carotenoids, phycocyanin, proteins, carbohydrates, lipids, polyhydroxyalkanoate (PHA) and antioxidant activity were determined. RESULTS: The proportionality of the biochemical composition varied among species, as well as the growth rates. Leptolyngbya sp. and Nostoc sp. (CCIBt3249) showed growth rates in the range of 0.7-0.8 d-1, followed by Rhabdorderma sp. (~ 0.6 d-1), and Phormidium sp. (~ 0.5 d-1). High carotenoid content was obtained in Rhabdoderma sp. (4.0 µg mL-1) and phycocyanin in Leptolyngbya sp. (60 µg mL-1). Higher total proteins were found in the genus Geitlerinema (75% DW), carbohydrates in Microcystis navacekii (30% DW) and lipids in Phormidium sp. (15% DW). Furthermore, Aphanocapsa holsatica showed the highest antioxidant activity (65%) and Sphaerocavum brasiliense, Microcystis aeruginosa, Nostoc sp. (CCIBt3249) and A. holsatica higher levels of PHA (~ 2% DW). CONCLUSIONS: This study reports on the biochemical composition of cyanobacteria that can impact the biotechnology of their production, highlighting potential strains with high productivity of specific biomolecules.

Environmentally relevant copper concentrations stimulate photosynthesis in Monoraphidium sp.

Microalgae require copper (Cu) in trace levels for their growth and metabolism, it is a vital component of certain metalloproteins. Although this element has been widely studied concerning microalgae physiology, the effects of environmentally relevant levels have been less studied. We studied the photosynthesis and growth of the Chlorophyte Monoraphidium sp. exposed to Cu ranging from low (1.7 nM) to high (589.0 nM) free Cu ions (Cu2+) concentrations. The growth rate was unaffected by Cu concentrations in the range of 1.7-7.4 nM Cu2+, but decreased beyond it. The relative maximum electron transport rate (rETRm), saturation irradiance (Ek), photochemical quenching (qP and qL), and PSII operating efficiency [Formula: see text] were stimulated in the 3.4-7.4 nM Cu2+ range, concentrations slightly higher than the control, whereas non-photochemical quenching (NPQ) gradually increased with increasing Cu2+. The photosystem II antenna size [Sigma (II)440] increased under high Cu (589.0 nM), which resulted in a decrease in the quinone A (QA) reduction time (tau). In contrast, the QA re-oxidation time was unaffected by Cu exposure. These findings show that a slight increase in Cu stimulated photosynthesis in Monoraphidium sp., whereas high Cu reduced photosynthesis and increased the dissipation of captured light energy. This research is a contribution to the understanding of the dynamic photo-physiological responses of Monoraphidium sp. to Cu ions.

Differential Effects of Atrazine on Chlorophyceae Species and Association with Morphology, Photosynthesis, Chlorophyll Content, and Glutathione-S-Transferase Activity.

Atrazine is a herbicide widely used in the control of weeds in crops such as corn, sugar cane, and sorghum. It is often found in aquatic environments, where it can potentially endanger nontarget organisms such as microalgae. The present study evaluated atrazine toxicity to seven different species of Chlorophyceae and the tolerance of the species to the herbicide was related to morphological, photosynthetic, chlorophyll-a content and the activity of the glutathione-S-transferase enzyme (GST). The comparison of median effect concentration (EC50) values for growth inhibition indicates higher toxicity of atrazine for Pseudopediastrum boryanum and Desmodesmus communis, intermediate toxicity for Ankistrodesmus densus, Chlamydomonas puliminiorfes, and Raphidocelis subcapitata, and lower toxicity for Kirchneriella lunaris and Ankistrodesmus falcatus (EC50: 38, 42, 66, 103, 248, 1004, and 1585 µg L-1 atrazine, respectively). Principal component analysis (PCA) with algal characteristics suggested that the atrazine-sensitive algae P. boryanum and D. communis were positively associated with photosynthetic levels and negatively associated with GST activity and chlorophyll-a concentration. The PCA also suggested that the atrazine-tolerant algae A. falcatus and K. lunaris were positively associated with morphological parameters, where the larger the cell size, the more tolerant. Although it is difficult to associate a single characteristic of algae as the key factor determining the tolerance to atrazine, results presented in this work indicate that the cell area, the photosynthetic parameters (mainly saturating irradiance), chlorophyll-a content, and the biotransformation by GST in combination may be potential predictors for the differential tolerance of Chlorophyceae species to the herbicide. Environ Toxicol Chem 2022;41:1675-1685. © 2022 SETAC.

Bioprospecting and selection of tolerant strains and productive analyses of microalgae grown in vinasse.

In order to contribute to the biotechnology of microalgae cultivated in vinasse, we carried out the bioprospection of tolerant species and synthesized biomolecules of the total biomass (microalgae and bacteria), recovered from cultures. To use vinasse as a culture medium for the microalgae, waste was centrifuged and used in concentrations from 5 to 50%. Daily cell densities, growth rates, and EC50 values were obtained. After defining the best pair of vinasse concentration/microalgae strain, dry biomass, and composition (proteins and carbohydrates) were determined in 96 h cultures, considering the associated community (bacteria and yeast). The microalgae tested were Chlamydomonas sp., Chlorella sorokiniana, Chlorella vulgaris, Desmodesmus spinosus, Haematococcus pluvialis, Monoraphidium sp., Scenedesmus quadricauda, and Tetraselmis gracilis. The results showed that although the microalgal growth rates in vinasse were similar to controls in BG11, the cells in vinasse had higher biovolumes, dry biomass, and total proteins. The species H. pluvialis, S. quadricauda, and T. gracilis showed the best productivity parameters in vinasse, despite lower growth rates than the other species. Using low concentrations of centrifuged vinasse as a culture medium, only 22% of biological contaminants were present, thus most of the processed biomass was mainly composed of microalgae. Thus, Chlamydomonas sp., D. spinosus, S. quadricauda, and H. pluvialis microalgae have attributes such as resistance and biomolecules that make them candidates for further optimization in production systems, combining the environmental benefits of using waste with the production of biomolecules and/or biomass of commercial interest.

Electron & Biomass Dynamics of Cyanothece Under Interacting Nitrogen & Carbon Limitations.

Marine diazotrophs are a diverse group with key roles in biogeochemical fluxes linked to primary productivity. The unicellular, diazotrophic cyanobacterium Cyanothece is widely found in coastal, subtropical oceans. We analyze the consequences of diazotrophy on growth efficiency, compared to NO3 --supported growth in Cyanothece, to understand how cells cope with N2-fixation when they also have to face carbon limitation, which may transiently affect populations in coastal environments or during blooms of phytoplankton communities. When grown in obligate diazotrophy, cells face the double burden of a more ATP-demanding N-acquisition mode and additional metabolic losses imposed by the transient storage of reducing potential as carbohydrate, compared to a hypothetical N2 assimilation directly driven by photosynthetic electron transport. Further, this energetic burden imposed by N2-fixation could not be alleviated, despite the high irradiance level within the cultures, because photosynthesis was limited by the availability of dissolved inorganic carbon (DIC), and possibly by a constrained capacity for carbon storage. DIC limitation exacerbates the costs on growth imposed by nitrogen fixation. Therefore, the competitive efficiency of diazotrophs could be hindered in areas with insufficient renewal of dissolved gases and/or with intense phytoplankton biomass that both decrease available light energy and draw the DIC level down.

Optimization and qualitative comparison of two vinasse pre-treatments aiming at microalgae cultivation / Otimização e comparação qualitativa de dois pré-tratamentos da vinhaça visando ao cultivo de microalgas

ABSTRACT The cultivation of microalgae is a possible destination for vinasse, a residue from the sugar and alcohol industry. This use can help reduce the costs of microalgae production and remediate this residue rich in nutrients. However, the physicochemical characteristics of vinasse limit its use for microalgae growth at low concentrations, except when the residue is pretreated. This work aimed at optimizing the vinasse pretreatments of centrifugation and adsorption by smectite clay and activated charcoal on laboratory scale in terms of amounts of materials used and time spent, making them more viable on larger scales. The optimized processes were then compared in productive, economic, and environmental terms. The dilution of treated vinasse with distilled water resulted in similar growth of Chlorella vulgaris to those obtained with the dilution in BG11 medium, indicating that the addition of nutrients in culture media is not necessary. Although microalgae growth occurs in higher concentrations of vinasse treated by adsorption, the results show that centrifugation required less processing time, has cheaper processing costs, and generated much less residue. Centrifugation treatment has greater economic and environmental viabilities and was more sustainable than the adsorption, even though the algae did not grow in the centrifuged residue in concentrations as high as it did after the adsorption treatment. Therefore, this article brings a new view about the economic and environmental aspects on the use of pretreated vinasse for microalgal growth, giving a lucrative destination for a highly polluting waste.

RESUMO O cultivo de microalgas é um possível destino para a vinhaça, um resíduo da indústria sucroalcooleira. Esse uso pode ajudar a reduzir custos da produção de microalgas, além de remediar esse resíduo rico em nutrientes, entretanto as características físico-químicas da vinhaça limitam seu uso para o crescimento de microalgas a baixas concentrações, exceto quando o resíduo é pré-tratado. Este trabalho objetivou otimizar os pré-tratamentos da vinhaça de centrifugação e de adsorção por argila esmectita e carvão ativado em escala laboratorial quanto à quantidade de materiais utilizados e tempo gasto, tornando-os mais viáveis em escalas ampliadas. Os processos otimizados foram então comparados em termos produtivos, econômicos e ambientais. A diluição das vinhaças tratadas com água destilada resultou em crescimento de Chlorella vulgaris semelhante ao obtido com diluições com meio BG11, indicando que a adição de nutrientes nos meios de cultura não é necessária. Embora o crescimento de microalgas ocorra em concentrações mais altas de vinhaça tratada por adsorção, os resultados mostram que a centrifugação exigiu menos tempo de processamento, tem custos mais baratos e gerou menos resíduos. De maneira comparativa, o tratamento por centrifugação possui maior viabilidade econômica e ambiental e foi mais sustentável do que a adsorção, embora as algas não tenham crescido no resíduo centrifugado em concentrações tão altas quanto após o tratamento de adsorção. Portanto, este artigo traz uma nova visão sobre os aspectos econômicos e ambientais do uso da vinhaça pré-tratada para o crescimento de microalgas, proporcionando um destino lucrativo para um resíduo altamente poluente.

Combination of P-limitation and cadmium in photosynthetic responses of the freshwater microalga Ankistrodesmus densus (Chlorophyceae).

In the environment, microalgae are exposed to a multitude of stressors simultaneously, inducing physiological adjustments. It is well documented that both phosphorus (P) limitation and trace metals exposure affect microalgal physiology. However, investigations regarding the combination of both P limitation and excess trace metals still deserve attention. In the present study, we evaluated the changes in photosynthetic parameters in the green microalga Ankistrodesmus densus acclimated to different P concentrations prior to exposure to Cd. Our results indicate that different concentrations of P in the medium were responsible for significant changes in some parameters, especially those related to photoprotection mechanisms. Cadmium also altered some of these variables in all P scenarios, and greater damage (i.e., synergism) was observed in the combination P-limited and high Cd, with all the evaluated parameters affected under the adverse scenario. Among the parameters analyzed, rapid light curves were the most sensitive to exposure of one or the combination of both stressors (Cd and P limitation). Based on our data, we suggest that P-limited algae activated photoprotective mechanisms as a response to nutrient limitation, especially at the most limited condition. The addition of Cd did not change linearly the parameters related to photoprotection mechanisms under P-limitation, i.e., synergism was observed in the intermediate P-limitation combined with Cd, while in the most P-limited, P seems to be the driving force affecting these mechanisms. Based on our results, we suggest the use of rapid light curves as a tool to complement the assessment of the impacts of stressors, such as metals, in ecotoxicological studies.

Temporal Patterns and Intra- and Inter-Cellular Variability in Carbon and Nitrogen Assimilation by the Unicellular Cyanobacterium Cyanothece sp. ATCC 51142.

Unicellular nitrogen fixing cyanobacteria (UCYN) are abundant members of phytoplankton communities in a wide range of marine environments, including those with rapidly changing nitrogen (N) concentrations. We hypothesized that differences in N availability (N2 vs. combined N) would cause UCYN to shift strategies of intracellular N and C allocation. We used transmission electron microscopy and nanoscale secondary ion mass spectrometry imaging to track assimilation and intracellular allocation of 13C-labeled CO2 and 15N-labeled N2 or NO3 at different periods across a diel cycle in Cyanothece sp. ATCC 51142. We present new ideas on interpreting these imaging data, including the influences of pre-incubation cellular C and N contents and turnover rates of inclusion bodies. Within cultures growing diazotrophically, distinct subpopulations were detected that fixed N2 at night or in the morning. Additional significant within-population heterogeneity was likely caused by differences in the relative amounts of N assimilated into cyanophycin from sources external and internal to the cells. Whether growing on N2 or NO3, cells prioritized cyanophycin synthesis when N assimilation rates were highest. N assimilation in cells growing on NO3 switched from cyanophycin synthesis to protein synthesis, suggesting that once a cyanophycin quota is met, it is bypassed in favor of protein synthesis. Growth on NO3 also revealed that at night, there is a very low level of CO2 assimilation into polysaccharides simultaneous with their catabolism for protein synthesis. This study revealed multiple, detailed mechanisms underlying C and N management in Cyanothece that facilitate its success in dynamic aquatic environments.

Environmental concentrations of copper nanoparticles affect vital functions in Ankistrodesmus densus.

Nanoparticles (NPs) have unique properties, leading to their widespread application in industry, consequently increasing their concentration in aquatic ecosystems. Although environmentally significant concentrations are still low, they tend to increase because of the intense use, posing into risk microalgae communities. Microalgae are primary producers that support food chains in aquatic ecosystems; thus factors that interfere with their physiology can be propagated throughout the food web. The present research investigated the effects of copper nanoparticles (Cu-NPs) in the physiology of a cosmopolitan green microalgae, Ankistrodesmus densus. Here, we focused on environmental NPs levels, so an ample Cu-NPs range was used, 0.3-635 µg L-1. Considering that NPs dissolve into the medium releasing their constituent material, free Cu2+ ions were determined and considered as surrogate for NPs concentration, which varied from 2.1 × 10-9 to 8.4 × 10-9 mol L-1. The experiment was based in 72 h Cu-NPs exposure, and to access the physiology of A. densus, we monitored population growth, photochemistry of photosynthesis and the content of cell biomolecules (total proteins, carbohydrates and lipids). The results showed that 2.1 × 10-9 mol L-1 free Cu2+ was enough to decrease growth rate, but 2.5x higher Cu was necessary to affect the photosynthetic parameters. Inorganic carbon fixation rate calculated by absolute electron transport rates was affected. Considering cell biomolecules, total proteins accumulated at 6.5 × 10-9 and kept increasing up to 8.4 × 10-9 mol L-1 free Cu2+. Because this was not related to biomass formation, we suggest a possible association with cell detoxification mechanisms. The most clear finding that emerged from this study is that environmental Cu-NPs concentrations affect vital functions in the green microalgae A. densus. An implication of this is the possibility of facing problems related to a increase of NPs in aquatic ecosystems in the near future.

Photosynthetic, morphological and biochemical biomarkers as tools to investigate copper oxide nanoparticle toxicity to a freshwater chlorophyceae.

Copper oxide nanoparticles (CuO NP) have been produced on a large scale due to their economically interesting thermophysical properties. This heightens the concern about risks they may pose on their release into the environment, possibly affecting non-target organisms. Microalga are important organisms in ecotoxicological studies as they are at the base of the aquatic food chain, but information about their biochemical and photosynthetic changes in response CuO NP are still scarce. We studied the effects of CuO NP in Raphidocelis subcapitata using morphological, photosynthetic and biochemical biomarkers. Our results showed that the NP affected microalgal population growth with 0.70 mg Cu L-1 IC50-96 h (inhibition concentration). Based on predicted environmental concentrations of Cu NPs in aquatic environments, our results indicate potential risks of the NP to microalgae. Algal cell size, granularity and photosynthetic efficiencies were affected by the CuO NP at 0.97 and 11.74 mg Cu L-1. Furthermore, lipid metabolism was affected mostly at the highest NP concentration, but at environmentally relevant values (0.012 and 0.065 mg Cu L-1) the production of sterols (structural lipids) and triacylglycerols (reserve lipid) increased. Moreover, we found evidence of cell membrane impairment at the highest CuO NP concentration, and, as a photosynthetic response, the oxygen evolving complex was its main site of action. To the best of our knowledge, this is the first study to date to investigate microalgal lipid composition during CuO NP exposure, showing that it is a sensitive diagnostic tool. This research demonstrated that CuO NP may affect the physiology of R. subcapitata, and because they were observed in a primary producer, we foresee consequences to higher trophic levels in aquatic communities.

Mixotrophy in green microalgae grown on an organic and nutrient rich waste.

Vinasse is a waste from the alcohol industry that can support microalgal growth because of its mineral and organic contents. Growing microalgae on waste can be a means of its remediation and decrease culture costs. The physiological knowledge of microalgae on waste can generate subsidies to increase biomass productivity. However, few studies have explored the photosynthetic metabolism of microalgae on complex matrices such as vinasse. In the present research, we investigated some physiological aspects related to photosynthesis and growth of several microalgae in centrifuged and diluted vinasse. The species investigated were Chlamydomonas sp., Chlorella sorokiniana, Chlorella vulgaris, Desmodesmus spinosus, Haematococcus pluvialis, Monoraphidium sp., Scenedesmus quadricauda and Tetraselmis gracilis. Daily cell density, in vivo chlorophyll a and final cellular biovolumes (96 h) were monitored to infer about population growth. Maximum and operational photosynthetic yields (ϕM and ϕ'M), photochemical (qP) and non-photochemical (NPQ) quenchings, light saturation curves and related parameters (α, rETRm and EK), as well as theoretical carbon fixation were evaluated using pulse amplitude modulated fluorometry (PAM). The results showed that chlorophyll a, photochemical (qP) and non-photochemical (NPQ) quenchings were not affected in vinasse, but photosynthetic yields, light curves parameters and autotrophic carbon fixation decreased. Connecting these results to the increase in productivities in vinasse lead us to the rationale that mixotrophic metabolism was supporting microalgae growth on the waste. This study offers important insights into the understanding of microalgal photosynthetic physiology on complex organic wastes, generating subsidies for optimization of biotechnological use of vinasse related to microalgae production.

Physiological responses of Chlorella sorokiniana to copper nanoparticles.

Copper (Cu) nanomaterials have been increasingly researched and produced for many different consumer products. They have high reactivity and bactericidal properties, making them important in antifouling paints, which are thus directly introduced into aquatic ecosystems. However, studies are scarce on the behavior of Cu nanoparticles (Cu-NPs) in natural aquatic systems and their interactions with primary producers such as microalgae. We investigated the effects of NPs on some physiological responses of the freshwater phytoplankton Chlorella sorokiniana. The cells were exposed to nominal concentrations ranging from 2.50 to 635.00 µg L-1 Cu-NPs for 96 h under laboratory-controlled conditions. The cultures were monitored daily for population growth and maximum photosynthetic quantum yield. Total lipids, proteins, and carbohydrates were quantified at 72 h of Cu-NP exposure. The results showed a positive correlation between nominal Cu-NPs and Cu in the biomass (0.97 correlation coefficient) and that this was inversely proportional to total carbohydrates, with a -0.64 correlation coefficient. At the higher end of the Cu-NP concentrations tested, higher total proteins and reduced growth rates were obtained in comparison with controls; we suggest that metal-binding proteins/antioxidants and nonstructural proteins were preferentially produced under these conditions. Our results contribute to an understanding of the interaction between Cu-NPs and a cosmopolitan phytoplankton, C. sorokiniana, and we emphasize that the disposal and use of Cu-NPs requires monitoring because even at environmentally relevant concentrations, the composition of the algae was affected. Environ Toxicol Chem 2019;38:387-395. © 2018 SETAC.

An investigation onto Cd toxicity to freshwater microalga Chlorella sorokiniana in mixotrophy and photoautotrophy: A Bayesian approach.

Aquatic ecosystems are composed by a myriad of dissolved organic materials that can be assimilated by microalgae, while they can perform photosynthesis, this is refereed as mixotrophy. However, ecotoxicological tests usually consider only the photoautotrophic metabolism. This research investigated the ecotoxicological differences between photoautotrophy and mixotrophy in Chlorella sorokiniana exposed to cadmium (Cd). Chlorophyll a, photosynthetic efficiency (Fv/Fm), cell viability, biochemical composition and pH were used to monitor possible toxic effects at 72 h cultures. Glucose (1 g.L-1) was used as organic carbon source. To evaluate the probability of the photoautotrophic culture being more affected by Cd than the mixotrophic one, Bayesian statistical analysis was performed. The photoautotrophic cultures were more affected by Cd than the mixotrophic ones, with reduction of all evaluated parameters, except for protein concentration. However, in mixotrophic cultures, no changes in protein concentration and proteins:carbohydrates ratio were observed, and chlorophyll a, Fv/Fm and cell viability were only affected at the high Cd concentrations (range ln -11.5 to -9.4). However, both mixotrophy and photoautotrophy had the same probability of having the carbohydrates concentration affected by Cd. We conclude that the microalgae in mixotrophy were more resistant to the Cd than in photoautotrophy. In addition, we showed that under photoautotrophy Fv/Fm decreased linearly as Cd concentration increased, but in mixotrophy no effect was observed up to 10-5 molL-1 Cd, after which it decreased. We rationale that the reduced photosynthetic capacity under mixotrophy can end up reducing the release of oxygen gas, which can compromise the entire aquatic ecosystem.

Higher biomolecules yield in phytoplankton under copper exposure.

Copper is an important metal for industry, and its toxic threshold in natural ecosystems has increased since the industrial revolution. As an essential nutrient, it is required in minute amounts, being toxic in slightly increased concentrations, causing great biochemical transformation in microalgae. This study aimed at investigating the physiology of Scenedesmus quadricauda, a cosmopolitan species, exposed to copper concentrations including those that trigger intracellular biochemical modifications. The Cu exposure concentrations tested ranged from 0.1 to 25 µM, thus including environmentally important levels. Microalgae cultures were kept under controlled environmental conditions and monitored daily for cell density, in vivo chlorophyll a, and photosynthetic quantum yield (ΦM). After 24 h growth, free Cu2+ ions were determined, and after 96 h, cellular Cu concentration, total carbohydrates, proteins, lipids, and cell volume were determined. The results showed that both free Cu2+ ions and cellular Cu increased with Cu increase in culture medium. Microalgae cell abundance and in vivo chlorophyll a were mostly affected at 2.5 µM Cu exposure (3.8 pg Cu cell-1) and above. Approximately 31% decrease of photosynthetic quantum yield was obtained at the highest Cu exposure concentration (25 µM; 25 pg Cu cell-1) in comparison with the control. However, at environmentally relevant copper concentrations (0.5 µM Cu; 0.4 pg Cu cell-1) cell volume increased in comparison with the control. Considering biomolecules accumulation per unit cell volume, the highest carbohydrates and proteins yield was obtained at 1.0 µM Cu (1.1 pg Cu cell-1), while for lipids higher Cu was necessary (2.5 µM Cu; 3.8 pg Cu cell-1). This study is a contribution to the understanding of the effects of environmentally significant copper concentrations in the physiology of S. quadricauda, as well as to biotechnological approach to increase biomolecule yield in microalgae production.

Physiological and Ecological Aspects of Chlorella sorokiniana (Trebouxiophyceae) Under Photoautotrophic and Mixotrophic Conditions.

Mixotrophy is a metabolic strategy in which an organism is autotrophic and heterotrophic simultaneously. Considering that the aquatic environment provides several organic sources of carbon, it is probably common for microalgae to perform mixotrophy and not only photoautotrophy, but little is known about microalgae mixotrophy. The present work aimed at investigating the growth, photosynthetic activity, morphology, and biochemical composition of the microalga Chlorella sorokiniana in mixotrophic and photo-mixotrophic conditions, comparing it with photoautotrophy. The results showed pH changes after glucose addition, reaching pH 11.62 in mixotrophic and 10.47 in sequential photo-mixotrophic cultures, which limited the microalgal growth. Highest biomass was obtained in the mixotrophic culture in comparison with the sequential photo-mixotrophic one. Rapid light saturation curves showed that α (photosynthetic efficiency, 1.69) and relative electron transport rate (rETR; 565.61) were higher in the mixotrophic cultures, whereas the highest Ik (irradiance saturation, 386.68) was obtained in the photoautotrophic ones. In the sequential photo-mixotrophic cultures, photosynthetic activity varied during glucose consumption, decreasing the maximum quantum yield Fv/Fm after glucose addition, indicating change in metabolism, from photoautotrophy to mixotrophy by the microalga. The results showed that the mixotrophic cultures had higher production of chlorophyll a (6.26 mg mL-1), cell density (6.62 × 107 cell mL-1), and lipids (0.06 pg µm-3). Sequential photo-mixotrophic cultures showed the highest biovolume (360.5 µm3 cell-1) and total carbohydrates (0.026 pg µm-3). The protein concentration was 3.2 and 2.4 times higher in photoautotrophy and photo-mixotrophic growth, respectively, than in mixotrophy, but lipids were three times higher under mixotrophy. The biochemical changes we observed indicate that the microalga's plasticity in face of new environmental characteristics, such as the presence of organic carbon, can change the flow of energy through natural ecosystems.

Potential effects of fungicide and algaecide extracts of Annona glabra L. (Annonaceae) on the microalgae Raphidocelis subcapitata and on the oomycete Pythium.

Annona glabra L. is a semi-deciduous tree that contains several active substances, including secondary metabolites, with antifungal activity. Phytopathogenic strains of the genus Pythium cause billion dollar losses all over the world on natural and crop species. Searching for eco-friendly algaecides and fungicides, we analyzed the effects of acetone extracts of A. glabra leaves on the algae Rhaphidocelis subcapitata (Korshikov) and on the oomycete Pythium aphanidermatum (Edson). We evaluated ten extract concentrations for each organism - 0 to 400 mg L-1 for algae and 0-1000 µg disc1 for oomycete. The results showed no effect on algae up to 75 mg L-1, but a significant inhibitory effect at 125 mg L-1 and above, which reduced the growth rate and the final biomass of the algae. Extract concentrations above 200 mg L-1 were completely inhibitory. The half maximal inhibitory concentration for 72 and 96 h of exposure to our crude extracts are comparable to those obtained with commercial fungicides and herbicides used in aquatic ecosystems. The P. aphanidermatum inhibition concentrations have effects comparable to fungicides as Cycloheximide and Bifonazole. Some substances isolated from the extracts are described as antifungals, which could explain part of anti-oomycete activity. Our results highlight the importance of searching bioactive compounds from plants.

Potential effects of fungicide and algaecide extracts of Annona glabra L. (Annonaceae) on the microalgae Raphidocelis subcapitata and on the oomycete Pythium

ABSTRACT Annona glabra L. is a semi-deciduous tree that contains several active substances, including secondary metabolites, with antifungal activity. Phytopathogenic strains of the genus Pythium cause billion dollar losses all over the world on natural and crop species. Searching for eco-friendly algaecides and fungicides, we analyzed the effects of acetone extracts of A. glabra leaves on the algae Rhaphidocelis subcapitata (Korshikov) and on the oomycete Pythium aphanidermatum (Edson). We evaluated ten extract concentrations for each organism - 0 to 400 mg L-1 for algae and 0-1000 µg disc1 for oomycete. The results showed no effect on algae up to 75 mg L-1, but a significant inhibitory effect at 125 mg L-1 and above, which reduced the growth rate and the final biomass of the algae. Extract concentrations above 200 mg L-1 were completely inhibitory. The half maximal inhibitory concentration for 72 and 96 h of exposure to our crude extracts are comparable to those obtained with commercial fungicides and herbicides used in aquatic ecosystems. The P. aphanidermatum inhibition concentrations have effects comparable to fungicides as Cycloheximide and Bifonazole. Some substances isolated from the extracts are described as antifungals, which could explain part of anti-oomycete activity. Our results highlight the importance of searching bioactive compounds from plants.

Copper and cadmium complexation by Cylindrospermopsis raciborskii exudates.

Cylindrospermopsis raciborskii is a potentially toxic cyanobacterium that excretes organic materials which act as ligands for metals. Metal ligands may be characterized for their strength of association, e.g., stability constants, which can be either thermodynamic (K) or conditional (K'). In this research we examined K and K' for Cu and Cd complexes with three molecular weight fractions (>30 kDa; 30-10 kDa; 10-3 kDa) of the cyanobacteria EOM. Complexation capacities of the excreted organic materials (EOM) for metals were determined at several ionic strengths (1.0 × 10(-2), 5.0 × 10(-2), 1.0 × 10(-1), and 5.0 × 10(-1) mol L(-1)) at pH 6.6 ± 0.1, with ligands for which no data for their acidity constants are available; these constants are thus conditional for this specific pH. Bayesian statistics showed that with a probability of 95-100% the EOM have two different ligands for Cu but only one for Cd, that ligands for Cu were stronger than for Cd (94-100% probability), and that the smallest EOM fraction had the highest strength of association for Cu (logKCuL 13.5). The lowest affinity was obtained for Cd (logKCdL 8.6) complexed to any molecular weight fraction. The present findings have important ecological implications, since the metal-ligand association is dynamic, and together with a diversity of ligands it can act as an environmental metal buffer. As a result, higher metal loads may be necessary for the detection of toxicity.

Chemical behavior of Cu, Zn, Cd, and Pb in a eutrophic reservoir: speciation and complexation capacity.

This research aimed at evaluating cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn) speciation in water samples as well as determining water quality parameters (alkalinity, chlorophyll a, chloride, conductivity, dissolved organic carbon, dissolved oxygen, inorganic carbon, nitrate, pH, total suspended solids, and water temperature) in a eutrophic reservoir. This was performed through calculation of free metal ions using the chemical equilibrium software MINEQL+ 4.61, determination of labile, dissolved, and total metal concentrations via differential pulse anodic stripping voltammetry, and determination of complexed metal by the difference between the total concentration of dissolved and labile metal. Additionally, ligand complexation capacities (CC), such as the strength of the association of metals-ligands (logK'ML) and ligand concentrations (C L) were calculated via Ruzic's linearization method. Water samples were taken in winter and summer, and the results showed that for total and dissolved metals, Zn > Cu > Pb > Cd concentration. In general, higher concentrations of Cu and Zn remained complexed with the dissolved fraction, while Pb was mostly complexed with particulate materials. Chemical equilibrium modeling (MINEQL+) showed that Zn(2+) and Cd(2+) dominated the labile species, while Cu and Pb were complexed with carbonates. Zinc was a unique metal for which a direct relation between dissolved species with labile and complexed forms was obtained. The CC for ligands indicated a higher C L for Cu, followed by Pb, Zn, and Cd in decreasing amounts. Nevertheless, the strength of the association of all metals and their respective ligands was similar. Factor analysis with principal component analysis as the extraction procedure confirmed seasonal effects on water quality parameters and metal speciation. Total, dissolved, and complexed Cu and total, dissolved, complexed, and labile Pb species were all higher in winter, whereas in summer, Zn was mostly present in the complexed form. A high degree of deterioration of the reservoir was confirmed by the results of this study.