Abstract The environmental and health risks associated with the application of synthetic chemical inputs in agriculture increased the demand for technologies that allow higher performance and quality of vegetable crops by implementing synergistic materials with the principles of sustainability. In this work, the seed coating with the biomass of Dunaliella salina incorporated in a bioplastic film of Manihot esculenta (cassava) was evaluated as an initial growth and secondary compounds stimulator of Coriandrum sativum (coriander) plants. The obtained results demonstrated that the coating stimulated an increase in the germination percentage (28.75%) and also in concentration of bioactive compounds, such as the six-fold increment of caffeic acid (13.33 mg 100 g-1). The carbohydrates, lipids, and proteins present in the microalgae biomass seem to be responsible for these increments once they are known for providing energy to the seedling development and coordinating the secondary metabolites synthesis. As conclusion, we consider the coating with biomass of D. salina an alternative for crop improvement that contributes to the development of sustainable agricultural practices.

ABSTRACT This research aims to evaluate the effect of molasses as a source of organic carbon on the growth and lipid profile of Dunaliella salina cultivated under different light regimes to determine its strategy and potential use to produce biodiesel. Two sources of nutrients (F/2 medium, F/2; molasses, M) and two light conditions (12 h light: 12 h darkness, 12L:12D) conformed four treatments (F/2 12L:12D, F/2 D, M 12L:12D, and MD) that were investigated with three replicates each. The concentration of microalgae produced by the groups under light conditions was more than double that of those grown in darkness. The highest biomass produced (46.91 ± 6.47 g L-1) was obtained in the F/2 medium with 12L:12D treatment, while the lowest (18.99 ± 0.78 g L-1) was in the M 12L:12D group. Regardless of the culture conditions, the lipids were mainly composed of the C16:0 and C18 fatty acids. The use of molasses as a culture medium produced the highest amount of C18:1, regardless of the light regime. Molasses promoted the highest production of fatty acids methyl esters. Depending on the culture medium, D. salina cells showed different percentages of saturated (SFA), monounsaturated (MFA), and polyunsaturated (PUFA) fatty acids. The highest amount of saturated fatty acids was obtained in F/2 12L:12D. The highest amount of polyunsaturated fatty acids was presented in the mixotrophic culture (molasses and light). The cultures grown with molasses were rich in monounsaturated fatty acids. An inexpensive industrial by-product, Cane molasses could be a low-cost feedstock for biodiesel in cultivating D. salina.

ABSTRACT The objective of the present study was to evaluate production and egg quality as well as the intestinal morphometry of laying hens fed diets supplemented with marine microalga Dunaliella salina. Six hundred laying hens were allocated based on a completely randomized design into five treatments (0, 0.25, 0.50, 0.75, and 1% inclusion of D. salina biomass) with 12 replicates of 10 hens per treatment. The experiment was divided into three periods of four weeks each, totaling 84 days. During this period, the productive performance of laying hens, the physical-chemical quality of the eggs, and the morphometric alterations of the small intestine and liver were determined. The inclusion levels of D. salina biomass had a linear effect on the performance (egg weight, egg mass, and feed conversion), qualitative parameters (yolk weight and yolk index), and physicochemical parameters of eggs (total carotenoids, TBARS, and yolk color). At the same time, villi lengths and the villus:crypt ratio of the duodenum and ileum segments and the metabolization of carotenoids in the liver were increased as an effect of Dunaliella salina dietary supplementation. Thus, the inclusion of marine microalgae D. salina biomass in experimental diets for laying hens improves the performance, the intestinal health, the physical-chemical quality of the eggs, and at the same time increases carotenoid content and improves egg oxidative stability.

ABSTRACT The objective of this study was to evaluate the effects of algae and cottonseed meal (CSM) supplementation on the fatty acid (FA) profile in the rumen fluid (RF) of steers fed a low-quality forage. Five Bos indicus crossbred steers, 187±7.5 kg liveweight (LW; mean±SD), were fed a low crude protein speargrass (Heteropogon contortus) hay as the basal diet alone or supplemented with either Spirulina platensis, Chlorella pyrenoidosa , Dunaliella salina , or CSM in Latin square design. The proportion of individual FA in the RF of steers varied in response to supplement, and these were most likely due to differences in the FA profile in supplements. Steers supplemented with Chlorella pyrenoidosa and CSM had a higher concentration of linoleic acid (C18:2n-6) in RF than unsupplemented steers or steers offered the other supplements, but there was no difference in the concentration in RF in steers supplemented with Chlorella pyrenoidosa and CSM. The concentration of linolenic acid (C18:3n-3) was higher in the RF of unsupplemented steers compared with supplemented steers. Steers receiving Chlorella pyrenoidosa supplementation showed an increase in total unsaturated FA in the RF compared with other supplemented and unsupplemented steers, which if transferred to meat, could have health related benefits to consumers. None of the supplements led to the formation of isomers known to inhibit fat synthesis.

Abstract Microalgae are potential sources of a wide range of bioproducts. It is essential to choose the proper microalgae strain and culture condition to achieve an efficient production. The production yield of carotenoids by Dunaliella salina under the stress-induced culture conditions of nitrogen deprivation and excessive light intensity was evaluated. Also, a survey at laboratorial scale of the growth kinetics under different culture conditions of photoperiod, aeration, and agitation was performed for the seven species of green microalgae Ankistrodesmus fusiformis, Chlamydocapsa bacillus, Desmodesmus brasiliensis, Kirchneriella lunaris, Pseudokirchneriella subcapitata, and Scenedesmus obliquus. As a result, aeration of atmospheric air is enough to improve the growth kinetics of the seven species studied. Production of carotenoids was enhanced under stress by excessive light intensity. Although D. salina does not grow effectively under nitrogen deprivation, this stress condition may be used to quickly stimulate carotenoid production once the culture reaches a high cellular population.

Technological processes were developed for ß-carotene and glycerol extraction from Dunaliella sp. at "Las Cumaraguas" Saltworks. Dunaliella sp. microalgae was characterize. It was established the technological flow of the process from laboratory data; energy balances and necessary energetic calculations were done, in order to select and size the required equipment for a pilot plant scale to produce one ton of ß-carotene. Two possibilities were evaluated: extraction with organic solvents and extraction with supercritical fluids. The procedure showed that ß-carotene and glycerol extractions are possible by means of the presented technological diagram, with 15,4 % of biomass ß-carotene yield and 5 % of biomass glycerol yield, for a conventional extraction and a yield of 70 % for ß-carotene from pigments and 5 % of biomass glycerol yield, for extraction with supercritical fluid.

Se desarrollaron los procesos tecnológicos para la extracción de ß-caroteno y glicerol a partir de Dunaliella sp. en la Salina "Las Cumaraguas". Se caracterizó fisicoquímicamente esta microalga. Se estableció el flujo tecnológico del proceso partiendo de datos de laboratorio. Se realizó el balance de materiales y los cálculos energéticos necesarios. para poder dimensionar los equipos donde se lleva a cabo la separación a escala piloto que permita la producción de una tonelada de ß-caroteno. Se evaluaron dos alternativas: extracción con solventes orgánicos y extracción con fluido supercrítico. El procedimiento permitió establecer que es posible la extracción de ß-caroteno y glicerol mediante los esquemas tecnológicos presentados con un rendimiento de ß-caroteno de biomasa: 15,4 % y rendimiento de glicerol de la biomasa: 5 % para la extracción convencional y un rendimiento de ß-caroteno de los pigmentos: 70 % y rendimiento de glicerol de la biomasa: 5 % para fluido supercrítico.

ABSTRACT The present work investigated the effects of aqueous extracts of eucalyptus ( Eucalyptus globulus) and elderberry ( Sambucus ebulus) leaves on β-carotene productivity in Dunaliella salina, a green microalga. Leaf extracts from eucalyptus have greater amounts of phenolics and flavonoids, as well as greater ferric reducing antioxidant potential than elderberry. The extracts of both species greatly inhibited growth of algal suspensions. However, chlorophyll and β-carotene concentration increased in cells treated with leaf extracts, and the highest values were detected in 1 % eucalyptus and 2 % elderberry extracts. Fresh weight, total sugar, and protein content significantly increased following exposure of cells to different doses of leaf extracts. However, in doses containing more than 2 % eucalyptus, the upward trend for total sugar and protein ceased and remained statistically unchanged. These results suggest that metabolic modifications enable D. salina cells to tolerate the stress induced by the leaf extracts through allocating carbon flux to the synthesis of osmolytes and putative antioxidant molecules (e.g. sugars and β-carotene). Therefore, the use of leaf extracts holds potential to be a promising and effective way to improve D. salina cultivation for β-carotene production and other biotechnological and industrial applications.

ABSTRACT Effect of salt stress on biomass, cell number, contents of total lipid, omega-3 fatty acids, including ALA (Alpha Linolenic Acid), EPA (Eicosapentaenoic Acid) and DHA (Docosahexaenoic Acid) and their biosynthetic pathway intermediates (palmitic acid, stearic acid, oleic acid and linoleic acid) of two microalgae Dunaliella salina and Chlorella vulgaris were investigated. Dilution stress from 1.5 to 0.5 M NaCl and salt stress from 1.5 to 3 M NaCl were incorporated into the D. salina medium. Salt stress of 200 mM NaCl was also applied to C. vulgaris culture. Results indicated that increasing salt concentration resulted in the reduced growth rate of C. vulgaris and substantial increase of the total lipid content in both species. Proper growth rate of D. salina observed at 1.5 M of NaCl, but higher and lower concentrations led to the decreased growth rate of D. salina. In addition, considerable increase in the degree of fatty acid unsaturation and thereby the total omega 3 fatty acid content of D. salina was observed under salt stress. Salt stress had little positive effect on the amount of total omega-3 fatty acid of C. vulgaris due to the slight increase of the EPA content. Results showed that salt stress is an effective way for enhancing the total lipid and omega-3 fatty acid production in D. salina.

Dunaliella salina es una microalga utilizada para la producción de metabolitos de alto valor industrial y farmacéutico. Se evaluó el efecto combinado de la irradiancia y salinidad sobre el crecimiento, producción de pigmentos y composición bioquímica de una cepa D. salina aislada de las salinas de Araya, a fin de contribuir con el conocimiento de las respuestas fisiológicas de esta cepa ante cambios en las condiciones de su entorno. Se buscó, además, incrementar la variedad de especies microalgales nativas que puedan servir como fuentes de bioproducción para compuestos bioactivos, biomasa y metabolitos de aplicación en la industria de alimentos funcionales, farmacéuticos y biomedicina. Se realizaron cultivos discontinuos de la cepa de D. salina durante 22 d en medio f/2 Guillard (0,88 mmol.L-1 de nitrógeno) a 27 ± 2°C, pH de 7,8; fotoperíodo 12 h luz: 12 h oscuridad, aireación continua (150 mL.L-1) y en dos diferentes salinidades (40 y 250 UPS) e irradiancias (195 y 295 μmolphotons.m-2.s-1). Diariamente, se evaluó el crecimiento poblacional y al final del ensayo se determinaron los contenidos de biomasa, pigmentos, proteínas, lípidos y carbohidratos, así como el tamaño celular. La tasa de crecimiento de la nueva cepa de D. salina disminuyó al aumentar la salinidad y la irradiancia. Sin embargo, el tamaño celular, biomasa, contenidos de proteínas, lípidos, carbohidratos, carotenoides totales y β-caroteno aumentaron al incrementarse la irradiancia y la salinidad. Además, se evidenció la potencialidad que posee esta cepa para ser cultivada para la producción de β-caroteno.

Dunaliella salina is a microalga used for the production of metabolites of high industrial and pharmaceutical value. The combined effect of irradiance and salinity on growth, pigment production and biochemical composition of this strain were evaluated in order to contribute to the knowledge of the physiological responses of this new isolate of D. salina from Araya saltworks, to changes in the conditions of its environment. It was aslo intendend to increase the variety of native microalgae species which can serve as sources of bioproduction for bioactive compounds, biomass and metabolites of application in pharmaceuticals and biomedical industries. Batch cultures of the strain of D. salina were conducted for 22 d at f/2 medium Guillard (0.88 mmol.L-1 of nitrogen) at 27 ± 2°C, pH 7.8; photoperiod 12 h light: 12 h darkness, continuous aeration (150 mL.L-1) in two salinities (40 and 250 UPS) and irradiances (195 and 295 μmolphotons.m-2.s-1). Population growth was evaluated daily and at the end of the trial the contents of biomass, pigments, proteins, lipids, carbohydrates and cell size were determined. The growth rate of the new strain of D. salina decreased with increasing salinity and irradiance. However, cell size, biomass, content of proteins, lipids, carbohydrates, total carotenoids and β-carotene increased with increasing irradiance and salinity. In addition, the potential of this strain for the production of β-carotene was shown.

Abstract Dunaliella salina, a single-celled marine alga with extreme salt tolerance, is an important model organism for studying fundamental extremophile survival mechanisms and their potential practical applications. In this study, two-dimensional differential in-gel electrophoresis (2D-DIGE) was used to investigate the expression of halotolerant proteins under high (3 M NaCl) and low (0.75 M NaCl) salt concentrations. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and bioinformatics were used to identify and characterize the differences among proteins. 2D-DIGE analysis revealed 141 protein spots that were significantly differentially expressed between the two salinities. Twenty-four differentially expressed protein spots were successfully identified by MALDI-TOF/TOF MS, including proteins in the following important categories: molecular chaperones, proteins involved in photosynthesis, proteins involved in respiration and proteins involved in amino acid synthesis. Expression levels of these proteins changed in response to the stress conditions, which suggests that they may be involved in the maintenance of intracellular osmotic pressure, cellular stress responses, physiological changes in metabolism, continuation of photosynthetic activity and other aspects of salt stress. The findings of this study enhance our understanding of the function and mechanisms of various proteins in salt stress.

El uso creciente del biodiésel ha impulsado la búsqueda de nuevas materias primas, dentro de las que se destacan las microalgas. En esta investigación se estudió el cultivo de las microalgas Chlorella sp. y Dunaliella salina bajo diferentes condiciones de pH y concentración de nitrógeno (mgL-1), y la caracterización de los aceites obtenidos, con el fin de evaluar su potencial uso como materia prima en la producción de biodiésel. La D. salina alcanzó una máxima concentración celular de 1.15x10(6)células mL-1 en 6 días de cultivo en unas condiciones de 8.5 pH y 0.1 mg L-1 concentración de nitrógeno, mientras que Chlorella sp. Presentó una máxima concentración celular de 2.6x10(7) células mL-1en 14 días de cultivo en unas condiciones de 7.5 pH y 0.1 mg L-1 concentración de nitrógeno. La extracción de aceite intracelular de las microalgas cultivadas bajo las mejores condiciones de crecimiento celular se realizó empleando el método modificado de Bligh & Dyer. Al determinar el perfil de ácidos grasos de los aceites extraídos de ambas microalgas, se encontraron en mayor proporción dos ácidos grasos insaturados: ácido linolénico y ácido oleico. En el aceite de D. salina se encontró una concentración de 51% p/p de ácido linolénico, mientras que para el aceite de Chlorella sp. Fue de 39% p/p. Con relación al ácido oleico, el aceite de Chrorella sp. Presentó una concentración de 35% p/p, superior al aceite de D. salina, con 25% p/p.

The increasing use of biodiesel has prompted the search for new raw materials, among them are highlighted the microalgae. In this research the cultivation of microalgae Chlorella sp. and Dunaliella salina at different conditions of pH and nitrogen is reported; and the oils obtained from them characterization, in order to assess the biodiesel potential production. D. Salina reached the maximun cells concentration, during a 6 days cultivation, at pH 8.5 and 0.1 mg L-1 of nitrogen, obtaining values of 1.15x10(6) cells mL-1, while the Chlorella sp. under a pH value of 7.5 and 0.1 mg L-1 of nitrogen conditions, achieved the maximun concentrations of 2.6x10(7) cells mL-1. Analysis of the fatty acid profiles revealed that the most abundant were linolenic, and then, oleic; this implies a large number of unsaturations that would have negative implications on the physicochemical properties and performance of biodiesel combustion.

O uso crescente do biodiesel tem impulsado a busca de novas matérias primas, dentro das quais se destacam as microalgas. Nesta pesquisa se estudou o cultivo das microalgas Chlorella sp. e Dunaliella salina sob diferentes condições de pH e concentração de nitrogênio (mgL-1), e a caracterização dos azeites obtidos, com o fim de avaliar seu potencial uso como matéria prima na produção de biodiesel. A D. salina alcançou uma máxima concentração celular de 1.15x10(6) células mL-1 em 6 dias de cultivo em umas condições de 8.5 pH e 0.1 mg L-1 concentração de nitrogênio, enquanto que Chlorella sp. apresentou uma máxima concentração celular de 2.6x10(7) células mL-1 em 14 dias de cultivo em umas condições de 7.5 pH e 0.1 mg L-1 concentração de nitrogênio. A extração de azeite intracelular das microalgas cultivadas sob as melhores condições de crescimento celular se realizou empregando o método modificado de Bligh & Dyer. Ao determinar o perfil de ácidos graxos dos azeites extraídos de ambas as microalgas, encontraram-se em maior proporção dois ácidos graxos insaturados: ácido linolênico e ácido oleico. No azeite de D. salina se encontrou uma concentração de 51% p/p de ácido linolênico, enquanto que para o azeite de Chlorella sp. foi de 39% p/p. Com relação ao ácido oleico, o azeite de Chrorella sp. apresentou uma concentração de 35% p/p, superior ao azeite de D. salina, com 25% p/p.

The southern Chilean Patagonia is being incorporated into the productive activity through their water bodies applied in aquaculture. This makes it necessary to establish guidelines for sediment quality. The aim of this study was to analyze the environmental quality of sediments collected during an oceanographic cruise from Estrecho de Magallanes (52°41.1'S) to Cabo de Hornos (55°0.9'S), through testing of survival, fertilization and growth. The testing organisms were Tisbe longicornis a benthic arthropoda, Arbacia spatuligera an echinoderm and 3 microalgae: Dunaliella tertiolecta, D. salina and Isochrysis galbana. The principal component analysis (PCA) and dendrogram allowed verify in the first place the sensitivity of the assays applied. Furthermore, PCA allowed observing clusters of stations based on the growth of the 3 microalgae, showing that the first 2 components accounted for almost 80% of the total variance. The stations that behaved differently from the control groups were two in the southern sector and one in the northwestern sector, due to their oceanic influence. These result showed that the slight difference between fertilization trials, survival and growth of microalgae on the controls, allow us to suggest that this area is in pristine condition even from the standpoint of sediment quality.

La zona sur de la Patagonia chilena se está incorporando a la actividad productiva, a través del uso de sus cuerpos de agua en la acuicultura, lo que hace necesario establecer líneas bases de calidad de sus sedimentos. El objetivo de este trabajo fue analizar la calidad de los sedimentos recolectados en el sector entre el Estrecho Magallanes (52°41,1') y Cabo de Hornos (55°0,9'S), a través de ensayos de supervivencia, fecundación y crecimiento. Los organismos utilizados fueron un artrópodo bentónico, Tisbe longicornis;gametos de un equinodermo, Arbacia spatuligera y 3 microalgas, Dunaliella tertiolecta, D. salina e Isochrysis galbana. El análisis de componentes principales y dendograma confirmó la sensibilidad de los ensayos utilizados y permitió observar agrupaciones de estaciones sobre la base del crecimiento de las 3 microalgas, observándose que las primeras componentes representaron casi un 80% de la varianza total. Las estaciones que se comportaron diferentes de los grupos control fueron dos en el sector sur y una en el sector noroeste, debido a su mayor influencia oceánica. Se concluye que la escasa diferencia entre los ensayos de fecundación, supervivencia y crecimiento de las microalgas respecto de los controles, permite considerar a esta área, como prístina desde el punto de vista de la calidad del sedimento.

We studied the effects of n-propyl gallate, which is a plastid terminal oxidase inhibitor involved in chlororespiration, on photosynthesis and physiological parameters in Dunaliella salina grown under different salinities and under low or high irradiance using chlorophyll a fluorescence transient measurements and pigment analysis. The inhibitor up to 1 mM had an additive significant effect on the photosynthetic efficiency in the cell suspensions grown under low salinity and irradiance. However, in the presence of high n-propyl gallate concentration (4 mM), there was a negative effect on all physiological aspects. In contrast, this high concentration of the inhibitor could enhance efficiency of electron transport and growth parameters under high irradiance. On the other hand, with salinity increase, the unfavorable effects of high inhibitor concentration on the efficiency of photosystem II were less evident than of low salinity. Interestingly, n-propyl gallate high concentration had a positive effect on fluorescence and on physiological parameters when high salinities-grown cells were exposed to high irradiance. The results suggest that there is a rational correlation between increase of salinity and algae ability to bypass n-propyl gallate inhibited plastid terminal oxidase function and also direct influence of its lethal concentration on photosystem II compartment. The ability is especially substantial when the increase of salinity is accompanying high irradiance. Furthermore, these data show that algal responses to inhibitor concentrations are different under various environmental conditions.

We analysed the ecotoxicological quality of sediments in Moraleda, Puyuguapi and Elefantes Channels and Aysén Fjord, located between 44º and 46.5ºS, using non-specific tests in order to evaluate their base conditions in terms of possible pollutants that may have been introduced in the area. The survival tests with Ampelisca araucana and Tisbe longicornis and fertility test with Arbacia spatuligera indicate that the sediments do not present problems of toxicity. Nevertheless, the increased cellular density observed in the tests with microalgae (Dunaliella tertiolecta, Dunaliella salina and Isochrysis galbana) indicates the accumulation of sediments rich in nutrients within the area.