Microalgal extract from thermotolerant Coelastrella sp. F50 retards the liver tumor progression by targeting hepatic cancer stem cells.

Microalgae extracts have shown antitumor activities. However, the antitumor mechanism of them is not yet completely clear, especially the effect on cancer stem cells (CSCs). This study aimed to elucidate the antitumor activity and mechanism of microalgal extract from thermotolerant Coelastrella sp. F50 (F50) in hepatocellular carcinoma (HCC). Oncogenic behaviors were analyzed using cell proliferation, colony formation, invasion, sphere formation, and side population cells (SPCs) assays in HCC cells after F50 treatment. The molecular mechanism was further studied by quantitative real-time PCR, immunoblot, and immunofluorescence analyses. The chemopreventive efficacy of F50 was evaluated in rat orthotopic hepatoma, and the hepatic pathologies were investigated by immunohistochemical, immunoblot, and immunofluorescence analyses. F50 specifically suppressed hepatic CSCs (tumor spheres, drug efflux, CD133/ABCG2 CSCs markers) with no cytotoxicity in vitro. In the animal experiments, prophylactic F50 administration significantly attenuated tumor progression and improved liver function in HCC-bearing rats. In the mechanistic analysis, F50 potentially inhibited cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2 ) axis in HCC cells and rat hepatoma, and exogenous PGE2 restored CSCs properties in F50-treated HCC cells. In summary, F50 extract inhibits hepatic CSCs by COX-2/PGE2 downregulation and may facilitate a novel phytotherapy for HCC prevention.

Mechanisms protect airborne green microalgae during long distance dispersal.

Viable microalgae occur in the air. Whether they can survive the stresses such as UV, desiccation and freezing temperatures at high altitudes during long distance dispersal is rarely studied. If yes, what mechanisms confer the tolerance? Four freshwater airborne green microalgae were isolated from Dongsha Atoll in the South China Sea, classified as Scenedesmus sp. DSA1, Coelastrella sp. DSA2, Coelastrella sp. DSA3 and Desmodesmus sp. DSA6 based on their morphologies and ITS sequences. Their survival rates under UV stress were tightly correlated with their cell wall thickness. All the four airborne green microalgae survived the air-dry stress on benchtop followed by - 20 °C freeze-desiccation stress for 4 weeks, but not the two waterborne green microalgae Desmodesmus sp. F5 and Neodesmus sp. UTEX 2219-4 used as controls. Three of the four airborne microalgae survived the lyophilization treatment, excluding Desmodesmus sp. DSA6 and the two waterborne microalgae. The four airborne microalgae produced carotenoids under prolonged stress conditions, which might help detoxify the reactive oxygen species generated under environmental stresses and shield from the high-light stress in the air. Characterization of these airborne microalgae may help answer how the descendants of green algae survived on the land about 450 MYA.

Disruption of thin- and thick-wall microalgae using high pressure gases: Effects of gas species, pressure and treatment duration on the extraction of proteins and carotenoids.

Industrial scale microalgal cell disruption requires low cost, high efficiency and structural conservation of biomolecules for biorefinery. Many cultivated microalgae have thick walls and these walls are barriers for efficient cell disruption. Until recently, despite the high biodiversity of microalgae, little attention has been paid to thin-wall microalgal species in the natural environment for the production and recovery of valuable biomolecules. Instead of developing high power cell disruption devices, utilization of thin-wall species would be a better approach. The present paper describes a simple device that was assembled to evaluate the viability and effectiveness of biomolecule extraction from both thin- and thick-wall species as a proof of concept. This device was tested with high-pressure gases including N2, CO2 plus N2, and air as the disruption force. The highest nitrogen pressure, 110 bar, was not able to disrupt the thick-wall microalgal cells. On the other hand, the thin-wall species was disrupted to different degrees using different pressures and treatment durations. In the same treatment duration, higher nitrogen pressure gave better cell disruption efficiency than the lower pressure. However, in the same pressure, longer treatment duration did not give better efficiency than the shorter duration. High pressure CO2 treatments resulted in low soluble protein levels in the media. The best conditions to disrupt the thin-wall microalgal cells were 110 bar N2 or air for 1 min among these tests. In these conditions, not only were the disruption efficiencies high, but also the biomolecules were well preserved.

Optimization of a simple, accurate and low cost method for starch quantification in green microalgae.

BACKGROUND: Lipids and starch are important feedstocks for bioenergy production. Genetic studies on the biosyntheses of lipids and starch in green microalgae have drawn significant attention recently. In these studies, quantifications of lipids and starch are required to clarify the causal effects. While lipids are assayed with similar procedures worldwide, starch in green microalgae has been measured using various methods with deficiencies in accuracy or high cost. RESULTS: A simple, accurate and low cost procedure for routine quantification of starch in green microalgae was developed. This procedure consists of quick-freezing of the cells, solvent extraction of the pigments, 134 °C autoclaving and glucoamylase double digestions of starch, followed by a glucose assay using the dinitrosalicylic acid reagent. This procedure was optimized to quantify starch in small volumes of green microalgal culture. The accuracy of starch quantification using this procedure was 102.3 ± 2.5% (mean ± SD, n = 6), as indicated by using cornstarch as internal controls. The working protocol is available at http://dx.doi.org/10.17504/protocols.io.2mhgc36 . CONCLUSIONS: This quantification approach overcomes the current problems in the starch quantification of green microalgae such as inaccuracy and high cost. This approach would provide an opportunity to compare the effects of genetic, physiological or cultivation manipulations on the productivity of starch in green microalgae elucidated in different labs, which is essential in the enhancement of lipid productivity studies in microalgae.

Long Distance Dispersal Potential of Two Seagrasses Thalassia hemprichii and Halophila ovalis.

The wide distribution of many seagrasses may be attributable to exploitation of currents. However, many species have seeds heavier than seawater, limiting surface floating, and thus, deep water becomes a potential barrier between suitable habitats. In this investigation, we studied the dispersal potential of various life history stages of two species of seagrasses, Thalassia hemprichii and Halophila ovalis, at Dongsha Atoll and Penghu Islands in Taiwan Strait, west Pacific. The adult plants of both species, often dislodged naturally from substrate by waves, could float, but only that of T. hemprichii could float for months and still remain alive and potentially able to colonize new territories. The seedlings of T. hemprichii could also float for about a month once failing to anchor to substrate of coral sand, but that of H. ovalis could not. The fruits and seeds of T. hemprichii could both float, but for too short a duration to enable long distance travel; those seeds released from long floating fruits had low germination rates in our tests. Obviously, their seeds are not adaptive for long distance dispersal. Fruits and seeds of H. ovalis do not float. The potential of animals as vectors was tested by feeding fruits and seeds of both species to a goose, a duck, and two fish in the laboratory. The fruits and seeds of T. hemprichii were digested and could no longer germinate; those of H. ovalis could pass through the digestive tracts and have a much higher germination rates than uningested controls. Therefore, birds could be important vectors for long distance dispersal of H. ovalis. The two seagrasses adopted very different dispersal mechanisms for long distance travel, and both exploited traits originally adaptive for other purposes.

Cultivation of two thermotolerant microalgae under tropical conditions: Influences of carbon sources and light duration on biomass and lutein productivity in four seasons.

Biomass and lutein productivities of two thermotolerant microalgae were assessed in tropical outdoor conditions in all four seasons. Generally, addition of bicarbonate at 0.2g/L every two days or 2% CO2 did not enhance the productivities compared to the controls, and the productivities in the spring were higher than in the fall. Durations of effective irradiance positively correlated to the productivity of Coelastrella sp. F50 well, but not for Desmodesmus sp. F2. The ineffective light intensity was below 5000 lux (about 106µmol/m(2)s). The productivities produced in the 17cm diameter bottles were 1.5- to 1.9-fold higher than that in the 27cm ones. Lutein content, about 0.5% in biomass on average, did not change significantly grown in different carbon sources or seasons. The annual productivities of the microalgal biomass and lutein in one hectare were estimated to be 33tons and 180kg, respectively, using the non-optimized photobioreactor cultivation.

Production of long chain omega-3 fatty acids and carotenoids in tropical areas by a new heat-tolerant microalga Tetraselmis sp. DS3.

Demand for long chain ω-3 fatty acids from non-fish source for vegetarians has increased recently. Marine microalgae are the primary producers of EPA/DHA and promising alternatives for fish oil. Tropical areas have abundant sunlight throughout the year for microalgal cultivation but this practice can be hindered by high temperature. Discovery of heat-tolerant marine microalgae that can synthesize EPA/DHA will solve these problems. A new species of microalga was isolated from a high temperature lagoon and identified as Tetraselmis sp. DS3. These cells could grow at 40 °C, the highest temperature for marine microalgal growth ever reported. Its ω-3 fatty acids and EPA accounted for 33 and 10% of total lipids, respectively, grown in nitrogen-depleted conditions. These cells also accumulated more than 5% ß-carotene and 0.48% lutein in biomass. This new microalga can be cultivated for long chain ω-3 fatty acids and lutein production in the tropical areas.

Exploring the high lipid production potential of a thermotolerant microalga using statistical optimization and semi-continuous cultivation.

A recently isolated thermotolerant microalga Desmodesmus sp. F2 has the traits of becoming potential biodiesel feedstock, such as high growth rate, high lipid content, and quick precipitation. Its overall lipid productivity was 113 mg/L/d when grown under non-optimal conditions using batch cultivation. A two-step response surface methodology was adopted to optimize its cultivation conditions. The overall lipid productivity was increased to 263 mg/L/d when the cells were grown under the optimized conditions of 6.6mM initial nitrogen level and 6 days nitrogen depletion treatment in 700 µmol/m(2)/s light intensity at 35°C using batch cultivation. Fed-batch and semi-continuous cultivations were employed to further increase its lipid productivity to 213 and 302 mg/L/d, respectively. The 302 mg/L/d is the highest overall lipid productivity of microalgae ever reported in the literature. This study provides the information required for the design and operation of photobioreactors for large scale outdoor cultivation of this species.

AlgaePath: comprehensive analysis of metabolic pathways using transcript abundance data from next-generation sequencing in green algae.

BACKGROUND: Algae are important non-vascular plants that have many research applications, including high species diversity, biofuel sources, and adsorption of heavy metals and, following processing, are used as ingredients in health supplements. The increasing availability of next-generation sequencing (NGS) data for algae genomes and transcriptomes has made the development of an integrated resource for retrieving gene expression data and metabolic pathway essential for functional analysis and systems biology. In a currently available resource, gene expression profiles and biological pathways are displayed separately, making it impossible to easily search current databases to identify the cellular response mechanisms. Therefore, in this work the novel AlgaePath database was developed to retrieve transcript abundance profiles efficiently under various conditions in numerous metabolic pathways. DESCRIPTION: AlgaePath is a web-based database that integrates gene information, biological pathways, and NGS datasets for the green algae Chlamydomonas reinhardtii and Neodesmus sp. UTEX 2219-4. Users can search this database to identify transcript abundance profiles and pathway information using five query pages (Gene Search, Pathway Search, Differentially Expressed Genes (DEGs) Search, Gene Group Analysis, and Co-expression Analysis). The transcript abundance data of 45 and four samples from C. reinhardtii and Neodesmus sp. UTEX 2219-4, respectively, can be obtained directly on pathway maps. Genes that are differentially expressed between two conditions can be identified using Folds Search. The Gene Group Analysis page includes a pathway enrichment analysis, and can be used to easily compare the transcript abundance profiles of functionally related genes on a map. Finally, the Co-expression Analysis page can be used to search for co-expressed transcripts of a target gene. The results of the searches will provide a valuable reference for designing further experiments and for elucidating critical mechanisms from high-throughput data. CONCLUSIONS: AlgaePath is an effective interface that can be used to clarify the transcript response mechanisms in different metabolic pathways under various conditions. Importantly, AlgaePath can be mined to identify critical mechanisms based on high-throughput sequencing. To our knowledge, AlgaePath is the most comprehensive resource for integrating numerous databases and analysis tools in algae. The system can be accessed freely online at http://algaepath.itps.ncku.edu.tw.

Achieving high lipid productivity of a thermotolerant microalga Desmodesmus sp. F2 by optimizing environmental factors and nutrient conditions.

The optimal conditions for cultivating the thermotolerant lipid-rich microalga Desmodesmus sp. F2 to achieve maximal lipid productivity were determined in this study. The conditions were light intensity, 700µmol/m(2)s; temperature, 35°C; cultivation nitrogen source, nitrate; initial nitrogen level, 6.6mM nitrogen. Carbon dioxide (2.5%, 0.2 vvm) was pumped into the cultures continuously. In the pre-optimized conditions, the maximal lipid productivity of this microalga was 113mg/L/d, which was raised to 263mg/L/d in the optimized conditions. This level of lipid productivity of microalgae is the highest ever reported in the literature. Fatty acid composition of the lipid produced by Desmodesmus sp. F2 in the optimal conditions was determined, in which C16 and C18 species accounted for 95% of the fatty acids. Saturated, monounsaturated and polyunsaturated fatty acids accounted for 38.9%, 33.1% and 22.6%, respectively. Based on the analysis, this lipid quality makes it a good feedstock for biodiesel production.

Novel seed adaptations of a monocotyledon seagrass in the wavy sea.

Returning to the sea, just like invasion of land, has occurred in many groups of animals and plants. For flowering plants, traits adapted to the terrestrial environments have to change or adopt a new function to allow the plants to survive and prosper in the sea where water motion tends to rotate and move seeds. In this investigation, how seeds of the seagrass Thalassia hemprichii (Hydrocharitaceae), a common monocotyledon in the Indo-Pacific, adapt to the wavy environment was studied. Mature seeds were collected from Dongsha Atoll in South China Sea. The effects of light qualities on seed germination, the seed morphology, the unipolar distribution of starch granules in the endosperms and growth of root hair-like filamentous cells from basal surface of the seeds were all found to differ from those of terrestrial monocotyledons. Physiologically, germination of the seeds was stimulated by blue light rather than red light. Morphologically, the bell-shaped seeds coupled with the unipolar distribution of starch granules in the enlarged bases helped maintain their upright posture on the tidal seafloor. Growth of root hair-like filamentous cells from the basal surface of the seeds prior to primary root growth served to attach onto sediments, providing leverage and attachment required by the primary roots to insert into sediments. These filamentous cells grasped coral sand but not silicate sand, demonstrating a habitat preference of this species.

Selection of elite microalgae for biodiesel production in tropical conditions using a standardized platform.

Four thermotolerant microalgae were isolated from tropical Taiwan and classified as members of Desmodesmus based on morphological and molecular studies. A platform was established to evaluate their biodiesel production-related traits, including thermotolerance, lipid productivity, lipid oxidative stability and auto-sedimentation. The findings demonstrated thermotolerance of all four species was at the same level, as all could live at 45 °C for 24 h and 50 °C for 8 h with mortality rates below 5% of cells. The lipid productivity of Desmodesmus sp. F2 reached 113 mg/L/d. Its saturated and monounsaturated fatty acids accounted for 75% of the FAMEs, and it required only 3.1 h to achieve 85% sedimentation. Comparing these traits to those of the other three Desmodesmus and microalgae in the literature, Desmodesmus sp. F2 is one of the best candidates for biodiesel production in tropical and subtropical areas. This platform effectively assessed traits of microalgae related to biodiesel production.

Phototrophic cultivation of a thermo-tolerant Desmodesmus sp. for lutein production: effects of nitrate concentration, light intensity and fed-batch operation.

Four indigenous thermo-tolerant Desmodesmus sp. strains were examined for their ability to produce lutein. Among them, Desmodesmus sp. F51 was the best strain for this purpose. The medium composition, nitrate concentration and light intensity were manipulated to improve the phototrophic growth and lutein production of Desmodesmus sp. F51. It was found that a nitrogen-sufficient condition was required for lutein accumulation, while a high light intensity enhanced cell growth but caused a decrease in the lutein content. The best cell growth and lutein production occurred when the light intensity and initial nitrate concentration were 600 µmol/m(2)/s and 8.8 mM, respectively. The fed-batch cultivation strategy was shown to further improve lutein production. The highest lutein productivity (3.56±0.10 mg/L/d) and content (5.05±0.20 mg/g) were obtained when pulse-feeding of 2.2 mM nitrate was employed. This study demonstrated the potential of using Desmodesmus sp. F51 as a lutein producer in practical applications.

Pigment production by a new thermotolerant microalga Coelastrella sp. F50.

Microalgae are good crops to produce natural pigments because of their high growth rates. Tropical zones are better locations than temperate areas for microalgal cultivation because they have longer duration of daylight and more stable temperatures throughout the year, but the high temperatures pose a challenge to microalgal cultivation. A newly isolated thermotolerant microalga produces reddish pigments under environmental stress. Morphological and molecular evidence including meridional ribs on the cell wall, pigment production, and its 18S rDNA sequence suggests that this microalga belongs to the genus Coelastrella. Salt stress and high light intensity accelerated biosynthesis of the pigments, and significant quantities of oil accumulated as the cells experienced stress due to nutrient deficiency. This microalga could withstand temperature of 50°C for more than 8h, which is a necessary trait for outdoor cultivation in tropical areas. The pigments contain astaxanthin, lutein, canthaxanthin, and ß-carotene as analysed by using HPLC.

Cultivation of a thermo-tolerant microalga in an outdoor photobioreactor: influences of CO2 and nitrogen sources on the accelerated growth.

A photobioreactor was designed to evaluate the performance of a newly isolated thermo-tolerant microalga Desmodesmus sp. F2 in municipal wastewater under tropical outdoor conditions. The environmental parameters, levels of nutrients, and growth rates were monitored during the cultivations to elucidate the factors that contributed to accelerated growth after lag phase. Cultures bubbled with CO(2)-air had about 20% higher yields than the air-bubbled culture, and 2% of CO(2) at a flux rate of 5L/min was sufficient to reach this increased yield. In the cultures bubbled with CO(2)-air, the microalgal cells preferentially utilized ammonium and nitrate, while the air-bubbled culture made greater use of ammonium and organic nitrogen. In conclusion, the factors required for microalga Desmodesmus sp. F2 to achieve accelerated growth in tropical outdoor conditions include (1) 2% CO(2) bubbling; (2) a level of ammonium higher than 100 µM; and (3) a level of nitrate higher than 400 µM.

Isolation of thermo-tolerant and high lipid content green microalgae: oil accumulation is predominantly controlled by photosystem efficiency during stress treatments in Desmodesmus.

Discoveries of new microalgae with thermo-tolerance, high growth rate, and high lipid content are crucial to algal biodiesel production in tropical and subtropical zones. Four new green microalgae were isolated in southern Taiwan. All four species are members of the genus Desmodesmus under the family Scenedesmaceae based on molecular and morphological analyses. Two of the four species survived at 45 °C for 24 h, with 5-13% of mortality rates caused by the heat. Total lipid contents of the two species reached over 50% in dry biomass under nitrogen starvation, and their triacylglycerols constituted around 75% of the total lipids. Thus the two species are good potential feedstocks for biodiesel production. Oil accumulation in the four species positively correlates with their photosystem II efficiencies during stress treatments (R2=0.90). This finding further supports that photosynthesis is essential for oil body formation under nitrogen starvation in green microalgae.

Autophagy is enhanced and floral development is impaired in AtHVA22d RNA interference Arabidopsis.

Autophagy is an intracellular process in which a portion of cytoplasm is transported into vacuoles for recycling. Physiological roles of autophagy in plants include recycling nutrients during senescence, sustaining life during starvation, and the formation of central digestive vacuoles. The regulation of autophagy and the formation of autophagosomes, spherical double membrane structures containing cytoplasm moving toward vacuoles, are poorly understood. HVA22 is a gene originally cloned from barley (Hordeum vulgare), which is highly induced by abscisic acid and environmental stress. Homologs of HVA22 include Yop1 in yeast, TB2/DP1 in human, and AtHVA22a to -e in Arabidopsis (Arabidopsis thaliana). Reverse genetics followed by a cell biology approach were employed to study the function of HVA22 homologs. The AtHVA22d RNA interference (RNAi) Arabidopsis plants produced small siliques with reduced seed yield. This phenotype cosegregated with the RNAi transgene. Causes of the reduced seed yield include short filaments, defective carpels, and dysfunctional pollen grains. Enhanced autophagy was observed in the filament cells. The number of autophagosomes in root tips of RNAi plants was also increased dramatically. The yop1 deletion mutant of Saccharomyces cerevisiae was used to verify our hypothesis that HVA22 homologs are suppressors of autophagy. Autophagy activity of this mutant during nitrogen starvation increased in 5 min and reached a plateau after 2 h, with about 80% of cells showing autophagy, while the wild-type cells exhibited low levels of autophagy following 8 h of nitrogen starvation. We conclude that HVA22 homologs function as suppressors of autophagy in both plants and yeast. Potential mechanisms of this suppression and the roles of abscisic acid-induced HVA22 expression in vegetative and reproductive tissues are discussed.