Microalgal biotechnology: Carotenoid and glycerol production by the green algae <Emphasis Type="Italic">Dunaliella</Emphasis> isolated from the Gave-Khooni salt marsh,Iran

In this study, carotenoid and glycerol production in two unicellular green algae (Dunaliella salina and D. viridis) isolated from the Gave-Khooni salt marsh grown in media containing five different salt concentrations (0.17, 1, 2, 3, and 4 M NaCl) were evaluated under sterile conditions. Algae growth decreased as the medium salinity increased. Optimum growth of D. salina and D. viridis were obtained at 2 and 1 M NaCl, respectively. As salinity increased, glycerol and carotenoid production were increased in D. salina, whereas lower values for these products were produced in D. viridis under the same conditions. Furthermore, the cell color of D. salina changed from green to orange-red following accumulation of carotenoid, but the color of D. viridis was not changed. Thereby, it seems that the Iranian D. salina may be suitable for carotenoid production (betacarotene) on a large scale. In addition, since carotenoid compounds enhance the efficiency of photosynthesis and glycerol synthesis, it appears that the pathway for glycerol production and mechanisms of salt tolerance in D. viridis are unique from those of D. salina.     

Mixotrophy in the sand-dwelling dinoflagellate Thecadinium kofoidii

Thecadinium kofoidii is a marine sand-dwelling dinoflagellate that sometimes forms dense blooms. This species was previously thought to be an exclusively autotrophic dinoflagellate, and its mixotrophic ability has not been explored yet. By investigating its ecophysiology, its trophic mode should be revealed. We explored the mixotrophic ability of T. kofoidii by examining its protoplasm under light and transmission electron microscopes with diverse algal prey species. Furthermore, the feeding mechanism of T. kofoidii and prey species on which it feeds were investigated. In addition, the growth and ingestion rates of T. kofoidii as a function of prey concentration were determined when feeding on the benthic cryptophyte Rhodomonas salina. Thecadinium kofoidii was able to feed on R. salina and the dinoflagellate Symbiodinium voratum, which had equivalent spherical diameters (ESDs) ≤ 10.1?µm, while it did not feed on the benthic dinoflagellates Levanderina fissa, Prorocentrum concavum or Ostreopsis cf. ovata, which had ESDs ≥ 15?µm. Thecadinium kofoidii fed on the edible prey cells using the peduncle. The maximum ingestion rate of T. kofoidii on R. salina was 1.3 cells predator^?1 d^?1. However, feeding on R. salina did not significantly increase the growth rate of T. kofoidii. The low ingestion rate of T. kofoidii on R. salina may have partially resulted in the lack of significant increase in its growth rate due to mixotrophy. The present study discovered predator–prey relationships between T. kofoidii and R. salina and S. voratum, which may change our view of the energy flow and carbon cycling in marine benthic food webs.     

Inhibitory effects of microalgae on the activation of hyaluronidase

The inhibitory effects of seven microalgae, Nostoc flagelliforme, Spirulina platensis, Porphyridium purpureum, Rhodosorus marinus,Chlorella pyrenoidosa, Dunaliella salina and Pleurochrysiscarterae on the activation of hyaluronidase were evaluated. Theinhibitory effect of the ethanol-insoluble fraction of each water extract frommicroalgae was stronger than that of the ethanol-soluble fraction. The50% inhibitory concentration (IC₅₀) of the ethanol-insolublefraction of S. platensis, P. purpureum, R. marinus, C.pyrenoidosa, D. salina and P. carterae was 0.15, 0.18, 0.26,0.94, 0.15 and 0.41 mg mL^-1, respectively. The IC₅₀ ofN .flagelliforme was not calculated, because there was no detectableinhibitory effect of this alga. The IC₅₀ of disodium cromoglycate(DSCG) used as the anti-allergic medicine was 0.14 mg mL^-1. The IC₅₀ of S. platensis, P. purpureum and D. salinawere almost the same as that of DSCG. This suggests that theethanol-insoluble fraction of S. platensis, P. purpureum and D. salina might be an anti-allergic substance. The ethanol-insoluble fractionof S. platensis and D. salina was ultrafiltered through a membranehaving a molecular exclusion limit of 20 kDa. The IC₅₀ of theresidue was stronger than that of the filtrate. These results suggest that theanti-allergic substance(s) of these microalgae may be polysaccharides.     

颗石藻Pleurochrysis carterae抗捕食特征

颗石藻Pleurochrysis carterae是沿海水域中常见钙化微藻,易形成高密度水华,也是养殖环境致害种之一。抗捕食防御能力可能是其种群增殖优势的一个重要原因。以卤虫作为捕食者,分析了颗石藻P.carterae抗捕食现象,以及在捕食压力下的重要生理生化响应特征,以期为颗石藻P.carterea抗捕食机制研究及其高密度增殖机理提供参考。研究结果显示:(1)当颗石藻P.carterae比例增加时,卤虫对微藻的摄食率显著降低,且存活率显著下降,显示该藻具抗捕食能力。(2)以卤虫饵料微藻球等鞭金藻(Isochrysis galbana)为对照,比较研究发现,相同的捕食压力下,饵料金藻的叶绿素荧光参数(电子传递速率ETR和最大量子产率Fv/Fm)显著降低,但颗石藻P.carterae的ETR和Fv/Fm没有显著变化,显示颗石藻P.carterae对卤虫抗捕食作用。(3)相对于没有捕食压力的对照组,捕食压力下,饵料金藻I.galbana的脂类组成没有显著差异。但是,颗石藻P.carterae的脂类组成则发生了显著变化,主要表现在对细胞叶绿体有重要作用的单半乳糖甘油二酯(MGDG),双半乳糖甘油二酯(DGDG),磷脂酰甘油二酯(PG)含量上升,与促细胞分裂相关的二酰甘油(DAG)和磷脂酰肌醇(PI)也上升。这些脂类代谢物的变化可能在其种群水平上抵抗捕食并实现种群增殖中发挥作用。(4)培养介质中磷的状态对颗石藻P.carterae细胞二甲基巯基丙酸(Dimethyl sulfonio propionate,DMSP)含量有显著影响,且影响颗石藻P.carterae对卤虫的致害效应:缺磷条件下生长的颗石藻P.carterae首先使卤虫受害。当培养液中仅以ATP为磷源时,颗石藻P.carterae的卤虫致害效应则降低。研究证明,颗石藻P.carterae具有抗捕食能力,细胞的脂类代谢物质以及DMSP可能在抗捕食防御中发挥作用。     

Improvement of efficiency of genetic transformation for <Emphasis Type="Italic">Dunaliella salina</Emphasis> by glass beads method

Dunaliella salina has been exploited as a new type of bioreactor due to its unique advantages. However, this bioreactor application was restricted for absence of a high-efficiency and stable transformation method at present. In the present study, the cells of D. salina were transformed by glass beads. The results of histochemical staining revealed that the GUS gene was successfully expressed in the positive transformants, and PCR and PCR-Southern blot analysis further demonstrated that the bar gene was integrated into the D. salina genome. Moreover, the three transformation methods, including glass beads, bombardment particle and electroporation, were compared for screening a high-efficiency transformation method for gene engineering of D. salina. The results showed that transformation efficiency of the glass beads was the highest, approximately 10² transformants/μg DNA. It is concluded that the established glass beads method has been demonstrated to be an optimal transformation way for D. salina.     

Growth and ultrastructure of the marine unicellular alga <Emphasis Type="Italic">Dunaliella salina</Emphasis> (Chlorophyta) after chronic selenium intoxication

The effects of selenium (0.01, 0.5, 1, 5 and 10 mg/liter) on the growth and ultrastructure of the microalga Dunaliella salina were investigated following its transfer into clean water. Selenium concentrations of 5 and 10 mg/liter were toxic to D. salina, and reinoculation of microalga into clean water did not prevent it from total mortality. When reinoculated from medium with 0.01 mg Se/liter, the cell population density of D. salina was restored in 14 days. The number of ultrastructural alterations in cells was the same as in the control, while the excretory activity of microalga between days 4 and 10 of this experiment was higher. Cell population growth of D. salina transferred from 0.5 and 1 mg Se/liter was lower than in the control. No ultrastructural defects were observed in microalga reinoculated from medium with a selenium concentration of 0.5 mg/liter and the excretion level corresponded to that at 0.01 mg/liter. Various types of ultrastructural damage were found in microalga from medium with 1 mg Se/liter, which was previously reported to be threshold for D. salina; however, the number of cell injuries decreased with increasing time in clean medium. Excretory activity was decreased at the beginning of experiment; but after 7 days, it was restored to the control level. Though there were no ultrastructural alterations in microalgal cells from medium with 0.5 mg Se/liter, we assume that they had molecular defects that could inhibit the cell population growth. The study of microalgae following their reinoculation from medium containing toxicants into clean medium can be a useful method for evaluating algal survival after toxic exposure.     

Cloning and characterization of the 14-3-3 protein gene from the halotolerant alga Dunaliella salina

Previous studies have demonstrated that 14-3-3 proteins exist in all the eukaryotic organisms studied; however, studies on the 14-3-3 proteins have not been involved in the halotolerant, unicellular green alga Dunaliella salina so far. In the present study, a cDNA encoding 14-3-3 protein of D. salina was cloned and sequenced by PCR and rapid amplification of cDNA end (RACE) technique based on homologous sequences of the 14-3-3 proteins found in other organisms. The cloned cDNA of 1485 bp in length had a 29.2 kDa of molecular weight and contained a 774 bp of open reading frame encoding a polypeptide of 258 amino acids. Like the other 14-3-3 proteins, the deduced amino acid sequences of the D. salina 14-3-3 protein also contained two putative phosphorylation sites within the N-terminal region (positions 62 and 67). Furthermore, an EF hand motif characteristic for Ca²⁺-binding sites was located within the C-terminal part of this polypeptide (positions 208–219). Analysis of bioinformatics revealed that the 14-3-3 protein of D. salina shared homology with that of other organisms. Real-time quantitative PCR demonstrated that expression of the 14-3-3 protein gene is cell cycle-dependent.     

The presence of glycollate oxidase and dehydrogenase in Dunaliella primolecta

Homogenates of Dunaliella primolecta, D. salina and D. tertiolecta were assayed for glycollate oxidase and glycollate dehydrogenase. Both D. primolecta and D. salina but not D. tertiolecta showed substantial glycollate-dependent O₂-uptake which is characteristic of glycollate oxidase. L-Lactate was an alternative substrate and both glycollate- and L-lactate-dependent O₂ uptake were insensitive to 2 mM cyanide. Glycollate dehydrogenase, measured by following the glycollate-dependent reduction of 2,6-dichlorophenolindophenol under aerobic conditions, was present in D. primolecta, D. salina and D. tertiolecta. In the presence of glycollate and D-lactate, rates were additive so both glycollate and D-lactate dehydrogenases are present in the homogenates. Glycollate and D-lactate oxidation were both inhibited by 2 mM cyanide. Organelles released from phototrophically grown cells of D. primolecta were separated by isopycnic centrifugation on sucrose gradients. Glycollate oxidase was present in the peroxisome fraction at an equilibrium density of 1.25 g/cm³, while the major peak of glycollate dehydrogenase activity was in the mitochondrial fraction at an equilibirium density of 1.22 g/cm³.     

Increased expression of transgene in stably transformed cells of <Emphasis Type="Italic">Dunaliella salina</Emphasis> by matrix attachment regions

Nuclear matrix attachment regions (MARs) are known to bind specifically to the nuclear scaffold and are thought to influence expression of the transgenes. In our previous studies, a new deoxyribonucleic acid fragment isolated from Dunaliella salina could bind to the nuclear matrix in vitro and had the typical characteristics of MARs. In this study, to investigate effects of MARs on expression of transgenes in the stably transformed cells of D. salina, expression vectors with and without MARs, which contained chloramphenicol acetyltransferase (CAT) reporter gene driven by D. salina ribulose 1,5-bisphosphate carboxylase/oxygenase promoter, were constructed and delivered, respectively, into cells of D. salina by electroporation. Twenty stably transformed colonies of D. salina were randomly picked out, and CAT gene expression was assayed. The results showed that the CAT enzyme of the colonies of D. salina transformed with the expression vector containing MARs averaged out about 4.5-fold higher than those without MARs, while the transgene expression variation among individuals of transformants decreased threefold. The CAT enzyme in the stably transformed lines was not significantly proportional to the gene copy numbers, suggesting that the effects of MARs on transgene expression may not be through increasing the transgene copy numbers.     

Physiological responses of marine Dendryphiella species from different geographical locations

The saprobic, cosmopolitan, marine fungi Dendryphiella arenaria and Dendryphiella salina, isolated from various plant and algal substrates from different geographical locations and climatic zones, were studied for their adaptations to the abiotic and biotic parameters commonly found in their natural marine habitats. All the tested strains of D. arenaria and D. salina grew optimally on culture media with added marine salts, at pH values between 6.5 and 8.0 and at an incubation temperature of 25°C. The D. arenaria strains had faster mean colony extension rates under all conditions of culture. All strains exhibited an increased salt optimum with increasing incubation temperature. The TLC profiles of strains of the two species were similar. The culture extracts were antimicrobial, though production of the biologically active metabolites was strain-specific. There were no significant correlations between source of origin and responses to the investigated parameters. These results demonstrate phenotypic plasticity and the ability of each isolate to adapt to diverse biotopes.     

Application of suppression subtractive hybridization (SSH) to cloning differentially expressed cDNA inDunaliella salina (chlorophyta) under hyperosmotic shock

Two subtracted cDNA libraries ofDunaliella salina (Volvocales, Chlorophyceae) under different hyperosmotic shock were constructed using the suppression subtractive hybridization (SSH) method. The mRNA isolated from algae grown without stress was used as a “driver”, and the mRNAs isolated from algae 16 h (short-term treatment) or 7 d (long-term treatment) after salt stress were used as “testers”. The differentially expressed cDNA fragments inD. salina under salt stress were identified by screening these 2 libraries. Two cDNA fragments,D27 andD114, were identified from clones pL27 and pL114 after the long-term treatment. Three cDNA fragments,D21, D39, andD88, were identified from clones pSh21, pSh39, and pSh88 after the short-term treatment. The homology analysis revealed that D27 was highly similar (91%) to the subunit V of PS I reaction center inChlamydomonas reinhardtii. D21 was similar to fructose-1,6-diphosphate aldolase (78.4%). After searching GenBank with the sequences ofD39, D88, andD114, no similar sequences were found. Northern analysis revealed that the expression levels of all 5 cDNAs were increased significantly after salt stress. This means that SSH can be used in cloning differentially expressed cDNAs inD. salina under salt stress. The expression ofD27, D21, andD88 wasde novo induced by salt stress, and the expression ofD114 andD39 was increased from a relatively lower level; this indicates that all 5 cDNAs might exert an influence on the alga under hyperosmotic shock.     

Abundance and growth response of microalgae at Megalon Embolon solar saltworks in northern Greece: An aquaculture prospect

There is continuous interest in many countries in maintaining and manipulating the rich ecological value of hypersaline ecosystems for aquaculture. The Megalon Embolon solar saltworks (northern Greece) were studied in sites of increasing salinity of 60–144 ppt to evaluate Dunaliella salina abundance and microalgal composition, in relation to physical and chemical parameters. Cluster and ordination analyses were performed based on the biotic and abiotic data matrices. Using fresh aliquots from 60 and 140 ppt salinity waters, phytoplankton performance was appraised with flask cultures in the laboratory by varying the inorganic PO₄-P concentration at 23 °C and 30 °C. At the saltworks, among the most abundant microalgae identified were species of the genera Dunaliella, Chlamydomonas, Amphora, Navicula, and Nitzschia. Dunaliella salina populations were predominant comprising 5–22% of the total microalgal assemblages during spring, but only 0.3–1.0% during the summer, when grazing by Artemia parthenogenetica and Fabrea salina was intense. D. salina cell density in April–July was in the range of 0.4–12.5 × 10⁶ L⁻¹ with typical densities of 1.5–4.5 × 10⁶ L⁻¹. Overall, microalgal densities were high in salinities of ≥100 ppt when inorganic-P concentrations were ≥0.20 mg L⁻¹ within saltworks waters. Multivariate analysis of species abundance showed that algal growth responses were primarily related to variation in salinity and inorganic-P concentrations, but also to NO₃-N concentration. In the laboratory, experiments indicated effective fertilization and denser microalgal growth under high inorganic PO₄-P applications (4.0 and 8.0 mg L⁻¹) at 60 ppt salinity and 23 °C. The lower PO₄-P applications (0.6–2.0 mg L⁻¹) were more effective at 60 ppt salinity and 30 °C. At 140 ppt salinity, microalgal growth response was less obvious at any of the corresponding phosphorus concentrations or temperatures. In both salinity experiments, Dunaliella salina bloomed easily and was predominant among the microalgae. Our observations indicate that Dunaliella salina populations and the overall rich microalgal profile of the saltworks, along with their performance in laboratory mono–and mixed cultures hold promise for mass cultivation within the M. Embolon saltworks basins.     

Cloning and expression study of a putative high-affinity nitrate transporter gene from Dunaliella salina

A nitrate transporter gene, named DsNRT2.1 (GeneBank accession number AY621079), from Dunaliella salina has been cloned by screening a cDNA library, which was constructed with mRNAs from D. salina after 60 min treatment with 5 mM nitrate, with a 342 bp NRT2 cDNA fragment from D. salina as a probe. DsNRT2.1 exhibits sequence similarity to those known nitrate transporters of the NRT2 family. A hydrophobicity blot indicated that DsNRT2.1 belongs to the major facilitator superfamily (MFS). Northern analysis showed that an mRNA species of 1.9 kb can be rapidly induced by NO^– ₃, but not by NH⁺ ₄. Northern analysis also showed that NaCl could significantly increase the expression of DsNRT2.1.     

The survival of <Emphasis Type="Italic">Sicyopterus stimpsoni</Emphasis>, an endemic amphidromous Hawaiian gobiid fish,relies on the hydrological cycles of streams: evidence from changes in algal composition of diet through growth stages fish

Gut contents of larval, juvenile, and adult specimens of the Hawaiian gobiid fish Sicyopterus stimpsoni were examined to catalog the algal flora ingested by this species. The developmental stages of S. stimpsoni examined represented hallmark points in the fish’s life cycle corresponding with major migratory and metamorphic transitions. The algal flora was dominated by diatom species and shifted from taxa representative of a marine, planktonic community in larval fish to a freshwater, benthic community in juvenile and adult fish. This change in diet corresponds with the migration of larval fish to freshwater streams just prior to juvenile development in which rapid modification in mouth anatomy makes ingestion of planktonic algal species difficult. Benthic diatoms from juvenile and adult fish assemblages represented multiple genera that live in a narrow set of environmental conditions. These algae grow during a specific period in the development of the benthic algal community in Hawaiian streams. This suggests a highly specialized dietary behavior that depends heavily on continually restarting the benthic algal successional pattern, which appears to be regulated by the hydrological cycles of streams on the island.     

LINKING BENTHIC ALGAL BIOMASS TO STREAM SUBSTRATUM TOPOGRAPHY1

The physical properties of substrata significantly influence benthic algal development. We explored the relationships among substratum surface texture and orientation with epilithic microphytobenthic biomass accumulation at the whole‐substratum and micrometer scales. Unglazed clay tiles set at three orientations (horizontal, vertical, and 45°), and six substrata of varying surface roughness were deployed in a prairie stream for 3 weeks. Substrata were analyzed for loosely attached, adnate, and total benthic algal biomass as chl a, and confocal laser scanning microscopy was used to measure substrata microtopography (i.e., roughness, microscale slope angles, and three‐dimensional surface area). At the whole‐substratum level, vertical substrata collected significantly (P < 0.05) less algal biomass, averaging 34% and 36% less than horizontal and 45° substrata, respectively. Benthic algal biomass was also significantly less on smoother surfaces; glass averaged 29% less biomass than stream rocks. At the microscale level, benthic algal biomass was the greatest at intermediate values, peaking at a mean roughness of approximately 17 μm, a mean microscale slope of 50°, and a projected/areal surface area ratio of 2:1. The proportion of adnate algae increased with surface roughness (26% and 67% for glass and brick, respectively), suggesting that substratum type changes the efficiency of algal removal by brushing. Individual substrata and microsubstrata characteristics can have a strong effect on benthic algae development and potentially affect reach scale algal variability as mediated by geomorphology.     

Structure and properties of agar from two unexploited agarophytes from Venezuela

The red seaweeds Gelidiella acerosa and Gracilaria mammillaris growing along the coast of Venezuela were investigated as potential economic sources of agar. Agar extracted from Gracilaria mammillaris accounted for 27% of the algal dry weight but had quite a poor gelling ability because of the presence of alkali-stable sulphate groups on the D-galactose residue. However, the gel strength of its aqueous solutions was considerably enhanced by the addition of potassium, sodium and calcium ions (up to 1N). On the contrary, the galactan from Gelidiella acerosa (yield of about 20% w/w) exhibited quite good properties. In particular, the gel strength was comparable to that of commercial agaroses. Sulphate esters were not detectable by chemical methods and NMR spectroscopy revealed an agarose backbone with a high degree of methylation on both D and L-galactose residues.     

The mass culture of Dunaliella viridis (Volvocales,Chlorophyta) for oxygenated carotenoids : laboratory and pilot plant studies

The biology, and hence the mass culture, of Dunaliella viridis closely follows that of Dunaliella salina, which is successfully mass cultured for the production of -carotene. Both algae can grow at extremely high salinities and light intensities. They co-exist in the coastal salt lake, Hutt Lagoon, Western Australia. In contrast to D. salina, D. viridis does not accumulate large amounts of -carotene, producing only up to 0.7% of mixed carotenoids (lutein, zeaxathin, other oxygenated carotenoids and -carotene), compared to D. salina's ca 10% dry wt of mainly -carotene. However, in laboratory experiments, D. viridisgrew much faster and to much higher cell densities than D. salina, and attained levels of mixed carotenoids similar to those of D. salina (ca 13 mg L^–1 carotenoid). Preliminary experiments in outdoor ponds were much less promising. Harvesting by chemical flocculation was as effective as with D. salina, but extraction of carotenoids directly into vegetable oil proved inefficient. When incorporated into feed, caretonoids derived from D. viridis pigmented hen eggs acceptably. Extrapolating from laboratory results, and using costing derived from D. salina technology, the cost of production of mixed oxygenated carotenoids from D. viridis was similar to that for the production of -carotene from D. salina, at ca $A500 kg^–1.     

Microphytobenthic Primary Production and Sedimentary Carbohydrates Along Salinity Gradients in the Lagoons of Grado and Marano (Northern Adriatic Sea)

Primary production of the microphytobenthic community and carbohydrates concentrations were studied in the lagoonal system of Grado and Marano, located in the Northern Adriatic coast. Sediment samples were collected along a salinity gradient. Abundance and species composition of the microphytobenthic communities were analysed and the benthic microalgal biomass was estimated as Chlorophyll a (Chl a). Primary production of benthic diatoms was estimated using ¹⁴C-tracer. Extracellular carbohydrates were extracted from the sediment and separated in two operationally defined fractions (colloidal and EDTA-extractable). Salinity was higher in the Grado lagoon, where the benthic microalgal community was mainly composed of marine diatoms. In the Marano lagoon, which has a lower salinity, freshwater species were also found. In both lagoons, photosynthetic efficiency showed an inverse relationship with salinity and a direct relationship with the main biological variables. Photosynthetic activity was directly related to Chl a and abundance of benthic microalgae, suggesting that in the benthic system microalgal community is responsible for primary production. Overall, salinity was also influent on the microphytobenthic primary production, which was greater in the more saline Grado lagoon.     

Cloning and heterologous expression of nitrate reductase genes from <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame of 2,703 bp encoding 900 amino acids, a 5′-untranslated region of 151 bp and a 3′-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme assay, confirming that the cloned gene from D. salina is indeed NR.