The effect of mixotrophy on microalgal growth, lipid content, and expression levels of three pathway genes in Chlorella sorokiniana

Nannochloropsis oculata CCMP 525, Dunaliella salina FACHB 435, and Chlorella sorokiniana CCTCC M209220 were compared in mixotrophic and photoautotrophic cultures in terms of growth rate, protein, and lipid content. Growth improved in glucose, and the biomass productivities of N. oculata, D. salina, and C. sorokiniana were found to be 1.4-, 2.2- and 4.2-fold that observed photoautotrophically. However, biomass and lipid production decreased at the highest glucose concentrations. Meanwhile, the content of protein and lipid were significantly augmented for mixotrophic conditions at least for some species. C. sorokiniana was found to be well suited for lipid production based on its high biomass production rate and lipid content reaching 51% during mixotrophy. Expression levels of accD (heteromeric acetyl-CoA carboxylase beta subunit), acc1 (homomeric acetyl-CoA carboxylase), rbcL (ribulose 1, 5-bisphosphate carboxylase/oxygenase large subunit) genes in C. sorokiniana were studied by real-time PCR. Increased expression levels of accD reflect the increased lipid content in stationary phase of mixotrophic growth, but expression of the acc1 gene remains low, suggesting that this gene may not be critical to lipid accumulation. Additionally, reduction of expression of the rbcL gene during mixotrophy indicated that utilization of glucose was found to reduce the role of this gene and photosynthesis.     

The ascorbic acid content of eleven species of microalgae used in mariculture

The ascorbic acid (vitamin C) concentrations in 11 species of microalgae commonly used in mariculture were determined. The species examined were 4 diatoms (Chaetoceros calcitrans (Paulsen) Takano,Chaetoceros gracilis Schütt,Skeletonema costatum (Greville) Cleve,Thalassiosira pseudonana (Hustedt, clone 3H) Hasle and Heimdal); 2 prymnesiophytes (Isochrysis sp. (clone T.ISO) Parke,Pavlova lutheri (Droop) Green); 1 prasinophyte (Tetraselmis suecica (Kylin) Butcher); 2 chlorophytes (Dunaliella tertiolecta Butcher,Nannochloris atomus Butcher); 1 eustigmatophyte (Nannochloropsis oculata (Droop) Green); and 1 cryptophyte (Chroomonas salina (Wislouch) Butcher). Duplicate cultures of each species were grown under defined conditions and analysed during both logarithmic and stationary phase of growth.Average values for ascorbic acid ranged from 9.4 fg cell^–1 (N. oculata, stationary phase) to 700 fg cell^–1 (S. costatum, stationary phase). This value was generally related to cell size. Levels of ascorbic acid cell^–1 increased during the stationary growth phase forS. costatum andD. tertiolecta and decreased forC. gracilis, T. pseudonana, C. salina andN. oculata. Levels did not change significantly for the remaining species.Average values for per cent ascorbic acid ranged from 0.11% (T. pseudonana, stationary phase) to 1.62% of dry weight (C. gracilis, logarithmic phase). The per cent ascorbic acid was not related to algal class. Also, the percentage between logarithmic and stationary phase cultures differed for many of the species, but differences were unrelated to algal class.Chaetoceros gracilis, T. pseudonana, N. oculata andIsochrysis sp. (T.ISO) had higher per cent ascorbic acid during the logarithmic phase, whereasD. tertiolecta andN. atomus contained more per cent ascorbic acid during the stationary phase.Despite the differences in the composition of the different microalgae (0.11–1.62% ascorbic acid), all species would provide a rich source of ascorbic acid for maricultured animals, which can require 0.003–0.02% of the vitamin in their diet.     

The effect of ultrasound on the growth and viability of microalgae cells

Ultrasound has shown potential for both increasing microalgal lipid extraction yields and for the control of microalgal blooms through cell disruption. The effect of ultrasound on the viability of microalgae was investigated on the following species: Dunaliella salina, Chlamydomonas concordia and Nannochloropsis oculata. Sonication with a 20 kHz probe (0.086 W cm^?3) caused complete cell disruption of D. salina after 4 min. This microalgae species does not have a true cell wall. In the case of C. concordia which has a thin cell wall complete cell disruption under the same conditions took 16 min. Under the same conditions, there was no visible disruption of N. oculata, a species which has a thick cell wall. However spectro-fluorophotometer analysis of the sonicated suspension of N. oculata showed that although the cells were intact, the level of intracellular chlorophyll was reduced by ~10 %. This clearly indicated damage to the microalgal cell wall. After 16 min, treatment cultures of all three species remained viable. Programmed cell death (PCD) has been induced in some microalgal species to terminate algal blooms; ultrasonic application did not induce PCD in any species tested. The supernatant of sonicated D. salina and C. concordia has also been shown to be able to boost the growth of established cultures. These results provide important information concerning the uses of ultrasound in both the microalgal biofuels industry and for the control of microalgal blooms.     

Microalgal growth enhancement by levoglucosan isolated from the green seaweed <Emphasis Type="Italic">Monostroma nitidum</Emphasis>

Microalgal growth was enhanced by the addition of levoglucosan to the culture medium. The growth-enhancing compound levoglucosan was isolated from the green seaweed Monostroma nitidum using water extraction, molecular fractionation, DEAE-cellulose column chromatography, and high-performance liquid chromatography. Yield of the compound from seaweed powder was 5 × 10⁻³% (w/w). At 10 mM concentration, levoglucosan enhanced cell growth and the specific growth rate of all feed microalgal species tested (Chaetoceros gracilis, Chlorella ellipsoidea, Dunaliella salina, Isochrysis galbana, Nannochloris oculata, Navicula incerta, Pavlova lutheri, Tetraselmis suecica) in most culture media by approximately 150%. Cellular fatty acid profiles and cell size differed marginally between cultures with and without levoglucosan.     

Effects of carbon source and light intensity on the growth and total lipid production of three microalgae under different culture conditions

We attempted to enhance the growth and total lipid production of three microalgal species, Isochrysis galbana LB987, Nannochloropsis oculata CCAP849/1, and Dunaliella salina, which are capable of accumulating high content of lipid in cells. Low nitrogen concentration under photoautotrophic conditions stimulated total lipid production, but a decreasing total lipid content and an increasing biomass were observed with increasing nitrogen concentration. Among the different carbon sources tested for heterotrophic cultivation, glucose improved the growth of all three strains. The optimal glucose concentration for growth of I. galbana LB987 and N. oculata CCAP849/1 was 0.02 M, and that of D. salina was 0.05 M. Enhanced growth occurred when they were cultivated under heterotrophic or mixotrophic conditions compared with photoautotrophic conditions. Meanwhile, high total lipid accumulation in cells occurred when they were cultivated under photoautotrophic or mixotrophic conditions. During mixotrophic cultivation, biomass production was not affected significantly by light intensity; however, both chlorophyll concentration and total lipid content increased dramatically with increasing light intensity up to 150 µmol/m²/s. The amount and composition ratio of saturated and unsaturated fatty acids in cells were different from each other depending on both species and light intensity. The highest accumulation of total fatty acid (C16–C18) among the three strains was found from cells of N. oculata CCAP849/1, which indicates that this species can be used as a source for production of biodiesel.     

Differential Effects of the Substituted Pyridazinone Herbicide Sandoz 9785 on Lipid Composition and Biosynthesis in Photosynthetic and Non-Photosynthetic Marine Microalgae: II. FATTY ACID COMPOSITION

The effect of the substituted pyridazinone, 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone(Sandoz 9785), on fatty acid synthesis in two photosyntheticspecies (Chroomonas salina and Nannochloropsis oculata) andone non-photosynthetic species (Crypthecodiniun cohnii) of marinemicroalgae were examined. Effects were more obvious in C. salinathan in C. cohnii or N. oculata. In C. cohnii the relative distributionamongst polyunsaturated fatty acids (PUFA) of radioactivityincorporated from ¹⁴C-acetate was not influenced by the herbicideto any great extent and no major changes in the fatty acid compositionof lipid fractions were observed. In C. salina, Sandoz 9785reduced the proportions of radioactivity recovered in 20: 5(n–3) and 22: 6 (n–3) of the phospholipid fraction.The distribution pattern of radioactivity in the fatty acidsof monogalactosyldiacylglycerol (MGDG) in this species was notgreatly affected by the herbicide whereas its presence significantlyreduced the proportions recovered in 18: 4 and 20: 1 in digalactosyldiacylglycerol(DGDG). The level of 20: 5 (n–3) in DGDG of C. salinawas increased from 4.0 to 19.8% by growing the algae in thepresence of Sandoz 9785. The only notable effect of the herbicideon the synthesis of PUFA in N. oculata was a reduction from18.3% to 11.3% of the proportion of radioactivity recoveredin 20: 5 in phospholipids. The herbicide had no effect on thedistribution of radioactivity in PUFA of galactolipids or onthe fatty acid composition of lipid fractions. The results arediscussed in relation to the potential role of galactolipidsand phospholipids as substrates for desaturations involved inthe formation of long chain PUFA in marine microalgae. Key words: Microalgae, herbicide, fatty acids     

Treatment with Algae Extracts Promotes Flocculation, and Enhances Growth and Neutral Lipid Content in Nannochloropsis oculata—a Candidate for Biofuel Production

Marine microalgae represent a potentially valuable feedstock for biofuel production; however, large-scale production is not yet economically viable. Optimisation of productivity and lipid yields is required and the cost of biomass harvesting and dewatering must be significantly reduced. Microalgae produce a wide variety of biologically active metabolites, many of which are involved in inter- and intra-specific interactions (the so-called infochemicals). The majority of infochemicals remain unidentified or uncharacterised. Here, we apply known and candidate (undefined extracts) infochemicals as a potential means to manipulate the growth and lipid content of Nannochloropsis oculata—a prospective species for biofuel production. Five known infochemicals (β-cyclocitral, trans,trans-2,4-decadienal, hydrogen peroxide, norharman and tryptamine) and crude extracts prepared from Skeletonema marinoi and Dunaliella salina cultures at different growth stages were assayed for impacts on N. oculata over 24?h. The neutral lipid content of N. oculata increased significantly with exposure to three infochemicals (β-cyclocitral, decadienal and norharman); however the effective concentrations affected a significant decrease in growth. Exposure to particular crude extracts significantly increased both growth and neutral lipid levels. In addition, water-soluble extracts of senescent S. marinoi cultures induced a degree of flocculation in the N. oculata. These preliminary results indicate that artificial manipulation of N. oculata cultures by application of algae infochemicals could provide a valuable tool towards achieving economically viable large-scale algae biofuel production.     

The <Emphasis Type="Italic">Dunaliella salina</Emphasis> organelle genomes: large sequences,inflated with intronic and intergenic DNA

Background  

Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri.     

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.     

Population growth dynamics of the rotifer Brachionus plicatilis cultured in non-limiting food condition

Population growth parameters in batch culture of Brachionus plicatilis under a continuous supply of freeze-dried microalgae powder have been determined. Two B. plicatilis strains (L- and S-types) and four microalgae species (Nannochloropsis oculata, Nannochloropsis gaditana, Nannochloris oculata and Tetraselmis suecica) have been tested, establishing the dynamics of growth at different daily food rations. Cultures showed a short lag phase, an exponential growth phase, a long post-exponential growth phase and long decline with episodic increases. In both rotifer strains, the best growth was obtained with Nannochloropsis oculata and the poorest with Nannochloris oculata.     

The reproductive biology of two deep-water,reef-building scleractinians from the NE Atlantic Ocean

The reproductive ecology of colonies of Lophelia pertusa (Linné 1758) and Madrepora oculata Linné (1758) from the Porcupine Seabight (Thérèse Mound and South Porcupine Seabight site) and the Darwin Mounds (NE Rockall Trough—L. pertusa only) was investigated using histological techniques. Samples of L. pertusa exhibited seasonal reproduction, whereas the evidence for M. oculata is equivocal but suggests multiple cohorts of gamete production. L. pertusa produces a single cohort of around 3,000 oocytes, whereas M. oculata produces two cohorts, with a total fecundity of around 60 oocytes. The maximum observed oocyte size in L. pertusa was 140 μm and in M. oculata was 405 μm. From these oocyte sizes and the timing of reproduction, a lecithotrophic larva is expected, though not observed. This seasonality of reproduction fits with the phytodetrital food fall occurring around July in the Seabight area. L. pertusa was found to be non-reproductive at the Darwin Mound site. Though unable to be specifically tested, this may suggest that the increased trawling activity in this area might be keeping colonies below sexually viable sizes, as seen in numerous shallow water situations. All areas in the NE Atlantic are coming under threat from increased fishing and commercial exploration practices. This study shows that these highly seasonal reproducers could be sensitive to these fishing operations and care must be taken so as not to repeat the destruction that has occurred on shallower reefs.     

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.     

Intraspecific selectivity,compensatory feeding and flexible homeostasis in the phagotrophic flagellate <Emphasis Type="Italic">Oxyrrhis marina</Emphasis>: three ways to handle food quality fluctuations

The phagotrophic flagellate Oxyrrhis marina shows a strong stoichiometric plasticity when fed differently grown Rhodomonas salina. We tested whether differently pre-conditioned O. marina displayed selective feeding behaviour from a mixture of nitrogen and phosphorus depleted R. salina. We observed selective feeding of O. marina, always selecting phosphorus rich R. salina independent of the pre-conditioning of the protists. In a second experiment, O. marina was again pre-conditioned either with nitrogen- or phosphorus-depleted R. salina and was refed with either of the differently limited R. salina in single food treatments (not in a mixture). The phagotrophic flagellate displayed compensatory feeding which means that O. marina feed more on the food source which they were not given before. Due to its stoichiometric plasticity, O. marina might handle bad quality food by following the stoichiometry of its prey and additionally by active selective feeding towards P-rich algae to enhance growth. Post-ingestion selection might as well be an important feature which means that ingested elements in excess are quickly excreted and scarce elements are ingested through accelerated food uptake.     

Growth-Promoting Effect of Alginate Oligosaccharides on a Unicellular Marine Microalga,Nannochloropsis oculata

Effects of an alginate oligosaccharide mixture (AOM) on Nannochloropsis oculata, a unicellular marine microalga, were investigated. The growth of N. oculata was significantly promoted by AOM in a concentration-dependent manner. The maximum effect was attained at 20 mg/ml, at which the growth rate of the alga became nearly 5 times higher than that of control without AOM. The growth-promoting effect of AOM decreased slightly at 40 mg/ml. Furthermore, the algicidal effect of Cu²⁺ was profoundly alleviated by the addition of AOM. These results suggest that AOM is useful for promoting and/or improving the growth of N. oculata.     

A Novel Glyceraldehyde-3-Phosphate Dehydrogenase (<Emphasis Type="Italic">GAPDH</Emphasis>) Promoter for Expressing Transgenes in the Halotolerant Alga <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A major challenge for efficient transgene expression in Dunaliella salina is to find strong endogenous promoters to drive the transgene expression. In the present study, a novel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was cloned and used to drive expressions of the bialaphos resistance (bar) gene and of the N-terminal fragment of human canstatin (Can-N). The results showed that the bar gene was transcribed by the GAPDH promoter and integrated into the genome of the transformants of D. salina. Furthermore, the PCR identification, Southern and western blots indicated that Can-N was expressed in transgenic D. salina, demonstrating that the promoter of the D. salina GAPDH gene is suitable for driving expression of heterologous genes in transgenic D. salina.     

Allozyme comparison of three nemertean species of the genus Oerstedia (Nemertea: Monostilifera) from the Sea of Japan

Three species of hoplonemerteans, all very similar in external features (Oerstedia oculata, Oerstedia phoresiae, and Oerstedia zebra) were compared using 23 allozyme loci. The results of this study confirm the specific validity of O. phoresiae. At 20 of the 23 loci this species shares no common alleles with O. oculata (D = 2.718) and at 21 of the 23 loci O. phoresiae shares no common alleles with O. zebra (D = 2.730). Most likely O. zebra is a color variation of O. oculata, because these species are very similar in their allozyme markers (D = 0.000).     

ISOLATION OF THREE NOVEL LONG‐CHAIN POLYUNSATURATED FATTY ACID Δ9‐ELONGASES AND THE TRANSGENIC ASSEMBLY OF THE ENTIRE PAVLOVA SALINA DOCOSAHEXAENOIC ACID PATHWAY IN NICOTIANA BENTHAMIANA1

The transgenic aerobic synthesis of long‐chain polyunsaturated fatty acids (LC‐PUFA) will in most land plants commence with either a Δ6‐desaturation or a Δ9‐elongation. Numerous Δ6‐desaturases have been characterized, but only one Δ9‐elongase has been reported in peer‐reviewed literature. In the present study, we describe the isolation of three additional Δ9‐elongases from the class Haptophyceae and demonstrate that the Δ9‐elongase group contains highly conserved regions, which differentiate them from other ELO‐type elongases. One such important difference is the presence of an LQxFHH motif instead of the usual LHxYHH motif, a feature that should simplify further gene discovery efforts in this group of enzymes. Moreover, the identification of the Pavlova salina (N. Carter) J. C. Green Δ9‐elongase completes the isolation of the entire P. salina docosahexaenoic acid (DHA) pathway, and we describe the assembly of this pathway in Nicotiana benthamiana. Finally, we comment on possible explanations for the widespread presence of the Δ6‐desaturated fatty acid stearidonic acid (SDA, 18:4^Δ6,9,12,15) in the plastidial lipids of organisms using the Δ9‐elongase pathway.