Effects of deep seawater on the growth of several species of marine micro-algae

Deep seawater (DSW; seawater under the euphotic layer), obtained from Deep Seawater Laboratory in Muroto, Japan, was applied to the culture ofDunaliella tertiolecta, D. salina, Nannochloropsis oculata, N. salina, Porphyridium cruentum, Tetraselmis tetrahele, andChaetoceros ceratosporum. DSW supported the exponential growth of every species. The growth yields were at 14.7 (±2.3 SD) mg dry weight per liter, and could be heightened by the addition of nitrate to DSW.Author for correspondence     

Long chain polyunsaturated fatty acid production and partitioning to triacylglycerols in four microalgae

Gas chromatographic profiling of fatty acids was performed during the growth cycle of four marine microalgae in order to establish which, if any, of these could act as a reliable source of genes for the metabolic engineering of long chain polyunsaturated fatty acid (LC-PUFA) synthesis in alternative production systems. A high-throughput column based method for extraction of triacylglycerols (TAGs) was used to establish how much and at what stage in the growth phase LC-PUFAs partition to storage lipid in the different species. Differences in the time course of production and incorporation of docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n-3, EPA) into TAGs were found in the marine microalgae Nannochloropsis oculata (Eustigmatophyceae), Phaeodactylum tricornutum and Thalassiosira pseudonana (Bacillariophyceae), and the Haptophyte Pavlova lutheri. Differences were not only observed between species but also during the different phases of growth within a species. A much higher percentage of the total cellular EPA was partitioned to TAGs in stationary phase cells of N. oculata compared to P. tricornutum. Although P. tricornutum produces DHA it does not partition it to TAGs. Both T. pseudonana and P. lutheri produce EPA and DHA and partition these to TAGs during the stationary phase of growth. These two species are therefore good candidates for further biochemical and molecular analysis, in order to understand and manipulate the processes that are responsible for the incorporation of LC-PUFAs into storage oils.     

Total, free and conjugated sterolic forms in three microalgae used in mariculture

Total, free and conjugated forms (steryl esters, steryl glycosides and acyl steryl glycosides) of sterols from three microalgae that are extensively used in mariculture (Tetraselmis chuii, Nannochloropsis salina and Skeletonema costatum) were examined. The results revealed that cholesterol is the only common fraction detected in all investigated species and distributed in free and all conjugated forms. However, the total sterol content of T. chuii was about 325 microg/g dry wt, most of it was concentrated amongst 24-methylcholesta-5,24-diene-3beta-ol and 24-methylcholest-5-en-3beta-ol. On the other hand, the majority of the fractions were distributed in the free form. The total sterol content of N. salina was about 180 microg/g dry wt, cholesterol was the major fraction that was detected. Nevertheless, the dominant distribution forms were esterified. While in S. costatum, the total sterol content was 76 microg/g dry wt, approximately most fractions are quantitatively alike and dominated in the free form. Furthermore, our study shows clearly that most sterols are not distributed regularly within each form, a result that encouraged us to suggest a distribution of specific sterol fraction as a free or conjugated can be used as a serving tool in chemotaxonomic studies.     

STEROLIC BIOMARKERS IN MARINE PHYTOPLANKTON. II. FREE AND CONJUGATED STEROLS OF SEVEN SPECIES USED IN MARICULTURE

The sterols of seven unicellular algae widely used in mariculture and belonging to different classes (Prasinophyceae, Haptophyceae, Eustigmatophyceae, and Diatomophyceae) were examined for free and combined forms. Under standard culture conditions, all synthesized free sterols and combined forms (steryl esters, acyl steryl glycosides, and steryl glycosides). Free sterols were dominant in five of the species. In contrast, sterols were mostly esterified in the eustigmatophyte Nannochloropsis oculata (Droop) Hibberd and the diatom Thalassiosira pseudonana Hasle et Heimdal, whereas in another diatom, Chaetoceros calcitrans (Paulsen) Takano, glycosylated forms represented over 60% of total sterols. The pennate diatom Haslea ostrearia (Gaillon) Simonsen synthesized large amounts of steryl glycosides consisting mainly of an unusual sterol, 23,24-dimethylcholest-5-en-3β-ol, occurring in some dinoflagellates.     

ANALYSIS OF EXPRESSED SEQUENCE TAGS FROM THE MARINE MICROALGA NANNOCHLOROPSIS OCULATA (EUSTIGMATOPHYCEAE)1

Nannochloropsis oculata (Droop) D. J. Hibberd (Eustigmatophyceae), a marine eukaryotic unicellular alga, is widely used in mariculture as live feed. It is considered to be of high nutritional value owing to its high content of proteins; polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA, C20:5n3); and diverse pigments. Previous studies of this microalga focused on its taxonomy, culture, and biochemistry, but little is known at the molecular level. Establishing a molecular base is vital to understand the biological processes of this alga. Therefore, we constructed a cDNA library using algal cells grown at exponential growth phase and carried out expressed sequence tag (EST) analysis. A total of 1,960 nonredundant sequences (NRSs) were generated for N. oculata clone CS‐179. Only 32.5% of NRSs showed significant similarity (E < 1e‐04) to proteins registered in the GenBank nonredundant protein database. The KOG (clusters of euKaryotic Orthologous Groups) profile database returned significant hits for 490 NRSs. Analysis revealed that a large proportion of NRSs could be unique to this microalga.     

Microalgae for use in tropical aquaculture I: Gross chemical and fatty acid composition of twelve species of microalgae from the Northern Territory,Australia

Twelve species of microalgae, isolated from north Australian marine, freshwater and hypersaline environments, were grown under controlled conditions of temperature, pH, photon flux density and salinity, and analysed for ash, total protein, water soluble carbohydrates, chlorophylla, total lipids, total fatty acids and fatty acid composition. Highest levels of the polyunsaturated fatty acid eicosapentaenoic acid [20:5(n-3)] were found in the marine diatoms.Nitzschia (frustulum) andN. closterium (23.1% and 15.2% of total fatty acids, respectively). None of the species studied had levels of docosahexaenoic acid [22:6(n-3)] greater than 1.1 % of total fatty acids. None of the chlorophyte species contained significant levels of either 20:5(n-3) or 22:6(n-3). The highest total fatty acid concentration of all species in the study was found in the freshwater chlorophyte speciesScenedesmus dimorphus (105 mg g^–1 dry wt). The hypersaline speciesDunaliella salina had the highest total lipid content (28.1% dry wt), followed byN. closterium, N. (frustulum) andNavicula sp. (24.2–27.8% dry wt).Chlamydomonas sp. had the highest protein content (66.9% dry wt).N. (frustulum) was highlighted as a possible useful source of lipids and polyunsaturated fatty acids in mixed microalgal diets for mariculture organisms used in tropical aquaculture.Author for correspondence     

Triacylglycerol profiling of marine microalgae by mass spectrometry

We present a method for the determination of triacylglycerol (TAG) profiles of oleaginous saltwater microalgae relevant for the production of biofuels, bioactive lipids, and high-value lipid-based chemical precursors. We describe a technique to remove chlorophyll using quick, simple solid phase extraction (SPE) and directly compare the intact TAG composition of four microalgae species (Phaeodactylum tricornutum, Nannochloropsis salina, Nannochloropsis oculata, and Tetraselmis suecica) using MALDI time-of-flight (TOF) mass spectrometry (MS), ESI linear ion trap-orbitrap (LTQ Orbitrap) MS, and 1H NMR spectroscopy. Direct MS analysis is particularly effective to compare the polyunsaturated fatty acid (PUFA) composition for triacylglycerols because oxidation can often degrade samples upon derivatization. Using these methods, we observed that T. suecica contains significant PUFA levels with respect to other microalgae. This method is applicable for high-throughput MS screening of microalgae TAG profiles and may aid in the commercial development of biofuels.     

氮源种类及浓度对眼点拟微绿球藻生长密度、油脂产率和二十碳五烯酸含量的影响

氮源是影响微藻生长和油脂积累的重要因素,文中通过单因素试验比较了NaNO3、CO(NH2)2、NH4Cl、CH3COONH4及其浓度对眼点拟微绿球藻生长密度、生长速率、油脂产率、二十碳五烯酸(EPA)含量的影响。结果表明：NH4+更易被眼点拟微绿球藻利用,能更好地促进微藻生长和油脂积累;氮浓度的增加有利于微藻的生长和藻油脂肪酸的去饱和,但不利于微藻油脂的积累。在实验考察的氮源种类和浓度范围内,CH3COONH4是促进眼点拟微绿球藻生长和油脂积累、EPA生成的适宜氮源,其适宜的浓度为5.29 mmol/L。     

FATTY ACIDS AND HYDROXY FATTY ACIDS IN THREE SPECIES OF FRESHWATER EUSTIGMATOPHYTES

The monocarboxylic fatty acids and hydroxy fatty acids of three species of freshwater microalgae—Vischeria punctata Vischer, Vischeria helvetica (Vischer et Pascher) Taylor, and Eustigmatos vischeri (Hulbert) Taylor, all from the class Eustigmatophyceae— were examined. Each species displayed a very similar distribution of fatty acids, the most abundant of which were 20:5n-3, 16:0, and 16:1n-7; C₁₈ polyunsaturated fatty acids were minor components. These fatty acid distributions closely resemble those found in marine eustigmatophytes but are quite distinct from those found in most other algal classes. These microalgae also contain long-chain saturated and unsaturated monohydroxy fatty acids. Two distinct types of hydroxy fatty acids were found: a series of saturated α-hydroxy acids ranging from C₂₄ to C₃₀ with a shorter series of monounsaturated α-hydroxy acids ranging from C₂₆ to C₃₀ together with a series of saturated β-hydroxy acids ranging from C₂₆ to C₃₀. The latter have not previously been reported in either marine or freshwater microalgae, although C₃₀ to C₃₄ midchain (ω-18)-hydroxy fatty acids have been identified in hydrolyzed extracts from marine eustigmatophytes of the genus Nannochloropsis, and C₂₂ to C₂₆ saturated and monounsaturated α-hydroxy fatty acids have been found in three marine chlorophytes. These findings have provided a more complete picture of the lipid distributions within this little studied group of microalgae as well as a range of unusual compounds that might prove useful chemotaxonomic markers. The functions of the hydroxy fatty acids are not known, but a link to the formation of the lipid precursors of highly aliphatic biopolymers is suggested.     

PHOTOSYNTHETIC LIGHT-HARVESTING FUNCTION OF VIOLAXANTHIN IN NANNOCHLOROPSIS SPP. (EUSTIGMATOPHYCEAE)1

Whole cell absorption spectra of the Eustigmatophycean algae Nannochloropsis salina Bourrelly and Nannochloropsis sp. reveal the presence of a distinct absorption peak at 490 nm. The lack of chlorophylls b and c in these species indicates that this peak must be attributed to carotenoid absorption. In vivo fluorescence excitation spectra for chlorophyll a emission show a corresponding maximum at 490 nm. This peak is more clearly resolved than carotenoid maxima in other algal classes due to the absence of accessory chlorophylls. The carotenoid composition of the two Nannochloropsis species shows that violaxanthin and vaucheriaxanthin are the main contributors to 490 nm absorption. Violaxanthin accounts for approximately 60% of the total carotenoid in both clones. We conclude that light absorption by violaxanthin, and possibly by vaucheriaxanthin, is coupled in energy transfer to chlorophyll a and that violaxanthin is the major light-harvesting pigment in the Eustigmatophyceae. This is the first report of the photosynthetic light-harvesting function of this carotenoid.     

Lipid accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2 aeration

In order to produce microalgal lipids that can be transformed to biodiesel fuel, effects of concentration of CO(2) aeration on the biomass production and lipid accumulation of Nannochloropsis oculata in a semicontinuous culture were investigated in this study. Lipid content of N. oculata cells at different growth phases was also explored. The results showed that the lipid accumulation from logarithmic phase to stationary phase of N. oculata NCTU-3 was significantly increased from 30.8% to 50.4%. In the microalgal cultures aerated with 2%, 5%, 10% and 15% CO(2), the maximal biomass and lipid productivity in the semicontinuous system were 0.480 and 0.142 g L(-1)d(-1) with 2% CO(2) aeration, respectively. Even the N. oculata NCTU-3 cultured in the semicontinuous system aerated with 15% CO(2), the biomass and lipid productivity could reach to 0.372 and 0.084 g L(-1)d(-1), respectively. In the comparison of productive efficiencies, the semicontinuous system was operated with two culture approaches over 12d. The biomass and lipid productivity of N. oculata NCTU-3 were 0.497 and 0.151 g L(-1)d(-1) in one-day replacement (half broth was replaced each day), and were 0.296 and 0.121 g L(-1)d(-1) in three-day replacement (three fifth broth was replaced every 3d), respectively. To optimize the condition for long-term biomass and lipid yield from N. oculata NCTU-3, this microalga was suggested to grow in the semicontinuous system aerated with 2% CO(2) and operated by one-day replacement.     

Seasonal changes in lipid content and composition of the polychaete Nereis (Hediste) diversicolor

The lipid content and composition of Nereis (Hediste) diversicolor O. F. Müller (Annelida, Polychaeta, Nereidae) a mud-dwelling, intertidal errant polychaete in the Tagus estuary (Portugal), were examined on the monthly basis by lipid extraction, TLC and capillary GC. In this estuary, N. diversicolor is by far the dominant species among polychaeta and the main food item in the natural diet of several flatfishes. The biochemical elucidation of its lipid structure and distribution throughout the year, described in this study, provides information not only about the physiological role of lipids in the animal under consideration but also about dietary fatty acid requirements of some flatfishes in the wild and under laboratory conditions.The total lipid content varied between a maximum of 19.3% lyophilized dry weight in February (4.4% fresh weight) and a minimum of 6.6% in August (1.9% fresh weight). The major lipid classes were triacylglycerol, phospholipid, free sterol, free fatty acid, sterol ester/wax ester and alkyldiacylglycerol.The fatty acid composition was rather unsaturated with a 1:2 mean ratio of n-3: n-6. The major fatty acids were C160:0, C18:1n-9, C18:2n-6, and C20:5n-3; there were smaller amounts of C180:0, C18:1n-11, C18:1n-7, C18:3n-3, C20:1, C20:2n-6, C20:4n-6, C22:2, C22:5n-3, and many other fatty acids were detected at trace levels. The unsaturation ranged from 36.9 mg/g dry weight in summer to 107.4 mg/g in winter. An accumulation of fatty acids from plant origin was evident, in particular linoleic acid (C18:2n-6), which was quantitatively one of the major fatty acids throughout the year.     

BIOCHEMICAL COMPOSITION AND FATTY ACID CONTENT OF FILAMENTOUS NITROGEN-FIXING CYANOBACTERIA

The biochemical composition and fatty acid content of twelve strains of filamentous, heterocystous, nitrogen-fixing cyanobacteria have been determined. When grown under diazotrophic conditions, protein, carbohydrate, lipid, and nucleic acids comprised 37–52%, 16–38%, 8–13%, and 8–11% of the dry weight, respectively. The presence of a combined nitrogen source resulted in an increase in the protein content of the cells and a decrease in the levels of lipids and carbohydrates, although biomass productivity was not affected significantly. Biochemical composition also changed during culture growth, with the highest levels of proteins and lipids occurring as the culture entered stationary phase, whereas the highest levels of carbohydrate and nucleic acids were found during the exponential phase. Total fatty acid levels in the strains assayed ranged between 3 and 5.7% of the dry weight. With regard to fatty acid composition, all strains showed high levels of polyunsaturated fatty acids (PUFAs) and saturated fatty acids (SAFAs), with values of 24–45% and 31–52% of total fatty acids, respectively, whereas the levels of monounsaturated fatty acids (MUFAs) were in general lower (11– 32%). Palmitic acid (16:0) was the most prevalent SAFA, whereas palmitoleic (16:1n- 7) and oleic acid (18:1n-9) were the most abundant MUFAs in all the strains. Among PUFAs, γ-linolenic acid (GLA, 18:3n-6) was present at high levels (18% of total fatty acids) in Nostoc sp. (Chile) and at lower levels (3.6% of total fatty acids) in Anabaenopsis sp. The presence of GLA has not been previously reported in these genera of cyanobacteria. The rest of the strains exhibited high levels (12–35% of total fatty acids) of α-linolenic acid (ALA, 18:3n-3). Linoleic acid (18:2n-6) was also present at a substantial level in most of the strains. Eicosapentaenoic acid (EPA, 20:5n-3) was also detected in Nostoc sp. (Albufera). Some filamentous nitrogen-fixing cyanobacteria therefore represent potential sources of commercially interesting fatty acids.     

Characterization of the fatty acid composition of Nannochloropsis salina as a determinant of biodiesel properties

Nannochloropsis salina was cultured batch-wise to evaluate the potential of the alga to produce biodiesel. The cells were harvested at the end of the exponential growth phase when the concentration was 18 x 10(6) cells/mL culture. The growth estimated as dry weight from this cell number was (3.8 +/- 0.7) mg/L. The lipid and triglyceride contents were 40% and 12% on a dry weight basis, respectively. The amount of the ratio triglycerides/total lipids was approximately 0.3. The composition of triglyceride fatty acid methyl esters (biodiesel) was analysed by gas-liquid chromatography and identified as: C14:0, C16:0, C16:1, C18:0, C18:1, C18:2, C18:3, C20:1, and C20:5. The ratio of unsaturated to saturated fatty acid contents was approximately 4.4. Additionally, the characterization of each individual fatty acid ester was discussed with regard to the fuel properties of biodiesel produced by the alga.     

Cryptophyceae and rhodophyceae; chemotaxonomy, phylogeny, and application

The biochemical compositions of seven strains of marine cryptomonad and a rhodophyte were determined in logarithmic phase batch (1.4 L flask) and semi-continuous (10 L carboy) culture. Lipid ranged from 13% to 28%, protein ranged from 53% to 68%, and carbohydrate ranged from 9% to 24% of the organic weight. The major lipid classes in the species examined were polar lipids (78-88% of total lipid). The major sterol in the Cryptophyceae and the Rhodophyceae was 24-methylcholesta-5,22E-dien-3beta-ol (62-99% of total sterols); which is also the major sterol in some diatoms and haptophytes. Smaller proportions of cholest-5-en-3beta-ol (1-17.7%) were also found in the Cryptophyceae. Most cryptomonads contained high proportions of the n-3 polyunsaturated fatty acids (PUFA), 18:3n-3 (20.7-29.9% of the total fatty acids), 18:4n-3 (12.5-30.2%), 20:5n-3 (7.6-13.2%) and 22:6n-3 (6.4-10.8%). However, the blue-green cryptomonad Chroomonas placoidea was characterized by a low proportion of 22:6n-3 (0.2% of total fatty acids), and a significant proportion of 22:5n-6 (4.5%), and the presence of 24-ethylcholesta-5,22E-dien-3beta-ol (35.5% of total sterols). The fatty acid composition of the rhodophyte Rhodosorus sp. was similar to those of the Cryptophyceae except for lower proportions of 18:4n-3 and lack of C21 and C22 PUFA. It is postulated that the primary endosymbiosis of a photosynthetic n-3 C18 PUFA-producing prokaryote and a eukaryotic host capable of chain elongation and desaturation of exogenous PUFA, resulted in the Rhodophyceae capable of producing n-3 C20 PUFA. The secondary endosymbiosis of a photosynthetic n-3 C20 PUFA-producing eukaryote (such as a Rhodosorus sp. like-rhodophyte) and a eukaryotic host capable of further chain elongation and desaturation, resulted in the Cryptophyceae being capable of producing n-3 C20 and C22 PUFA de novo. Selected isolates were examined further in feeding trials with juvenile Pacific oysters (Crassostrea gigas). Rhodomonas salina CS-24(containing elevated 22:6n-3) produced high growth rates in oysters; equivalent to the microalga commonly used in aquaculture, Isochrysis sp. (T.ISO).     

The effect of growth phase on the lipid class,fatty acid and sterol composition in the marine dinoflagellate,Gymnodinium sp. in batch culture

We have studied the effects of growth phase on the lipid composition in batch cultures of Gymnodinium sp. CS-380/3 over 43 days of culturing. The lipid content increased two fold, from late logarithmic (day 6) to linear growth phase (day 22) then decreased at stationary phase (day 43) while the lipid yield (mg l(-1)) increased 30-fold from day 6 to 30 mg l(-1) at day 43. Changes in fatty acid content mirrored those observed for the total lipid, while the sterol content continued to increase with culture age through to stationary phase. The largest changes occurred in the lipid classes, especially the polar lipids and triacylglycerols (oil). The proportion of triacylglycerols increased from 8% (of total lipids) at day 6 to 30% at day 43, with a concomitant decrease in the polar lipid fraction. The proportions of 16:0 and DHA [22:6(n-3)] increased while those of 18:5(n-3) and EPA [20:5(n-3)] decreased with increasing culture age. The proportion of the major sterol, dinosterol, decreased from 41% (day 6) to 29% (day 43), while the major dinostanol epimer (23R,24R) increased from 33% (day 6) to 38% (day 22). Despite small changes in the proportion of the main sterols, the same sterols were present at all stages of growth, indicating their value as a chemotaxonomic tool for distinguishing between strains within the same genus. Growth phase could be a useful variable for optimising the oil and DHA content with potential for aquaculture feeds and a source of DHA-rich oils for nutraceuticals.     

PIGMENTS,FATTY ACIDS,AND STEROLS OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM1

Cultures and field samples of the toxic dinoflagellate Gymnodinium catenatum Graham from Tasmania, Australia, were analyzed for pigment, fatty acid, and sterol composition. Gymnodinium catenatum contained the characteristic pigments of photosynthetic dinoflagellates, including chlorophyll a, chlorophyll c₂, and the carotenoids peridinin, dinoxanthin, diadinoxanthin, diatoxanthin, and β,β-carotene. In midlogarithmic and early stationary phase cultures, the chlorophyll a content ranged 50–72 pg · cell^?1, total lipids 956–2084 pg · cell^?1, total fatty acids 426–804 pg · cell^?1, and total sterols 8–20 pg · cell^?1. The major fatty acids (in order of decreasing abundance) were 16:0, 22:6(n-3), and 20:5(n-3) (collectively 65–70% of the total fatty acids), followed by 16:1(n-7), 18:2(n-6), and 14:0. This distribution is characteristic of most dinoflagellates, except for the low abundance (<3%) of the fatty acid 18:5(n-3), considered by some authors to be a marker for dinoflagellates. The three major sterols were 4α-methyl-5α-cholest-7-en-3β-ol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (the dinoflagellate sterol, dinosterol), and 4α,23,24-trimethyl-5α-cholest-7-en-3β-ol. These three sterols comprised about 75% of the total sterols in both logarithmic and early stationary phase cultures, and they were also found in high proportions (22–25%) in natural dinoflagellate bloom samples. 4-Desmethyl sterols, which are common in most microalgae, were only present in trace amounts in G. catenatum. The chemotaxonomic affinities of G. catenatum and the potential for using specific signature lipids for monitoring toxic dinoflagellate blooms are discussed.     

THE FATTY ACID AND STEROL COMPOSITION OF FIVE MARINE DINOFLAGELLATES

The fatty acid and sterol compositions of five species of marine dinoflagellates (Scrippsiella sp. Symbiodinium microadriaticum Freud, Gymnodinium sp., Gymnodinium sanguineum Hirasaki, and Fragilidium sp.) are reported. All contained the major fatty acids that are considered common in dinoflagellates, but the proportions were quite variable, and some species contained low contents of some polyunsaturated fatty acids. Concentration ranges for the major fatty acids were: 16:0 (9.0%–24.8%), 18:4(n-3) (2.5%–11.5%), 18:5(n-3) (7.0%–43.1%), 20:5(n-3) (EPA) (1.8%–20.9%), and 22:6(n-3) (DHA) (9.9%– 26.3%). Small amounts of novel very-long-chain highly unsaturated C₂₈ fatty acids occurred in all species. Each dinoflagellate contained a complex mixture of 4-methyl sterols and 4-desmethyl sterols. Four species contained cholesterol, although the amounts were highly variable (from 0.2% of total sterols in Scrippsiella sp. to 45.6% in Fragilidium sp.). All but G. sanguineum contained the 4-methyl sterol dinosterol, and all species contained sterols lacking a double bond in the ring system (i.e. stanols); in Scrippsiella sp. cholestanol composed 24.3% of the total sterols. Other common features of the 4-methylsterol profiles were the presence of 23,24-dimethyl alkylation and unsaturation at Δ²² in the side chain. In Scrippsiella sp., four steroidal ketones were identified: cholestanone, dinosterone, 4α,23,24-trimethyl-5α-cholest-8(14)-en-3-one, and dinostanone. The structures of these corresponded to the major sterols in this species, suggesting that the sterols and steroidal ketones are biosynthetically linked. Steroidal ketones were not detected in the other species. Although fatty acid profiles can be used to distinguish among algal classes, they were not useful for differentiating among dinoflagellate species. In contrast, whereas some taxonomic groupings of dinoflagellates display similar sterol patterns, others, such as the gymnodinoids studied here, clearly do not. The combination of fatty acid, sterol, and steroidal ketone profiles may be useful complementary chemotaxonomic tools for distinguishing morphologically similar species. The identification of steroidal ketones supports earlier suggestions that certain dinoflagellates might be a significant source of such components in marine environments.     

Cryopreservation of a marine microalga, Nannochloropsis oculata (Eustigmatophyceae)

The cryopreservation of algae could prevent genetic drift and minimize labor costs compared to the current method of maintenance and subculturing. Clear, simple protocols for cryopreservation of marine microalga, Nannochloropsis oculata were developed and cryoprotectant choice and concentration optimized. The viability of the microalga was assessed directly after thawing, and algal concentration was measured after 2-30 days of growth. Five cryoprotectants (dimethyl sulphoxide, Me2SO; ethylene glycol, EG; glycerol, Gly; methanol, MeOH; and propylene glycol, PG) at five concentrations (10, 20, 30, 40, and 50%; v/v) were evaluated to determine the toxicity of various cryoprotectants to N. oculata. The toxicity of cryoprotectant (Me2SO, EG, MeOH, and PG) was observed only at higher concentrations of CPAs: > 20% for EG, > 30% for Me2SO and methanol, and > 40% for PG. Direct freezing of algae in liquid nitrogen resulted in a severe loss of viability and a modified cryopreservation protocol proved to be more appropriate for the preservation of N. oculata. Cryopreservation protocols developed and tested in the present study might be applied to cryopreserving other strains, or species, in this genus.