Maged P. Mansour

Microalgal biomarkers: A review of recent research developments

Abstract Microalgae are major sources of lipids in lacustrine and marine environments. This paper provides a review of some recent advances in our knowledge of the wide variety of lipid types that have been isolated from microalgae with an emphasis on those likely to be useful biomarkers for identifying sources of organic matter in sediments. Extensive data are now available on the fatty acids in all of the major classes of microalgae and some useful characteristic features have been observed in the abundance of particular polyunsaturated fatty acids. Despite several decades of study, it is now apparent that some of the biosynthetic steps leading to the formation of these unsaturated fatty acids are still not known with certainty as shown by the occurrence of C 28 polyunsaturated fatty acids in some dinoflagellates and the likely involvement of chain-shortening reactions. Considerable data have also been obtained on the sterols in microalgae, but some classes of organisms are still not well documented (e.g. cryptomonads, eustigmatophytes, xanthophytes and raphidophytes). Diatoms show a great variety of sterol compositions and no sterol appears to be either unique or representative. However, 24-methylenecholesterol in sediments is probably derived in most cases from diatoms. High contents of C 25 highly branched isoprenoid (HBI) alkenes have been identified in the diatom Haslea ostrearia and both C 25 and C 30 HBI alkenes have been found in diatom strains thought to be Rhizosolenia setigera . Genetic and environmental factors appear to be important controls on the relative abundances of the various homologues identified. Microalgae are also suspected to be a source of long-chain saturated fatty acids having an even carbon number predominance and of long-chain alkanes with no odd over even carbon number predominance, although the available data are not conclusive. An exciting development in recent years is the identification of highly aliphatic biopolymers (algaenans) in some species of marine and freshwater green algae and eustigmatophytes. This material persists in sediments and may be a source of the alkyl chains in ancient kerogens and crude oil constituents. Algaenans do not occur in all algal species and may be absent from some classes, such as diatoms. This implies that the organic matter preserved in sediments is strongly influenced by a subset of the microalgal contributors of organic matter. Although reasonable sources have been identified for many of the lipids in sediments, there are still many gaps in our knowledge and further studies are clearly required.

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.

Lipid and fatty acid yield of nine stationary-phase microalgae : Applications and unusual C24-C28 polyunsaturated fatty acids

Nine microalgal species from the classes Bacillariophyceae, Cryptophyceae, Prymnesiophyceae and Dinophyceae were isolated from Australian waters, cultured to stationary phase and analyzed for their lipid and fatty acid composition and yield. Five species (Pavlova pinguis, Heterocapsa niei, Proteomonas sulcata, Navicula jeffreyi and Thalassiosira pseudonana) produced high proportions of triacylglycerol (TAG: 22–57% total lipid). An unidentified Navicula-like diatom (CS-786), despite having a low TAG content, had the highest EPA yield (5.8 mg L−1), due to high biomass and a high relative proportion of EPA. Heterocapsa niei had the highest DHA yield (2.9 mg L−1), due to a high cellular lipid and DHA content (171 pg cell−1 and 13.7 pg cell−1, respectively) despite its relatively low biomass. The desirable PUFA composition and yield of both diatom CS-786 and H. niei make them potential candidates for optimization of biomass and PUFA production for use as live-feeds in aquaculture. In addition, H. niei may have potential as a source of DHA for other uses. Low proportions (< 1.2%) of 24:6(n−3) accompanied by trace proportions of 24:5(n−6) were detected in most strains, while 28:8(n−3) was found in dinoflagellates and also in the prymnesiophyte P. pinguis. All non-diatomaceous species contained 26:7(n−3) in minor quantities. This is the first time these unusual C24 and C26 PUFA have been reported in microalgae and the first report of C28 PUFA in a microalga other than dinoflagellates. Possible biosynthetic reasons why these might occur in stationary phase cultures are considered and the likely dietary transfer of these PUFA to higher aquatic life is discussed.

Metabolic Engineering Camelina sativa with Fish Oil-Like Levels of DHA

Background Omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) such as eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) are critical for human health and development. Numerous studies have indicated that deficiencies in these fatty acids can increase the risk or severity of cardiovascular, inflammatory and other diseases or disorders. EPA and DHA are predominantly sourced from marine fish although the primary producers are microalgae. Much work has been done to engineer a sustainable land-based source of EPA and DHA to reduce pressure on fish stocks in meeting future demand, with previous studies describing the production of fish oil-like levels of DHA in the model plant species, Arabidopsis thaliana. Principal Findings In this study we describe the production of fish oil-like levels (>12%) of DHA in the oilseed crop species Camelina sativa achieving a high ω3/ω6 ratio. The construct previously transformed in Arabidopsis as well as two modified construct versions designed to increase DHA production were used. DHA was found to be stable to at least the T5 generation and the EPA and DHA were found to be predominantly at the sn-1,3 positions of triacylglycerols. Transgenic and parental lines did not have different germination or seedling establishment rates. Conclusions DHA can be produced at fish oil-like levels in industrially-relevant oilseed crop species using multi-gene construct designs which are stable over multiple generations. This study has implications for the future of sustainable EPA and DHA production from land-based sources.

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.

Very-long-chain (C28) highly unsaturated fatty acids in marine dinoflagellates

Very-long-chain (C28) highly unsaturated fatty acids (VLC-HUFA), octacosaheptaenoic acid (28:7(n-6)(4,7,10,13,16,19,22)) and octacosaoctaenoic acid (28:8(n-3)(4,7,10,13,16,19,22,25)) were identified in seven marine dinoflagellate species: Prorocentrum mexicanum, P. micans, Scrippsiella sp., Symbiodinium microadriaticum, Gymnodinium sp., G. sanguineum and Fragilidium sp. The proportion of these fatty acids accounted for less than 2.3% of the total fatty acids in these species. The main VLC-HUFA in S. microadriaticum was 28:7(n-6); in the other species it was 28:8(n-3), with Scrippsiella sp., Gymnodinium sp. and Fragilidium sp. containing only 28:8(n-3) and Gymnodinium sanguineum containing a small proportion of 28:7(n-6) in addition to 28:8(n-3). These findings show that some marine dinoflagellates have the ability to produce VLC-HUFA, but precursors of these VLC-HUFA with chain lengths between 22 and 28 carbon atoms were below the detection level. Both 28:7(n-6) and 28:7(n-3), as well as other VLC-HUFAs, have been detected previously in Baltic herring; our results suggest that they may have originated from microalgae.

Sterols of four dinoflagellates from the genus Prorocentrum

Abstract The compositions of 4-desmethyl sterols and 4-methyl sterols in four species of marine dinoflagellates of the genus Prorocentrum (viz., P. micans Ehrenberg, P. minimum (Pavillard) Schiller, P. balticum (Lev.) Lemm and P. mexicanum Tafall) were identified by capillary gas chromatography–mass spectrometry as part of a study to identify signature lipids for dinoflagellates in marine organic matter. Complex mixtures were found in each species with over 20 sterols identified in all. All species contained the same core group of sterols, but there were significant differences in the proportions of the various sterols. Two distinct groupings could be discerned in the sterol patterns. The 4-methyl sterol 4α,23,24-trimethyl-5α-cholest-22 E -en-3β-ol (dinosterol), which is common in many dinoflagellates, predominated in P. balticum and in P. minimum whereas in the closely related species P. micans and P. mexicanum the major sterol was cholesterol. A novel monounsaturated C 23 sterol having a much shortened side-chain was found in P. balticum and P. minimum and both P. balticum and P. minimum contained peridinosterol (4α,23,24-trimethyl-5α-cholest-17(20)-en-3β-ol). 24-Methylenecholesterol was only found in P. minimum , where it comprised over one-third of the sterols. The steroid ketone dinosterone occurred in P. balticum , but none of the other species contained steroid ketones. Although all the sterol distributions were broadly similar, the presence or absence of specific components might be a useful chemotaxonomic tool for distinguishing between closely related species.