Susan I. Blackburn

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.

Alkenones in Gephyrocapsa oceanica: Implications for studies of paleoclimate

Abstract Emiliania huxleyi is widely regarded as the most likely source of C 37 C 39 alkenones in present-day seawater and Recent sediments, but other sources are required to account for the presence of alkenones in sediments that predate the first occurrence of E. huxleyi about 265,000 years ago. Analysis of the lipids of a laboratory culture of the closely related marine coccolithophorid Gephyrocapsa oceanica (strain JB02) isolated from a massive bloom in Jervis Bay, eastern Australia showed that this species also synthesizes C 37 C 39 alkenones and esters of di- and tri-unsaturated C 36 fatty acids. This confirms earlier predictions based on the fossil record that species of Gephyrocapsa should contain these distinctive biomarkers. In this strain of G. oceanica the sum of the C 38 ethyl ketone concentrations is similar to, or greater than, that of the C 37 methyl ketones over the temperature range 11–29°C, whereas the reverse is true for Emiliania huxleyi . It should thus be possible to determine whether there is a contribution from Gephyrocapsa to the alkenones in seawater and sediments. The concentrations per cell of the major alkenones showed different responses to increasing growth temperature. The 37:3 and 38:3 methyl alkenones and 38:3 ethyl alkenone showed an approximately linear decrease in cellular concentration over the entire temperature range, whereas the 37:2 and 38:2 methyl alkenones and 38:2 ethyl alkenone concentrations showed almost no change from 11 to 20°C followed by a dramatic increase above 20°C. As a result, the ratio of di- to tri-unsaturated methyl alkenones as measured by U 37 k ′ changes greatly with growth temperature, but the is different from that found for E. huxleyi in culture. The temperature response can be approximated by the linear relationship U 37 k ′ = 0.049 T − 0.520 ( r 2 = 0.89), although a better fit can be obtained using polynomial expressions. These data might account for some of the apparent anomalies in predictions of sea surface temperature (SST) derived from Emiliania -based alkenone-SST calibrations in those sediments which contain contributions from Gephyrocapsa .

Eustigmatophyte microalgae are potential sources of C29 sterols, C22–C28 n-alcohols and C28–C32 n-alkyl diols in freshwater environments

The neutral lipids of three freshwater microalgae from the class Eustigmatophyceae were examined to investigate possible algal sources of lipids in lacustrine sediments. The major neutral lipids in Eustigmatos vischeri (Hulbert) Taylor, Vischeria helvetica (Vischer et Pascher) Taylor and Vischeria punctata Vischer were sterols, long-chain alcohols and alkyl diols. The distributions of long-chain n-alkanols and n-alkenols were very similar in each species, but there were small interspecies differences in the proportions of alkyl diols. Saturated alcohols ranged from 14:0 to 28:0 (both present in trace amounts), with 22:0 as the major alkanol. The latter has previously been reported to be the major n-alcohol in some lacustrine sediments. C16–C30 monounsaturated alcohols were more abundant: 26:1 and 28:1 were the major constituents. The sterol distributions consisted predominantly of 24-ethylcholesterol with small amounts of cholesterol, 24-methylcholesterol, 24-ethylcholesta-5,22E-dien-3β-ol and isofucosterol. These results add to the growing list of microalgae that contain a high proportion of 24-ethylcholesterol, which is more typically associated with higher plants. The long-chain alkyl diols ranged from C28 to C32: the major constituents were the C28 1,13-diol and C30 and C32 1,15 diols which occurred together with smaller amounts of other positional isomers. C30 and C32 alkyl diols have been found in marine eustigmatophytes of the genus Nannochloropsis, and these new results confirm that the same class of compounds occurs in freshwater eustigmatophytes.

Isolation and purification of Australian isolates of the toxic cyanobacterium Microcystis aeruginosa Kutz

Isolation and laboratory culture ofMicrocystis aeruginosa Kütz. using a growth medium (MLA medium) suitable for both non-axenic and axenic cultures is described. Seventeen established strains ofM. aeruginosa were subjected to one or more of three purification methods: centrifugation cleaning, sulphide gradient selection, and antibiotic treatment (Imipenem®). While each method purified only about half of the strains attempted, the selective application of each method, based on the morphological characteristics of the strains, succeeded in purifying 12 of the 17 strains. Three of the 5 strains not purified were contaminated with a sulphide-tolerant, Imipenem-resistant spirochaete,Spirochaeta cf.aurantia, which could not be detected on normal, broad spectrum bacterial test media. The presence of this bacterial species was detected only by phase contrast and DAPI (4,6-diamidino-2-phenylindole) stained fluorescence microscopy.

Vegetative reproduction and sexual life cycle of the toxic dinoflagellate Gymnodinium catenatum from Tasmania, Australia

The toxic, chain‐forming dinoflagellate Gymnodinium catenatum Graham was cultured from vegetative cells and benthic resting cysts isolated from estuarine waters in Tasmania, Australia. Rapidly dividing, log phase cultures formed long chains of up to 64 cells whereas stationary phase cultures were composed primarily of single cells (23‐41 pm long, 27‐36 pm wide). Vegetative growth (mean doubling time 3‐4 days) was optimal at temperatures from 14.5‐20° C, salinities of 23‐34% and light irradiances of 50‐300 μE·m−2·s−1.

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.

Phylogenetic and functional diversity of the cultivable bacterial community associated with the paralytic shellfish poisoning dinoflagellate Gymnodinium catenatum

Gymnodinium catenatum is one of several dinoflagellates that produce a suite of neurotoxins called the paralytic shellfish toxins (PST), responsible for outbreaks of paralytic shellfish poisoning in temperate and tropical waters. Previous research suggested that the bacteria associated with the surface of the sexual resting stages (cyst) were important to the production of PST by G. catenatum. This study sought to characterise the cultivable bacterial diversity of seven different strains of G. catenatum that produce both high and abnormally low amounts of PST, with the long-term aim of understanding the role the bacterial flora has in bloom development and toxicity of this alga. Sixty-one bacterial isolates were cultured and phylogenetically identified as belonging to the Proteobacteria (70%), Bacteroidetes (26%) or Actinobacteria (3%). The Alphaproteobacteria were the most numerous both in terms of the number of isolates cultured (49%) and were also the most abundant type of bacteria in each G. catenatum culture. Two phenotypic (functional) traits inferred from the phylogenetic data were shown to be a common feature of the bacteria present in each G. catenatum culture: firstly, Alphaproteobacteria capable of aerobic anoxygenic photosynthesis, and secondly, Gammaproteobacteria capable of hydrocarbon utilisation and oligotrophic growth. In relation to reports of autonomous production of PST by dinoflagellate-associated bacteria, PST production by bacterial isolates was investigated, but none were shown to produce any PST-like toxins. Overall, this study has identified a number of emergent trends in the bacterial community of G. catenatum which are mirrored in the bacterial flora of other dinoflagellates, and that are likely to be of especial relevance to the population dynamics of natural and harmful algal blooms.

Comparative study on paralytic shellfish toxin profiles of the dinoflagellate Gymnodinium catenatum from three different countries

Paralytic shellfish toxin profiles of the dinoflagellate Gymnodinium catenatum Graham were investigated as a possible biochemical marker to distinguish different geographic populations of this species. Isolates obtained between 1986 and 1988 from Japan, Tasmania (Australia) and Galicia (Spain) were cultured under similar conditions and the toxins produced were analyzed using HPLC. Variations in temperature, salinity, and nitrate and phosphate levels in the culture medium had no significant effect on the toxin profile, suggesting that toxins can be used as a stable biochemical marker for this dinoflagellate. All the isolates produced mainly toxins of the N-sulfocarbamoyl group (C1–C4, gonyautoxins 5 and 6) but their relative abundance differed according to their geographic origin. Furthermore, only the Australian population produced the newly found 13-deoxydecarbamoyl toxins, and these could readily be used to distinguish the Australian populations from those of the other two countries.

Metabolic engineering of Arabidopsis to produce nutritionally important DHA in seed oil

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are nutritionally important long-chain (≥ C20) omega-3 polyunsaturated fatty acids (ω3 LC-PUFA) currently obtained mainly from marine sources. A set of genes encoding the fatty acid chain elongation and desaturation enzymes required for the synthesis of LC-PUFA from their C18 PUFA precursors was expressed seed-specifically in Arabidopsis thaliana. This resulted in the synthesis of DHA, the most nutritionally important ω3 LC-PUFA, for the first time in seed oils, along with its precursor EPA and the ω6 LC-PUFA arachidonic acid (ARA). The assembled pathway utilised Δ5 and Δ6 desaturases that operate on acyl-CoA substrates and led to higher levels of synthesis of LC-PUFA than previously reported with acyl-PC desaturases. This demonstrates the potential for development of land plants as alternative sources of DHA and other LC-PUFA to meet the growing demand for these nutrients.

Dinoflagellate Gymnodinium catenatum as the source of paralytic shellfish toxins in Tasmanian shellfish

Paralytic shellfish toxins in both cultured cells and natural phytoplankton blooms of the dinoflagellate Gymnodinium catenatum from inshore Tasmanian waters (Australia) were analyzed by high performance liquid chromatography, thin layer chromatography and electrophoresis techniques. The dinoflagellate toxins were dominated by low potency sulfocarbamoyl saxitoxin derivatives (98-99 mole% in total), including gonyautoxin VIII (C2) and its epimer (C1) and sulfocarbamoyl gonyautoxins I and IV (C3 and C4). Mussels and oysters contaminated by the dinoflagellate showed similar toxins, but contained larger proportions of C3 (40-57 mole%) and more potent carbamate toxins (7-23 mole% total).