Characterisation and application of Australian thraustochytrids

Aspects of the taxonomy, lipid production and aquacultural application of several new polyunsaturated fatty acid (PUFA)-producing microheterotrophs were examined sing 18S ribosomal DNA sequencing and fatty acid, sterol and lipid class analyses. Thraustochytrid-like organisms were isolated from marine environments in southeastern Tasmania (Australia). A high proportion of strains produced PUFA, especially eicosapentaenoic acid (EPA, 20:5n3) and docosahexaenoic acid (DHA, 22:6n3). The phylogenetic relationships of Tasmanian and type strains were compared, using 18S rDNA sequences and fatty acid profiles. The effect of different extraction techniques on the recovery of fatty acids from two Tasmanian thraustochytrid strains (ACEM 6063 and ACEM 000A) was examined. Two procedures were used: the extraction of lipids from biomass followed by transesterification of the fatty acids (extraction-transesterification); and the direct transesterification of biomass to produce fatty acid methyl esters (i.e. without the initial extraction step). Results showed that the most efficient direct transesterification method yielded significantly more fatty acids than the most efficient extraction-transesterification method. Production of biomass and lipid by strain ACEM 6063 under different culture conditions was examined. Biomass production increased with increasing glucose and sea salt concentration, culture temperature, and with low fermenter impeller speeds. The predominant lipid classes were triacylglycerols and phospholipids. The proportion of triacylglycerols increased, and the proportion of phospholipids decreased, with culture age. Lipid content increased with increasing culture age, temperature, glucose and sodium glutamate concentration and dissolved oxygen (DO). Increasing DO levels substantially decreased the proportion of saturated fatty acids and substantially increased the proportion of monounsaturated fatty acids. Twenty sterols, 13 of which were identified, were detected. Predominant were: cholest-5-en-3-ol, 24-ethylcholesta-5,22E-dien-3-ol, 24-methylcholesta-5,22E-dien-3-ol, and two co-eluting sterols — one of which was 24-ethylcholesta-5,7,22-trien-3- ol. Culture age, temperature and DO influenced squalene and sterol content. Total sterols (as a proportion of total lipids) decreased with increasing culture age. The ability of PUFA-producing thraustochytrids to enrich rotifers (Brachionus plicatilis) in these PUFA was examined, and compared to that of two previously isolated PUFA-producing bacteria, which were rich in either EPA or DHA. Enrichment with the EPA-producing bacterium only resulted in rotifer EPA levels increasing from 0.1% to 1.2% of total dry weight (%dw). Similarly, following enrichment with the DHA-producing bacterium only, rotifer DHA levels increased from below detection to 0.1 %dw. When enriched with a mixture of the two bacterial strains, final rotifer EPA- and DHA-levels were 0.5 %dw and 0.3 %dw respectively. When thraustochytrid strains ACEM 6063 and ACEM 000A were fed to rotifers, no significant differences were found between the fatty acid profile of rotifers enriched with thraustochytrids preserved by different treatments. Significant differences were observed between rotifers fed biomass from individual thraustochytrid strains or a mixture of the two strains. With both strains, the sum of arachidonic acid (AA, 20:4n6), EPA and DHA in rotifers enriched for 6 h reached 1.5-2 %dw in most treatments. There was little change in fatty acid content of rotifers enriched for 24 h compared to 6 h. Rotifers enriched with thraustochytrids contained increased levels of PUFA compared to rotifers enriched with bacteria. Strain specific variation in the PUFA content of the thraustochytrid strains also allowed the PUFA content of thraustochytrid-enriched rotifers to be manipulated to a greater degree than that of bacteria-enriched rotifers. Following 24 h post-enrichment starvation, levels of AA, EPA and DHA in most thraustochytrid-enriched rotifers were still significantly higher than in continuously starved and yeast-fed rotifers, indicating that rotifers had digested and assimilated the microheterotroph fatty acids. The two thraustochytrid strains are considered to be suitable for use in Australian aquaculture, as they provide enrichment levels comparable to those achieved with currently available commercial enrichment diets.