Comparison of Thraustochytrids Aurantiochytrium sp., Schizochytrium sp., Thraustochytrium sp., and Ulkenia sp. for production of biodiesel, long-chain omega-3 oils, and exopolysaccharide.

Heterotrophic growth of thraustochytrids has potential in coproducing biodiesel for transportation, as well as producing a feedstock for omega-3 long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), especially docosahexaenoic acid (DHA) for use in nutraceuticals. In this study, we compared eight new endemic Australian thraustochytrid strains from the genera Aurantiochytrium, Schizochytrium, Thraustochytrium, and Ulkenia for the synthesis of exopolysaccharide (EPS), in addition to biodiesel and LC-PUFA. Aurantiochytrium sp. strains readily utilized glucose for biomass production, and increasing glucose from 2 to 4 % w/v of the culture medium resulted in increased biomass yield by an average factor of 1.7. Ulkenia sp. strain TC 010 and Thraustochytrium sp. strain TC 033 did not utilize glucose, while Schizochytrium sp. strain TC 002 utilized less than half the glucose available by day 14, and Thraustochytrium sp. strain TC 004 utilized glucose at 4 % w/v but not 2 % w/v of the culture suggesting a threshold requirement between these values. Across all strains, increasing glucose from 2 to 4 % w/v of the culture medium resulted in increased total fatty acid methyl ester content by an average factor of 1.9. Despite an increasing literature demonstrating the capacity of thraustochytrids for DHA synthesis, the production of EPS from these organisms is not well documented. A broad range of EPS yields was observed. The maximum yield of EPS was observed for Schizochytrium sp. strain TC 002 (299 mg/L). High biomass-producing strains that also have high lipid and high EPS yield may be better candidates for commercial production of biofuels and other coproducts.

High cell density cultivation of a novel Aurantiochytrium sp. strain TC 20 in a fed-batch system using glycerol to produce feedstock for biodiesel and omega-3 oils.

A recently isolated Australian Aurantiochytrium sp. strain TC 20 was investigated using small-scale (2 L) bioreactors for the potential of co-producing biodiesel and high-value omega-3 long-chain polyunsaturated fatty acids. Higher initial glucose concentration (100 g/L compared to 40 g/L) did not result in markedly different biomass (48 g/L) or fatty acid (12-14 g/L) yields by 69 h. This comparison suggests factors other than carbon source were limiting biomass production. The effect of both glucose and glycerol as carbon sources for Aurantiochytrium sp. strain TC 20 was evaluated in a fed-batch process. Both glucose and glycerol resulted in similar biomass yields (57 and 56 g/L, respectively) by 69 h. The agro-industrial waste from biodiesel production-glycerol-is a suitable carbon source for Aurantiochytrium sp. strain TC 20. Approximately half the fatty acids from Aurantiochytrium sp. strain TC 20 are suitable for development of sustainable, low emission sources of transportation fuels and bioproducts. To further improve biomass and oil production, fortification of the feed with additional nutrients (nitrogen sources, trace metals and vitamins) improved the biomass yield from 56 g/L (34 % total fatty acids) to 71 g/L (52 % total fatty acids, cell dry weight) at 69 h; these yields are to our knowledge around 70 % of the biomass yields achieved, however, in less than half of the time by other researchers using glycerol and markedly greater than achieved using other industrial wastes. The fast growth and suitable fatty acid profile of this newly isolated Aurantiochytrium sp. strain TC 20 highlights the potential of co-producing the drop-in biodiesel and high value omega-3 oils.

Evaluation of growth, nutrient utilization and production of bioproducts by a wastewater-isolated microalga.

Treatment of wastewater while producing microalgal biomass is receiving ever-increasing attention, particularly in the biofuels arena. In this study, a wastewater chlorophyte isolate, Kirchneriella sp., was tested for its ability to be mass cultivated, utilize nutrients from defined media and wastewater, and produce bioproducts of commercial interest. Growth studies were carried out in various systems at scales up to 60L, with Kirchneriella sp. showing an excellent amenability to being cultured. Biomass concentrations of greater than 1gL(-1) were consistently achieved, nitrogen and phosphorus uptake was rapid, and stable medium-term cultures were maintained. Nitrogen limitation affected biomass yield, fatty acid methyl ester (FAME) yield, and cetane index. In contrast, a low phosphorus condition had no effect. Kirchneriella sp. showed an ability to produce several products of commercial value, including carbohydrate-rich biomass, FAME/biodiesel and the pigments ß,ß-carotene and lutein.

Biodiscovery of new Australian thraustochytrids for production of biodiesel and long-chain omega-3 oils.

Heterotrophic growth of thraustochytrids has potential in co-producing a feedstock for biodiesel and long-chain (LC, ≥C(20)) omega-3 oils. Biodiscovery of thraustochytrids from Tasmania (temperate) and Queensland (tropical), Australia, covered a biogeographic range of habitats including fresh, brackish, and marine waters. A total of 36 thraustochytrid strains were isolated and separated into eight chemotaxonomic groups (A-H) based on fatty acid (FA) and sterol composition which clustered closely with four different genera obtained by 18S rDNA molecular identification. Differences in the relative proportions (%FA) of long-chain C(20), C(22), omega-3, and omega-6 polyunsaturated fatty acids (PUFA), including docosahexaenoic acid (DHA), docosapentaenoic acid, arachidonic acid, eicosapentaenoic acid (EPA), and saturated FA, as well as the presence of odd-chain PUFA (OC-PUFA) were the major factors influencing the separation of these groups. OC-PUFA were detected in temperate strains of groups A, B, and C (Schizochytrium and Thraustochytrium). Group D (Ulkenia) had high omega-3 LC-PUFA (53% total fatty acids (TFA)) and EPA up to 11.2% TFA. Strains from groups E and F (Aurantiochytrium) contained DHA levels of 50-61% TFA after 7 days of growth in basal medium at 20 °C. Groups G and H (Aurantiochytrium) strains had high levels of 15:0 (20-30% TFA) and the sum of saturated FA was in the range of 32-51%. ß,ß-Carotene, canthaxanthin, and astaxanthin were identified in selected strains. Phylogenetic and chemotaxonomic groupings demonstrated similar patterns for the majority of strains. Our results demonstrate the potential of these new Australian thraustochytrids for the production of biodiesel in addition to omega-3 LC-PUFA-rich oils.

Odd-chain polyunsaturated fatty acids in thraustochytrids.

A series of unusual odd-chain fatty acids (OC-FA) were identified in two thraustochytrid strains, TC 01 and TC 04, isolated from waters off the south east coast of Tasmania, Australia. FA compositions were determined by capillary GC and GC-MS, with confirmation of polyunsaturated fatty acids (PUFA) structure performed by analysis of 4,4-dimethyloxazoline derivatives. PUFA constituted 68-74% of the total FA, with the essential PUFA; eicosapentaenoic acid (20:5ω3, EPA), arachidonic acid (20:4ω6, AA) and docosahexaenoic acid (22:6ω3, DHA), accounting for 42-44% of the total FA. High proportions of the saturated OC-FA 15:0 (7.1% in TC 01) and 17:0 (6.2% in TC 04) were detected. The OC-FA 17:1ω8 was present at 2.8% in TC 01. Of particular interest, the C21 PUFA 21:5ω5 and 21:4ω7 were detected at 3.5% and 4.1%, respectively, in TC 04. A proposed biosynthesis pathway for these OC-PUFA is presented. It is possible that the unsaturated OC-PUFA found previously in a number of marine animals were derived from dietary thraustochytrids and they could be useful biomarkers in environmental and food web studies.

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).

Biochemical composition during growth and starvation of early larval stages of cultured spiny lobster (Jasus edwardsii) phyllosoma.

We examined biochemical changes accompanying feeding and starvation from hatch to Stage VI (day 74 after hatch) in spiny lobster, Jasus edwardsii, phyllosoma larvae. Larval dry weights (dw) increased 17-fold from hatch (80+/-1 microg) to Stage VI (1415+/-44 microg). Larvae starved for 6-11 days at Stages II, IV and VI were 14-40% lighter than their fed counterparts fed enriched Artemia. The increases and losses in total dry weight during feeding and starvation were associated with changes in the content of protein (constituting 31.4-41.7% of dw) and carbohydrate (constituting 2.6-5.3% of dw), while larger changes in lipid content indicated its greater importance as an energy substrate. Lipid content increased from 7.9% of dw at hatch to its highest of 12.5% at Stage IV, but declined by 50% or more during starvation. This suggests that protein, carbohydrate and lipid are all important energy stores, although lipids are catabolized at a greater rate during food deprivation. The principal lipid class was polar lipid (PL; 79-92% of total lipid), followed by sterol (ST; 6-20%), with triacylglycerol and other lipid classes at <2%. PL were catabolized and ST were conserved during starvation. Changes in the fatty acid (FA) profile had mostly occurred before the first moult at day 8 after hatch, with gradual changes thereafter to Stage VI, reflecting their abundance in the Artemia diet. There was some conservation of the major essential FAs, 20:4n-6, 20:5n-3, 22:6n-3, and the FA profile showed large gains in the C(18) polyunsaturated FA, 18:1n-9, 18:2n-6. Ascorbic acid content increased 10-fold from hatch to the end of Stage I (36 and 333 microgg(-1) dw, respectively), while the content at the end of Stage II was higher in fed than that in starved larvae (439 and 174 microgg(-1) dw, respectively). Our study will assist in the development of alternatives to nutritionally incomplete diets, such as live ongrown Artemia, to meet the requirements of phyllosoma in culture.