Graeme A. Dunstan

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, ≥C20) 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 C20, C22, 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 7xa0days of growth in basal medium at 20xa0°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%. β,β-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.

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 4xa0% 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 4xa0% w/v but not 2xa0% w/v of the culture suggesting a threshold requirement between these values. Across all strains, increasing glucose from 2 to 4xa0% 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 (299xa0mg/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 (2xa0L) bioreactors for the potential of co-producing biodiesel and high-value omega-3 long-chain polyunsaturated fatty acids. Higher initial glucose concentration (100xa0g/L compared to 40xa0g/L) did not result in markedly different biomass (48xa0g/L) or fatty acid (12–14xa0g/L) yields by 69xa0h. 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 56xa0g/L, respectively) by 69xa0h. 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 56xa0g/L (34xa0% total fatty acids) to 71xa0g/L (52xa0% total fatty acids, cell dry weight) at 69xa0h; these yields are to our knowledge around 70xa0% 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.

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.

Life cycle assessment: heterotrophic cultivation of thraustochytrids for biodiesel production.

This study provides a life cycle assessment of the energy balance and the potential greenhouse gas impacts of heterotrophic microalgal-derived biodiesel estimated from the upstream biomass production to the downstream emissions from biodiesel combustion. Heterotrophic microalgae can be cultivated using a by-product from biodiesel production such as glycerol as a carbon source. The oils within the algal biomass can then be converted to biodiesel using transesterification or hydroprocessing techniques. This approach may provide a solution to the limited availability of biomass feedstock for production of biorefined transportation fuels. The life cycle assessment of a virtual production facility, modeled on experimental yield data, has demonstrated that cultivation of heterotrophic microalgae for the production of biodiesel is comparable, in terms of greenhouse gas emissions and energy usage (90xa0gxa0CO2exa0MJ−1), to fossil diesel (85xa0gxa0CO2exa0MJ−1). The life cycle assessment identified that improvement in cultivation conditions, in particular the bioreactor energy inputs and microalgae yield, will be critical in developing a sustainable production system. Our research shows the potential of heterotrophic microalgae to provide Australia’s transportation fleet with a secure, environmentally sustainable alternative fuel.

Assessing near-infrared reflectance spectroscopy for the rapid detection of lipid and biomass in microalgae cultures

With intensification of interest in microalgae as a source of biomass for biofuel production, rapid methods are needed for lipid screening of cultures. In this study, near-infrared reflectance spectroscopy (NIRS) was assessed as a method for analysing lipid (specifically, total fatty acid methyl esters (FAME) obtainable from processing) and biomass in late logarithmic and stationary phase cultures of the green alga Kirchneriella sp. and the eustigmatophyte Nannochloropsis sp. Culture samples were filtered, scanned by NIRS and chemically analysed; by combining these sets of information, models were developed to predict total biomass, FAME content and FAME as a percentage of dry weight in samples. Chemically derived (actual) and NIRS-predicted data were compared using the coefficient of determination (R2) and the ratio of the standard deviation (SD) of actual data to the SD of NIRS prediction (RPD). For Kirchneriella sp. samples, models gave excellent prediction (R2u2009≥u20090.96; RPDu2009≥u20094.8) for all parameters. For Nannochloropsis sp., the model metrics were less favourable (R2u2009=u20090.84–0.94; RPDu2009=u20092.5–4.2), though sufficient to provide estimations that could be useful for screening purposes. This technique may require further validation and comparison with other species, but this study shows the potential of the NIRS as a rapid screening method (e.g. up to 200 sample analyses per day) for estimating FAME or other microalgal constituents and encourages further investigation.

A novel series of C 18 –C 22 trans ω3 PUFA from Northern and Southern Hemisphere strains of the marine haptophyte Imantonia rotunda

A series of novel C18–C22trans ω3 polyunsaturated fatty acids (PUFA) with a single trans double bond in the ω3 position was found in Northern and Southern Hemisphere strains of the marine haptophyte Imantonia rotunda. The novel ω3 PUFA were identified as 18:3(9c,12c,15t) (0.2–1.8xa0% of total fatty acids), 18:4(6c,9c,12c,15t) (1.9–4.1xa0%), 18:5 (3c,6c,9c,12c,15t) (0.7–8.8xa0%), 20:5(5c,8c,11c,14c,17t) (1.2–4.1xa0%) and 22:6(4c,7c,10c,13c,16c,19t) (0.3–4.3xa0%), and were accompanied by larger proportions of the all cis isomers: 18:3ω3(9,12,15) (2.7–3.5xa0%), 18:4ω3(6,9,12,15) (9.3–14.3xa0%), 18:5ω3(3,6,9,12,15) (7.8–18.5xa0%), 20:5ω3(5,8,11,14,17) (3.2–3.9xa0%), 22:5ω3(7,10,13,16,19) (0.1–0.3xa0%) and 22:6ω3(4,7,10,13,16,19) (2.3–5.2xa0%). GC analysis of FAME using a non-polar column did not reveal the trans isomers as they coeluted with the all cis PUFA. However, GC using a polar column resolved the trans PUFA from the all cis PUFA, with the trans isomers eluting before the all cis isomers. GC-MS of FAME fractionated by argentation solid-phase chromatography confirmed the molecular ions of all components. FAME were derivatized to form 4,4-dimethyloxazoline (DMOX) derivatives, and GC-MS revealed the same double bond positions for each trans and cis FAME. The results suggest that the ω3 trans double bond originated from the Δ15/ω3 desaturation of 18:2(9c,12c), suggesting that this desaturase has dual cis/trans activity in these species. These results indicate that 18:3(9c,12c,15u2009t) was the precursor trans isomer produced for the trans series and further desaturation by the common Δ6 desaturase to produce the trans tetraene and successive elongations and desaturations led to the subsequent series of trans ω3 PUFA isomers. To our knowledge, this is the first report of these trans ω3 isomers occurring in strains of I. rotunda. These trans ω3 PUFA may be used as biomarkers in marine food webs for this species and with their unique structure may be biologically active.