This work focussed on the optimisation of the volumetric productivity (mg EPA L⁻¹ d⁻¹) of eicosapentaenoic acid (EPA, 20:5 n-3) from a New Zealand strain of microalgae, Trachydiscus sp. LCR-Awa-9-2. EPA is a high value polyunsaturated omega-3 fatty acid which plays a beneficial role in the prevention and treatment of a variety of human health conditions such as arthritis and cardiovascular disease. Trachydiscus sp. LCR-Awa-9-2 was a recently isolated strain of microalgae and at the time of isolation the strain showed a high fraction of EPA in its fatty acids (42 g/100 g total fatty acids). Due to the high amount of EPA present in the cells the microalga was a promising contender for commercial production of EPA. Research was required to determine the optimal conditions for growth and EPA productivity of the microalga and to allow evaluation of whether EPA production would be commercially viable using this strain.

In this work, the growth of Trachydiscus sp. LCR-Awa-9-2 in a purpose-built 76 L tubular photobioreactor was investigated as a first milestone towards potential scale up of the culturing of this microalga. The influence of different culture conditions on growth and EPA content were investigated at small scale in shake flasks and airlift photobioreactors. Studies were also conducted into the lipidome of the microalga and the oxygen evolution rate was investigated under a variety of conditions as a proxy measure of photosynthesis rate. Conditions such as liquid velocity, light intensity, light wavelength, and semi-continuous culturing were investigated in the tubular photobioreactor. In shake flasks the effect of nutrients (NaCl, Co, B, lactose, galactose, CO2, and Bold’s Basal modified Awarua (BBMA) vs. Zehnder (Z) media) were investigated as well as the effect of diurnal light cycling (24:0, 12:12, 16:8, hours light : hours dark).

The highest maintainable EPA productivity in this study of 30 ± 2 mg L⁻¹ d⁻¹ was achieved with semi-continuous culturing in the tubular photobioreactor at a cell density of approximately 4.5 × 10⁷ cells mL⁻¹ and a dilution rate of 0.19 d⁻¹. The highest EPA productivity in batch cultures of 16 ± 2 mg L⁻¹ d⁻¹ was achieved in shake flasks with 10% CO2. In both cases BBMA medium was used. The highest EPA fraction was 46 ± 1 g/100 g fatty acids and the highest content was 6.1 ± 0.1 g/100 g dry biomass, both achieved under the same conditions in the tubular photobioreactor (warm white light, 1500 μmol m⁻² s⁻¹, late exponential phase, BBMA medium, 3% CO2). The EPA fraction achieved was the highest that had been reported for this microalga. The maximum cell density achieved was 1.0 ± 0.1 × 10⁸ cells mL⁻¹ in shake flasks with 3% CO2, BBMA medium and a light intensity of 500 μmol m⁻² s⁻¹. Light availability was identified as the key limiting factor to the further improvement of EPA productivity of cultures. Diurnal cycles in shake flasks showed that cell densities and EPA productivity decreased with increasing dark time, but EPA content increased. The fraction of the photobioreactor volume illuminated influenced the growth of cultures in the tubular photobioreactor with decreasing illuminated volume negatively influencing both the growth rate and maximum cell density.

Increasing NaCl concentration negatively impacted both the EPA fraction and growth of cultures, with cultures unable to grow at a NaCl concentration of 35 g L⁻¹; however, EPA content was greatest at a NaCl concentration of 1 g L⁻¹. Cultures were able to grow at CO2 concentrations up to 30% but growth was not observed at 100% CO2. Air without supplemental CO2 (0.04% CO2) lead to carbon limitation of cultures and high pH (> pH 9) which inhibited growth. Oxygen evolution rates were roughly equal over a pH range of 3 to 7 but decreased outside this range. Cells were able to photosynthesise over a temperature range of 5 to 40 °C but the maximum rate was achieved at 20 to 25 °C with the rate decreasing above and below these temperatures. Warm white was found to be the most efficient light colour for oxygen evolution rate compared to red, blue, and green. Blue, warm white, and combined red-green-blue-warm white (RGBWW) light colours did not significantly impact EPA content of cells in the tubular photobioreactor but warm white was the most efficient for growth based on cells produced per quantity of photons supplied. Lipidomic analysis of Trachydiscus sp. LCR-Awa-9-2 showed that the majority of EPA (50 ± 2 wt%) was located in the monogalactosyl diacylglycerides (MGDG), as had been reported for other species of microalgae. Significant degradation of lipid extracts occurred during extraction from freeze-dried biomass using a modified Bligh and Dyer method. Degradation of lipids was overcome by extraction from fresh biomass using a hot isopropanol extraction method to prevent degradation due to enzymatic activity. Results from thin layer chromatography (TLC) separations of lipid extracts indicated that an unconfirmed minor glycolipid class may be present and that this could potentially be monoglucosyl diacylglyceride (GlcDG); however, the class of the lipids was unable to be conclusively identified during this study. The potential GlcDG class of interest was not detected in similar analyses of lipids from Chlorella vulgaris. Presence of GlcDG would indicate a gene transfer event from cyanobacteria as GlcDG is not typically produced by eukaryotic microalgae.

The results of this study showed that Trachydiscus sp. LCR-Awa-9-2 was able to be grown in a tubular photobioreactor and that the microalga was likely to be well-suited to large-scale culturing. The microalga tolerated a wide range of CO2 concentrations, pH, temperature, and shear stress, and was able to be semi-continuously cultured; all of which are beneficial for large-scale culturing. Further small improvements in EPA productivity may be possible by reduction of dark volumes in future designs of tubular photobioreactors to improve growth rates, as well as increasing the EPA content of the cells. Photoautotrophic production of EPA from Trachydiscus sp. LCR-Awa-9-2 at the scale used in this study was unlikely to be commercially viable and so future work is required to improve the economics of culturing this microalga for EPA.