Isolation, characteristation and screening of New Zealand alpine algae for the production of secondary metabolites in photobioreactors.

This inter-disciplinary thesis is concerned with the production of polyunsaturated fatty acids (PUFAs) from newly isolated and identified alpine microalgae, and the optimization of the temperature, photon flux density (PFD), and carbon dioxide (CO2) concentration for their mass production in an airlift photobioreactor (AL-PBR). Thirteen strains of microalgae were isolated from the alpine zone in Canyon Creek, Canterbury, New Zealand. Ten species were characterized by traditional means, including ultrastructure, and subjected to phylogenetic analysis to determine their relationships with other strains. Because alpine algae are exposed to extreme conditions, and such as those that favor the production of secondary metabolites, it was hypothesized that alpine strains could be a productive source of PUFAs. Fatty acid (FA) profiles were generated from seven of the characterized strains and three of the uncharacterized strains. Some taxa from Canyon Creek were already identified from other alpine and polar zones, as well as non-alpine zones. The strains included relatives of species from deserts, one newly published taxon, and two probable new species that await formal naming. All ten distinct species identified were chlorophyte green algae, with three belonging to the class Trebouxiophyceae and seven to the class Chlorophyceae. Comparative study between the distribution of algae at Canyon Creek and Mount Philistine, another alpine region in New Zealand where algal distribution was studied in detail, revealed that algal distribution patterns in the New Zealand alpine zone are complex, with some taxa apparently widely distributed and others range restricted or rare (with the caveat that very few sites have been studied in detail). At least some of the differences between the two sites could be accounted for by geographic differences, resulting in contrasting environmental conditions such as rainfall. As hypothesized, alpine strains isolated from the Canyon Creek were rich in PUFAs. Eight among the ten strains have PUFA proportions higher than monounsaturated fatty acids and saturated FAs. In a comparison of FA profiles of Scenedesmaceae species from a hot environment (Algerian Sahara) with the Scenedesmaceae species from Canyon Creek, the latter revealed a much greater degree of unsaturation. In addition, the Canyon Creek strains contained some FAs (such as docosapentaenoic acid, DPA) that were absent from Saharan strains. Among the strains from Canyon Creek Lobochlamys segnis LCR-CC-5-1A was selected for optimization experiments on the basis of growth kinetics, temperature response and FA composition, of which 60% of total FAs were PUFAs. Of that 60%, the α-linolenic acid (ALA) content was 46%. Two identical 1.5 Liter AL-PBRs were used for culturing Lobochlamys segnis LCR-CC-5-1A to study the effect of CO2 concentration, PFD and temperature on specific growth velocity, production of PUFAs, omega-3 FAs and, specifically, the concentration of ALA. The concentrations of CO2 examined in this research were 1.5, 3.0 and 4.5% in air. Similarly, the reponses of the strain to seven different PFDs, namely 38, 77, 115, 178, 210, 236 and 253 µmol m-2 s-1 and six different temperatures, 5, 10, 15, 20, 25 and 30οC, were analyzed. The maximum specific growth velocities (µmax) of the cultures were calculated from the experimental data and the cell production rate was calculated from fitting logistic growth models to these data; the two were compared by converting the former to the latter. The significance of the tested parameters was assessed using ANOVA and Tukey tests. The optimum conditions assessed at lab scale for maximum production of biomass, PUFAs and ALA were found to be a CO2 concentration of 3.0%, temperature of 20°C, and PFD of 178 µmol m-2 s-1. Increasing biomass production has the effect of maximizing PUFA production because there was no significant increase in concentration of PUFAs, omega-3 FAs, or ALA under levels of CO2, temperature, and PFD differing from those under which maximum growth occurred