Nutrition and carotenogenesis in Haematococcus pluvialis

Abstract

This study examined various aspects of nutrition and carotenogenesis in the chlorophyte microalga, Haematococcus pluvialis. A feature of the H. pluvialis literature has been conflicting results relating to growth and carotenogenic productivities as well as in the reporting of nutritional and environmental requirements. The results presented in this thesis showed that these differences were due to strain variation. Variation in growth parameters was often due to strain variation in nutritional requirements. Strains were shown to differ in the ability to grow heterotrophically, the optimal sodium acetate concentration and the requirement for vitamins. As well the concentration of carotenoid and the proportion of different carotenoid species in the pigment profile also varied with strain. The results demonstrated that optimisation of culture conditions for growth and carotenoid production in H. pluvialis must be strain specific. Heterotrophic and mixotrophic growth was demonstrated in two strains using sodium acetate as the organic substrate. Parameters tested in mixotrophic and heterotrophic culture included source of inoculum, pH, nitrogen source and the requirement for vitamins. An interaction between pH and sodium acetate concentration was identified in mixotrophic and heterotrophic growth and was associated with the mechanisms of acetate uptake. The results confirmed that the relationship between heterotrophic, mixotrophic and photoautotrophic growth was variable depending on strain and sodium acetate concentration. Whilst none of the tested strains could grow heterotrophically on substrates other than sodium acetate, the utilisation of different organic substrates for mixotrophic growth was also strain dependent. One particularly versatile strain could utilise organic acids, alcohols, sugar, amino acids and carbohydrates for mixotrophic growth. When glucose and acetate were supplied together in heterotrophic culture this strain metabolised both carbon sources even though glucose alone was an ineffective heterotrophic substrate. A review of the literature suggested that the three factors most commonly reported to lead to the induction and stimulation of astaxanthin accumulation in H. pluvialis could be summarised as i) light, ii) inhibition of cell division by nutrient limitation (e g. nitrogen, phosphorus, magnesium) or growth inhibiting conditions (high temperature, salinity) and iii) addition of a supplementary carbon supply (usually organic acids such as acetate). Astaxanthin accumulation was therefore examined in relation to these factors. The results showed that illumination, inhibition of cell division and/or formation of aplanospores were not essential for astaxanthin synthesis to begin. although astaxanthin concentration was higher with increased irradiance, conditions which prevented cell division (nitrogen limitation, incubation at 35°C) and/or the addition of supplementary carbon as sodium acetate. The results suggest that all cells of H. pluvialis carry the functioning enzymes for astaxanthin synthesis, and the concentration of carotenoid was defined by the availability of carbon which can be directed to pigment synthesis. Growth conditions which improve the supply of carbon to the cell will therefore improve the rate of astaxanthin accumulation. Examples of such conditions may include •Mixotrophic culture when compared to heterotrophic culture. •Increased photosynthetic rate associated with increasing but not photoinhibitory irradiance. •Addition of supplementary organic carbon. As well, a change in the metabolic status of the cell (eg inhibition of cell division) may divert the dominant metabolic pathways for available carbon, such that more carbon was available for pigment synthesis. With manipulation of culture conditions to stimulate the formation of astaxanthin-rich and astaxanthin-poor aplanospores and flagellate cells, the different cell-types were exposed to different stress conditions to elucidate possible functions for astaxanthin accumulation in H. pluvialis. The results showed that astaxanthin had multiple functions in the cell. Photoprotection was confirmed in both aplanospores and flagellate cells, however in the astaxanthin-rich flagellate cells high concentrations of astaxanthin conferred protection against high temperature and salinity. Also, the hypothesised relationship between carbon supply and astaxanthin concentration suggests a storage role for the pigment. Mutants produced by nitrosoguanidine mutation showed increased carotenoid concentrations when compared to the wild-type control. However, the variation in the mutants was less than was observed in the wild-type strains. Targeted strain selection appears to be the most effective strategy for increasing carotenogenesis in H. pluvialis