‘Algal blushing’ : characterization and bioprospecting of a novel alpine Haematococcus species from New Zealand for astaxanthin production.

In recent years there has been a growing interest in astaxanthin production from Haematococcus lacustris in the field of microalgal biotechnology. Astaxanthin is a keto-carotenoid pigment that has important applications in nutraceutical and cosmetics industries as a potent antioxidant. H. lacustris has been identified as a promising source of natural astaxanthin and is commercially cultivated. Culture conditions in Haematococcus have been demonstrated to be strongly strain specific; thus new strains in this genus needs detailed investigation of variables affecting growth and pigment production. This inter-disciplinary research study sought to find the optimal culture conditions for growth and astaxanthin production from a novel species: ‘Haematococcus alpinus’ isolated from an alpine zone in New Zealand. This species is morphologically distinguished from others by location, number, and thickness of cytoplasmic strands. Phylogenetic analysis based on 18S rDNA, internal transcribed spacer (ITS) rDNA, rbcL genes showed that our species is distinct with no known close relatives. Preliminary studies in shake flasks were performed to understand the effect of pH, nitrogen and carbon sources, vitamins and micronutrients on the vegetative growth of the strain. The influence of culture medium, aeration rates, CO₂ concentration, temperature and photon flux density (PFD) were also investigated using airlift photobioreactors. In addition, the effect of the spectral composition of light on growth and astaxanthin production was also explored. Evaluation of a range of different physical and chemical treatments on the disruption of the vegetative and encysted cells of H. alpinus in terms of chlorophyll and astaxanthin yield were also undertaken. Results of the study suggest that a pH window of 6.0 – 6.5 was optimal for growth. Supplementation of vitamins consisting of thiamine, biotin, and cyanocobalamin improved the cell density over the vitamin-free medium. The best nitrogen source tested was nitrate in the form of sodium nitrate (40 mM). Heterotrophic growth yielded much lower cell densities compared to autotrophic growth. Micronutrients like iron and manganese were essential for growth however, the best growth was obtained using a micronutrient mix consisting of iron, zinc, copper, cobalt, manganese and molybdenum. Modified MLA medium containing 40 mM of sodium nitrate was ideal for producing high density cultures. Temperature between 12 – 18°C, a CO₂ concentration of 3% and a narrow PFD range between 37 – 48 μmol m⁻² s⁻¹ were best suited for growth. The shear sensitive vegetative cells favored lower aeration rates in the photobioreactors. Different wavelengths of light had significant effect on cell growth and astaxanthin production. A mix of red-blue light at low PFD was ideal for promoting growth. A high cell density of 9.6 x105 cells mL⁻¹ was achieved using the red-blue LED mix. Blue light enhanced astaxanthin induction in the cells. The highest astaxanthin concentration of 185 μg mL⁻¹ was achieved when the cultures were stressed with blue LED at a high PFD. An additivity test was also performed under which cultures were grown in both vegetative and carotenoid phases under combined optimized conditions. The maximum cell density and astaxanthin concentration obtained were 12.0 x 10⁵ cells mL⁻¹ and 205 μg mL⁻¹ respectively, which were the highest values achieved throughout the study. Both dimethyl sulfoxide and methanol were effective in extracting chlorophyll from vegetative cells. Cell disruptions techniques such as sonication and vortexing facilitated rupturing of the fragile cells thereby increasing solvent efficiency. The rigid haematocysts were more resilient to cell rupturing techniques. Except dimethyl sulfoxide all the other solvents were particularly ineffective in extracting astaxanthin. Dimethyl sulfoxide showed the highest solvent efficiency in the extractability of astaxanthin, independent of the cell disruption technique used. Solvent choice was the dominant factor that impacted both chlorophyll and astaxanthin yield