Screening of a Thraustochytrid Strain Collection for Carotenoid and Squalene Production Characterized by Cluster Analysis, Comparison of 18S rRNA Gene Sequences, Growth Behavior, and Morphology.

Carotenoids and squalene are important terpenes that are applied in a wide range of products in foods and cosmetics. Thraustochytrids might be used as alternative production organisms to improve production processes, but the taxon is rarely studied. A screening of 62 strains of thraustochytrids sensu lato for their potential to produce carotenoids and squalene was performed. A phylogenetic tree was built based on 18S rRNA gene sequences for taxonomic classification, revealing eight different clades of thraustochytrids. Design of experiments (DoE) and growth models identified high amounts of glucose (up to 60 g/L) and yeast extract (up to 15 g/L) as important factors for most of the strains. Squalene and carotenoid production was studied by UHPLC-PDA-MS measurements. Cluster analysis of the carotenoid composition partially mirrored the phylogenetic results, indicating a possible use for chemotaxonomy. Strains in five clades produced carotenoids. Squalene was found in all analyzed strains. Carotenoid and squalene synthesis was dependent on the strain, medium composition and solidity. Strains related to Thraustochytrium aureum and Thraustochytriidae sp. are promising candidates for carotenoid synthesis. Strains closely related to Schizochytrium aggregatum might be suitable for squalene production. Thraustochytrium striatum might be a good compromise for the production of both molecule groups.

Development and validation of reliable astaxanthin quantification from natural sources.

Astaxanthin derived from natural sources occurs in the form of various esters and stereomers, which complicates its quantitative and qualitative analysis. To simplify and standardize astaxanthin measurement with high precision, an enzymolysis-based astaxanthin quantification method was developed to hydrolyze astaxanthin esters and determine free astaxanthin in all its diastereomeric forms. Astaxanthin standards and differently processed Haematococcus pluvialis biomass were investigated. Linear correlation of standards of all-E-astaxanthin was observed in a measurement range between extract concentrations of 1.0 µg/mL and 11.2 µg/mL with a coefficient of variation below 5%. The diastereomers 9Z-, and 13Z-astaxanthin, and two di-Z-forms were detected. In contrast to the measurement of standards, the observed measurement range was extended to 30 µg/mL in extracts from H. pluvialis. The nature of the sample had to be taken into account for measurement, as cell, respectively, sample composition altered the optimal concentration for astaxanthin determination. The measurement precision of all-E-astaxanthin quantification in dried H. pluvialis biomass (1.2-1.8 mg dried biomass per sample) was calculated with a coefficient of variation of maximum 1.1%, whereas it was below 10% regarding the diastereomers. Complete enzymolysis was performed with 1.0 to 2.0 units of cholesterol esterase in the presence of various solvents with up to 2.0 mg biomass (dry weight). The method was compared with other astaxanthin determination approaches in which astaxanthin is converted to acetone in a further step before measurement. The developed method resulted in a higher total astaxanthin recovery but lower selectivity of the diastereomers. The reliability of photometric astaxanthin estimations was assessed by comparing them with the developed chromatographic method. At later stages in the cell cycle of H. pluvialis, all methods yielded similar results (down to 0.1% deviation), but photometry lost precision at earlier stages (up to 31.5% deviation). To optimize sample storage, the shelf life of astaxanthin-containing samples was investigated. Temperatures below -20°C, excluding oxygen, and storing intact H. pluvialis cells instead of dried or disrupted biomass reduced astaxanthin degradation.

Optimization of Astaxanthin Recovery in the Downstream Process of Haematococcus pluvialis.

Astaxanthin derived from Haematococcus pluvialis is a valuable metabolite applied in a wide range of products. Its extraction depends on a sophisticated series of downstream process steps, including harvesting, disruption, drying, and extraction, of which some are dependent on each other. To determine the processes that yield maximum astaxanthin recovery, bead milling, high-pressure homogenization, and no disruption of H. pluvialis biomass were coupled with spray-drying, vacuum-drying, and freeze-drying in all possible combinations. Eventually, astaxanthin was extracted using supercritical CO2. Optimal conditions for spray-drying were evaluated through the design of experiments and standard least squares regression (feed rate: 5.8 mL/min, spray gas flow: 400 NL/h, inlet temperature: 180 °C). Maximal astaxanthin recoveries were yielded using high-pressure homogenization and lyophilization (85.4%). All combinations of milling or high-pressure homogenization and lyophilization or spray-drying resulted in similar recoveries. Bead milling and spray-drying repeated with a larger spray-dryer resulted in similar astaxanthin recoveries compared with the laboratory scale. Smaller astaxanthin recoveries after the extraction of vacuum-dried biomass were mainly attributed to textural changes. Evaluation of these results in an economic context led to a recommendation for bead milling and spray-drying prior to supercritical CO2 extraction to achieve the maximum astaxanthin recoveries.