Production of astaxanthin by xanthophyllomyces dendrorhous DSMZ 5626 using rapeseed meal hydrolysates as substrate.

Astaxanthin is one of the most important carotenoids in humans and has vast applications in the food, nutraceutical and aquaculture sectors. Currently, astaxanthin is primarily produced through chemical synthesis, whereas the microbial fermentation method for producing astaxanthin is hindered by its high production costs as compared to the synthetic route. Valorisation of rapeseed meal, a by-product of rapeseed oil processing industry holds potential to serve as an alternative route for the sustainable production of microbial astaxanthin. The aim of this thesis was to investigate the feasibility of producing microbial astaxanthin from rapeseed meal using a yeast species, Xanthophyllomyces dendrorhous DSMZ 5626 and its extraction strategies.

The preliminary study of X. dendrorhous growth in semi-defined media revealed that the yeast can consume a wide range of carbon sources including glucose, fructose, xylose, cellobiose, galactose, arabinose and glycerol with biomass values ranging from 10.7 g/l up to 13.3 g/l when 30 g/l of each carbon source was used individually. Suppression of astaxanthin production was observed when high glucose concentrations (> 30 g/l) were used due to Crabtree effects. The findings served as preliminary data to understand the biochemical behaviour of the selected yeast species.

Proximate analysis of the rapeseed meal used in this study demonstrated that it contained high protein (25 %, w/w), lignin (18%, w/w) and total carbohydrate (34%, w/w) contents, with the latter consisting primarily of glucose (20%, w/w) and to lesser extent arabinose (6%, w/w), galactose (3%, w/w) and also uronic acids (3%, w/w). Four commercial enzymes, namely (i) Viscozyme L, (ii) Accellerase 1500, (iii) pectinase and (iv) cellulase (from Aspergillus niger) were tested at different concentrations (1 – 15 %, v/v) for the individual assessment of their ability to break down the cellulosic and hemicellulosic compounds of rapeseed meal into monomeric fermentable sugars. Specifically, Viscozyme L and Cellulase treatments exhibited the highest glucose recovery yields (47 – 52% yield for 15 % (v/v) of enzyme used) and rapeseed meal derived total sugar concentration (74-77 g/l). A thermal pre-treatment step (126 oC, 30 min) prior to enzyme hydrolysis by Accellerase 1500 was also evaluated and was found to improve the hydrolysis rate of rapeseed sugars by 25%. Rapeseed meal hydrolysates were tested as fermentation media for microbial astaxanthin production using separate hydrolysis and fermentation (SHF) approach in batch and fed-batch fermentation modes. Batch fermentation with pectinase derived rapeseed meal hydrolysates supported both biomass (42 g/l) and astaxanthin production (11 mg/l) as the presence of glycerol from the enzyme formulation acted as additional carbon source for yeast growth.

Intracellular astaxanthin was extracted by using three cell disruption methods including glass beads, enzymatic cell lysis and supercritical fluid extraction. Results showed that highest astaxanthin extractability (>100 %) was obtained when enzymatic cell lysis with Glucanex, accompanied with acetone extraction was used under optimised conditions (pH 4.6 at temperature of 30.8 °C). Overall, the findings of the study can serve as basis towards the commercialisation potential of microbial astaxanthin using cheap and renewable substrate as fermentation feedstock (rapeseed hydrolysates) and gave useful insights on the extraction strategies that can be applied in future scaling up processes.