Fractionation and characterization of Dunaliella microalgal biomass and extraction of carotenoids

Abstract

This paper presents findings on the application of two fractionation protocols of Dunaliella microalgal biomass with the aim to separate and recover the lipid, pigment and protein fraction, leaving as “residue” the fraction that contains carbohydrates. The first protocol (LPC) is based on the initial extraction of lipids, followed by extraction of proteins, while in the second protocol (CLP), the pigments are first extracted followed by lipids extraction. With the LPC protocol the recovered lipid fraction was 34 and 32 wt%, while the recovered proteinic fraction was 46 and 50 wt% for the two samples under study, i.e. 1120 (D. granulata) and 1220 (D. minutissima). The CLP protocol led to the initial extraction of the pigment fraction with a percentage of 10–15 wt% while the subsequently recovered lipidic fraction was 16–24 wt%. The initial biomass was characterized for its moisture (<0.7 wt% for both samples), ash (approx. 45 wt% for both samples), organic matter, elemental composition and higher heating value (HHV) which was 23 and 30 MJ/kg (organic matter based-OMB). The composition of the lipid fraction was analyzed via esterification and gas chromatography (GC-FID) showing C16 and C18 as the predominant acids in the samples. The residue/carbohydrate fraction was analyzed via acid hydrolysis and analysis by high pressure liquid chromatography (HPLC) showing about 20 wt% (-OMB) content of carbohydrates. The high ash content of the biomass was found to be co-extracted during the fractionation protocols at a percentage of ≈49 % in the lipidic fraction and 45 % in the proteinic fraction. Furthermore, emphasis was given on pigment extraction and recovery using various solvents and quantification methods. Measurements of β-carotene, carotenoids, and chlorophyll content through UV and HPLC analyses is presented with chlorophyl and carotenoid content being ≈12 wt% and ≈9 wt% for samples 1120 and 1220 (-OMB). Additionally, a comparison between three solvents (THF, dichloromethane, acetone) was performed regarding their ability to extract the pigments from the microalgal biomass showing that acetone is capable of extracting pigments when concentration of the pigments is not high. The comparison of the HPLC with the UV method, suggested that UV spectroscopy is a reliable and rapid alternative for pigments determination, while HPLC enables identification and quantitative determination of specific pigments and chlorophyll species. The study provides insights for different fractionation protocols applied in two not yet fully studied Dunaliella species as well as the chlorophyl and carotenoid content.