Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

Wageningen University and Research Center

The process of the simultaneous production and extraction of carotenoids, milking, of Dunaliella salina was studied. We would like to know the selectivity of this process. Could all the carotenoids produced be extracted? And would it be possible to vary the profile of the produced carotenoids and, consequently, influence the type of carotenoids extracted? By using three different D. salina strains and three different stress conditions, we varied the profiles of the carotenoids produced. Between Dunaliella bardawil and D. salina 19/18, no remarkable differences were seen in the extraction profiles, although D. salina 19/18 seemed to be better extractable. D. salina 19/25 was not "milkable" at all. The milking process could only be called selective for secondary carotenoids in case gentle mixing was used. In aerated flat-panel photobioreactors, extraction was much better, but selectiveness decreased and also chlorophyll and primary carotenoids were extracted. This was possibly related to cell damage due to shear stress.

The selectivity of milking of Dunaliella salina.

Dunaliella salina is a halotolerant green alga that is well known for its carotenoid producing capacity. The produced carotenoids are mainly stored in lipid globules. For various research purposes, such as production and extraction kinetics, we would like to determine and/or localise the carotenoid globules in vivo. In this study, we show that the carotenoid-rich globules emit clear green fluorescence, which can be used in, for example, fluorescence microscopy (e.g. CLSM) to obtain pictures of the cells and their carotenoid content.

Carotenoid fluorescence in Dunaliella salina.

Algae are currently used for production of niche products and are becoming increasingly interesting for the production of bulk commodities, such as biodiesel. For the production of these goods to become economically feasible, production costs will have to be lowered by one order of magnitude. The application of two-phase systems could be used to lower production costs. These systems circumvent the costly step of cell harvesting, whilst the product is extracted and prepared for downstream processing. The mechanism of extraction is a fundamental aspect of the practical question whether two-phase systems can be applied for in situ extraction, viz, simultaneous growth, product formation and extraction, or as a separate downstream processing step. Three possible mechanisms are discussed; 1) product excretion 2) cell permeabilization, and 3) cell death. It was shown that in the case of product excretion, the application of two-phase systems for in situ extraction can be very valuable. With permeabilization and cell death, in situ extraction is not ideal, but the application of two-phase systems as downstream extraction steps can be part of a well-designed biorefinery process. In this way, processing costs can be decreased while the product is mildly and selectively extracted. Thus far none of the algal strains used in two-phase systems have been shown to excrete their product; the output has always been the result of cell death. Two-phase systems can be a good approach as a downstream processing step for these species. For future applications of two-phase in situ extraction in algal production processes, either new species that show product excretion should be discovered, or existing species should be modified to induce product excretion.

Two-phase systems: potential for in situ extraction of microalgal products.

During the in situ extraction of β-carotene from Dunaliella salina, the causal relationship between carotenoid extraction and cell death indicated that cell growth and cell death should be at equilibrium for a continuous in situ extraction process. In a flat-panel photobioreactor that was operated as a turbidostat cell numbers of stressed cells were kept constant while attaining a continuous well-defined light-stress. In this way it was possible to study the balance between cell growth and cell death and determine whether both could be increased to reach higher volumetric productivities of carotenoids. In the two-phase system a volumetric productivity of 8.3 mg β-carotene L(RV)(-1)d(-1) was obtained. In situ extraction contributed only partly to this productivity. The major part came from net production of carotenoid-rich biomass, due to a high growth rate of the cells and subsequent dilution of the reactor. To reach equilibrium between cell growth and cell death, sparging rates of dodecane could have been increased. However, already at the applied sparging rate of 286 L(dod)L(RV)(-1)min(-1) emulsion formation of the dodecane in the aqueous phase appeared. In a turbidostat without in situ extraction a volumetric productivity of 13.5 mg β-caroteneL(RV)(-1)d(-1) was reached, solely based on the continuous production of carotenoid-rich biomass.

Continuous production of carotenoids from Dunaliella salina.

In the so-called milking process of Dunaliella salina carotenoids are extracted and simultaneously produced by the culture, whilst the biomass concentration remains constant. Different theories exist about the extraction mechanisms although none have been proven yet. In this research, direct contact between dodecane and cells during the extraction process was studied microscopically and effects of direct contact were determined during in situ extraction experiments. Our results showed that water-solvent interphase contact resulted in cell death. This cell death and consequent cell rupture resulted in the release and concomitant extraction of the carotenoids. Furthermore, it has been suggested to add a small amount of dichloromethane to the biocompatible dodecane to create an organic phase with more extraction capacity. Our results showed that the addition of dichloromethane resulted in increased cell death and consequently the extraction rate increased. The improved solubility of carotenoids in an organic phase with dichloromethane did not significantly increase the extraction rate.

Phase toxicity of dodecane on the microalga Dunaliella salina.

The present work established an efficient staining method for fluorescence activated cell sorting (FACS) with Chlorococcum littorale maintaining cellular viability. The method was designed to detect high-lipid cells and to guarantee cellular viability. BODIPY505/515 (BP) was more suitable to FACS when compared to Nile red. The optimum concentrations were 0.4μgml(-1) of BP, 0.1% DMSO or 0.35% ethanol. Both ethanol and DMSO were equally efficient and assured cellular viability after the staining and sorting. Here a method is presented to rapidly screen and sort lipid rich cells of C. littorale with FACS, which can be used to produce new inoculum with increased cellular lipid content.

Dorinde M. M. Kleinegris

AlgaePARC,
Food and Biobased Research

Wageningen University and Research Center

Analysis of Fatty Acid Content and Composition in Microalgae

Dorinde M. M. Kleinegris

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}AlgaePARC,Food and Biobased Research

Wageningen University and Research Center

Analysis of Fatty Acid Content and Composition in Microalgae

AlgaePARC,
Food and Biobased Research