Microalgae are in general characterized by a high content of three main groups of biomolecules, proteins, polysaccharides, and lipids, according to the species and to the environmental growth conditions. Figure 2 reports the chemical composition of B. braunii strain used in the present study [30, 31]; the protein concentration and the polysaccharides content were determined by using the Lowry procedure [32] and the Dubois method [33], respectively; the lipid content was determined by weight after a solvent extraction procedure (n-hexane and chloroform/methanol mixture) [34], the ashes were determined by 5 h calcination at 550°C, and the intracellular water amount of the samples was evaluated by Karl-Fischer titration.

Figure 2 shows that B. braunii is characterized by a high amount of lipids (29%) and carbohydrates (22%), and a minor but relevant amount of proteins (7%); this composition is typical of this alga in its late growth phase (stationary phase) when the accumulation of lipids becomes much more relevant than cellular duplication.

The amount of ashes (22%) is in line with the typical ash content of microal­gae [35].

In addition to hydrocarbons, B. braunii also produces classic non-polar lipids as fatty acids, triacylglycerols, sterols, and polar lipids, as polyaldehydes, polyacetals, and non-polysaccharide biopolymers of very high molecular weight [36]. Thus, the oil extracted with n-hexane and chloroform/methanol mixture was initially analysed by GC-MS to qualitatively and quantitatively determine free fatty acid (after silyla- tion), triacylglycerol (after transesterification), and hydrocarbon (Tables 1 and 2) contents; then it was fractionated on a chromatographic column, thus separating hydrocarbons, non-polar, and polar lipids (Fig. 3).

□ Hydrocarbons

□ Non-polar lipids

□ Polar lipids

According to the literature [19 ] , the lipid oil of the A strain of B. braunii is mainly composed by non-polar lipids (62%) and hydrocarbons (29%), whereas polar lipids represent only a minor part (9%) of the composition.

By comparing the total amount of hydrocarbons calculated by GC-MS (7.8% on dry weight basis) and the percentage of the first fraction of the lipid oil (8.4% on dry weight basis), it is clear that the first fraction is entirely composed of hydrocarbons. This correspondence is not true in the case of non-polar lipids; the amount of fatty acids calculated by GC-MS (3.3% on dry weight basis) is much lower than the percentage of the second fraction of the lipid oil (17.8% on dry weight basis), indicating that bounded fatty acids and free fatty acids represent only a very small fraction of all the non-polar lipids. As reported in the literature, the main part of the non-polar fraction is in fact composed by a class of high molecular weight ether lipids, not GC-MS detectable ]37, 38] . These compounds are closely related to hydrocarbons, differently from what observed among the other vegetable ether lipids mainly based on glycerol (Fig. 4) [14]. the structures identified in the past years include alkadienyl-O-alkatrienyl ethers with an oxygen bridge between two C.7 hydrocarbon chains ] 37 ] . ether lipids with alkenylresorcinol linked by phenoxy bonds to one or two unsaturated hydrocarbon chains [39], or botryals, a-branched aldehydes originating from aldol condensation [38] .

Thanks to their hydrocarbon nature, these non-polar ethers could be processed by cracking to obtain biofuels, analogously to hydrocarbons, increasing the exploit­able fraction of B. braunii oil for energy and fuel purposes.