Microalgae are not the only option to produce biofuels from oily biomass. Multiple prokaryotes and eukaryotes can accumulate high amounts of lipids. But, as occurred with microalgae, not all species are suitable for biodiesel production owing to differences in the kind of storage lipids. Thus, as stated by Waltermann & Steinbuchel (2010), many prokaryotes synthesize polymeric compounds such as poly(3-hydroxybutyrate) (PHB) or other polyhydroxyalkanoates (PHAs), whereas only a few genera show accumulation of triacylglycerols (TAGs) and wax esters (WEs) in the form of intracellular lipid bodies. On the other hand, storage TAGs are often found in eukaryotes, while PHAs are absent, and WE accumulation has only been reported in jojoba (Simmondsia chinensis). All these lipids are energy and carbon storage compounds that ensure the metabolism viability during starvation periods. Similar to the formation of PHAs, TAGs and WE, synthesis is promoted by cellular stress and during imbalanced growth; for instance, by nitrogen scarcity alongside the abundance of a carbon source (Kalscheuer et al., 2004).

The most interesting prokaryote genera in terms of accumulation of TAGs are nocardioforms such as Mycobacterium sp., Nocardia sp., Rhodococcus sp., Micromonospora sp., Dietzia sp., and Gordonia sp, alongside streptomycetes, which accumulate TAGs in the cells and the mycelia. TAGs storage is also frequently shown by members of the gram-negative genus Acinetobacter (although, in this case, WE are the dominant inclusion bodies components) (Waltermann & Steinbuchel, 2010). Within eukaryotes, with the exception of algae, yeasts of the genera Candida (non albicans) (Amaretti et al., 2010), Saccharomyces (Kalscheuer et al., 2004; Waltermann & Steinbuchel, 2010) and Rhodotorula (Cheirsilp et al., 2011) are the most interesting ones to produce biodiesel feedstocks.

Steinbuchel and collaborators have worked on the heterologous expression of the non specific acyl transferase WS/DGAT from Acinetobacter calcoaceticus ADP1 in Saccharomyces cerevisiae H1246 (a mutant strain unable of accumulating TAGs) (Kalscheuer et al., 2004). These authors found that the yeast recovered the ability to accumulate TAGs, as well as fatty acid ethyl esters and fatty isoamyl esters. This finding showed that the Acinetobacter calcoaceticus transferase had a high potential for biotechnological production of a large variety of lipids, either in prokaryotic and eukaryotic hosts. From this basis, as will be discussed in detail in Section 4.3, they worked on Escherichia coli TOP 10 (Invitrogen) and obtained an engineered strain able to produce fatty acid ethyl esters (biodiesel) directly from oleic acid and glucose (Kalscheuer et al., 2006).

Another possibility is combining the biomass obtained from microalgae and yeast, as recently proposed by Cheirsilp et al. (2011). These authors studied a mixed culture of oleaginous yeast Rhodotorula glutinis and microalga Chlorella vulgaris in industrial wastes. The used effluents, including both a seafood processing wastewater and molasses from a sugar cane plant. They found a synergistic effect in the mixed culture. R. glutinis grew faster and accumulated more lipids in the presence of C. vulgaris, that acted as an oxygen generator for yeast, while the microalgae obtained surplus CO2 from yeast. The optimal conditions for lipid production were 1:1 microalga to yeast ratio initial pH of 5.0, molasses concentration at 1%, 200 rpm shaking, and light intensity at 5.0 klux under 16:8 hours light and dark cycles (Cheirsilp et al., 2011).