Microbial Oils

One concept currently under review is the use of algae as an oil producer for the manufacture of biodiesel. Oils of algae, fungi, and bacteria also have been investigated for biodiesel production (Schenk et al. 2008 and Strobel et al. 2008). Microalgae have a high potential as biodiesel precursors because many of them are very rich in oils, sometimes with oil contents over 80 % of their dry weight, although not all species are suitable as biodiesel production oils (Chisti 2008; Manzanera 2011). Besides, these microorganisms are able to double their biomass in less than 24 h, achieving a reduction between 49- and 132-fold in the medium culture time required by a rapeseed or soybean field. Furthermore, microalgae cultures require low maintenance and can grow in wastewaters, nonpotable water or water unsuitable for agriculture, as well as in seawater (Mata et al. 2010). The production of microalgae biodiesel could be combined with the CO2 removal from power generation facilities (Benemann 1997) or the synthesis of several valuable products, from bioethanol or biohydrogen to organic chemicals and food supple­ments (Banerjee et al. 2002; Chisti 2007; Harun et al. 2010). Research has shown that the oil content of algae per hectare can be a staggering 200 times more than the most productive land-based crop (algae are the fastest growing photosynthetic organisms and have the potential to produce 46 tons of oil/hectare/year). This is a promising lead for new generation biofuels, without compromising with food supply as these can be cultivated on nonagricultural lands. However, microalgae biomass-based biofuels have several problems ranging from the optimization of high density and large surface units of production to the location of the microalgae production unit. Anyway, the main decisions to take are the adoption of open or closed systems, and the election of batch or continuous operation mode. As will be discussed below, depending on the system and mode of operation choice, there will be different advantages and drawbacks.

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 and 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 and 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 and Steinbuchel 2010), and Rhodotorula (Cheirsilp et al.

2011) are the most interesting ones to produce biodiesel feedstock. Steinbuchel and collaborators have worked on the heterologous expression of the nonspecific acyl transferase WS/DGAT from Acinetobacter calcoaceticus ADP1 in S. cere- visiae H1246 (a mutant strain incapable 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 A. calcoaceticus transferase had a high potential for biotechno­logical production of a large variety of lipids, either in prokaryotic or eukaryotic hosts. From this basis, as will be discussed in detail in Sect. 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 sugarcane 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 h light and dark cycles (Cheirsilp et al. 2011).

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