1.1 Bacteria

The synthesis of intracellular lipids in oleaginous bacteria occurs during the log­arithmic phase and the beginning of the stationary growth phase (Gouda et al. 2008). However, only few species of bacteria can accumulate lipids suitable for biodiesel, as they mainly accumulate polyhydroxy alkanoates (PHA) and poly­hydroxy butyrate (PHB) (Kosa and Ragauskas 2011; Shi et al. 2011). The spe­cies that produce a large amount of lipids are those belonging to Streptomyces, Nocardia, Rhodococcus, and Mycobacterium (Alvarez and Steinbuchel 2002). The amount of triglycerides (TAG) and fatty acid composition differs depending on the species used for fermentation (Table 5). Gouda et al. (2008) tested Rhodococcus opacus and Gordonia sp. using different agroindustrial wastes (molasses, potato infusion, wheat bran, hydrolyzed barley, orange waste, tomato peel waste, arti­choke waste, and Na-gluconate) as carbon sources. Molasses provided the high­est percentage of lipid in cell, 93 and 96 % for R. opacus and Gordonia sp.,

respectively, while carob waste offered the best source for TAG accumulation, being 88.9 and 57.8 mg per liter of medium for R. opacus and Gordonia sp., respectively, and C17:1 the main fatty acid produced (20.7 %) by R. opaccus. When Gordonia sp. consumed molasses, they followed the same trend in terms of the accumulation of lipid in cell mass (96 %). However, the highest accumulation of TAG (57.8 mg/L) was achieved when orange waste was consumed, being C22:0 the predominant fatty acid, in a percentage close to 35 %. Two different strains of bacterium R. opacus, DSM 1069 and PD630, were inoculated in lignocellulosic compounds (4-hydroxybenzoic and vanillic acids) (Kosa and Ragauskas 2012). The experiments showed that both strains can consume these carbon sources and accumulate lipids close to 20 % of their own weight.

With regard to bacterial biodiesel properties and subsequent engine testing, only one analysis has been reported (Wahlen et al. 2012). In this study, the bacte­rium R. opacus was grown in sucrose and biodiesel properties were compared with those from microalgae and yeast oil-based biodiesel. Biodiesel bacterial molecu­lar properties differ considerably with the other biofuels in terms of carbon chain length. The physical properties were similar to other microbial biodiesel, with the exception of the heating value that was lower. When bacterial biodiesel was ran on a diesel engine, it provided the lowest power output, while NOx and HC emissions were higher and lower than other microbial biodiesel, respectively.

Bacteria that accumulate the highest proportion of triglycerides are providing neither sufficient oil yield under industrial conditions nor an economically sound process. For these reasons, genetic engineering is supporting this biotechnology to be considered a viable alternative for the biodiesel industry. Rucker et al. (2013) demonstrated the feasibility of the lipid metabolism of E. coli for TAG accumu­lation, but the yield achieved was below the threshold to be considered a viable source for biodiesel production. Authors propose two metabolic engineering steps, to increase either the supply of phosphatidic acid during late exponential and sta­tionary phase growth or the supply of acyl-CoA.

One of the most interesting uses of bacteria in the production of biodiesel was described by Kalscheuer et al. (2006). In this study, the genetically modified bac­teria E. coli was recombined with two different enzymes from Zymomonas mobilis and Acinetobacter baylyi. The target was to produce fatty acid ethyl esters (FAEE) in vivo, called “microdiesel.” Under fed-batch fermentation using renewable car­bon sources, they achieved a FAEE concentration of 1.28 g L-1, corresponding to a FAEE content of the cells of 26 % of the cellular dry mass. Gordonia sp. KTR9 may be considered among the suitable bacteria for in vivo synthesis of fatty acid ethyl esters from short-chain alcohols. This species has a large num­ber of genes dedicated to both the formation of fatty acids and lipid biosynthesis. Furthermore, it tolerates the addition of more than 4 % methanol, 4 % ethanol, and 2 % propanol in the medium (Eberly et al. 2013).

It may be concluded from above works that biodiesel produced from bacterial oil can be considered as an alternative to first — and second-generation biodiesel. However, more research is needed to both improve bacterial oil yield and provide economically viable substrates.

D. E. Leiva-Candia and M. P. Dorado