The cultivation of photosynthetic microalgae under dark conditions, supplied with organic carbon, closely resembles typical microbial fermentations. Because several bac­terial species are well known as accumulators of triglycerides (oils) and esters of fatty acids with long-chain alcohols (waxes), the logical conclusion was to combine these biosynthetic abilities with that of ethanol formation to generate the precursors of tri­glycerides in microbial production systems, that is, “microdiesel” produced without any need for a chemically or enzymatically catalyzed transesterification.9 The simple bacte­rium Escherichia coli was used as host for the Zymomonas mobilis pyruvate decarbox­ylase and alcohol dehydrogenase genes for ethanol production (chapter 3, section 3.3.2) together with the gene encoding an unspecific wax ester synthase/acyl-CoA: diacylg — lycerol transferase from a bacterial strain (Acinetobacter baylyi) known to accumulate lipid as an internal cell storage reserve. The resulting recombinant could accumulate ethyl esters of fatty acids at up to 26% of the cellular dry mass in fermentations fed with glucose. Insomuch as glucose is a fully renewable carbohydrate supply (via, e. g., cel­lulose or starch), microdiesel is a genuinely sustainable source of preformed transporta­tion fuel — although the chemical engineering aspects of its extraction from bacterial cells and the economics of its production systems require further definition.

A refinement preliminary to industrial feasibility studies would be to transfer to a host capable of higher endogenous accumulation of lipids; many of these are Gram-negative species (like E. coli), and ethanol production in such species is a well-understood area of biochemistry.1011