Because dependence on nutritional supplementation increases the process cost, the ideal biocatalyst should produce high amounts of ethanol in sim­ple mineral salts growth medium. While KO11 and LY01 both attained high ethanol yields and titers in rich media, these microbial biocatalysts perform poorly in minimal media. With nutritional supplementation, KO11 produced 45 g L-1 ethanol from 100 g L-1 glucose in 72 h; in minimal medium less than 30 gL-1 were produced in 96 h [31]. Results for LY01 were similarly disap­pointing: the final cell mass and ethanol titer attained in minimal medium were tenfold lower than in rich medium [32]. Considering that these strains were selected in rich media, this is not a surprising result. The low ethanol production by KO11 in minimal media has been attributed to suboptimal partitioning of pyruvate for biosynthesis [33,34]. Low acetyl CoA and high NADH levels result in inhibition of citrate synthase, limiting the availability of 2-oxoglutarate for biosynthesis. 2-Oxoglutarate is required for the biosynthe­sis of many amino acids and is an important compound for osmotic tolerance. This proposed inhibition of citrate synthase was supported by the finding that expression of a NADH-insensitive citrate synthase from Bacillus increased the growth and ethanol production of KO11 by about 75% [33].

The ability of microbial biocatalysts to retain ethanologenicity over time without dependence on antibiotics is important for minimizing production costs. While instability of KO11 has been reported [35,36], other reports have demonstrated maintenance of KO11 ethanologenicity for up to 27 days in continuous stirred tank and fluidized beds reactors [37].

In addition to the production of 48 gL-1 ethanol in rich media, KO11 also produced up to 192 mgL-1 of the undesirable co-product ethyl acetate. An es­terase with ethyl acetate hydroylase activity (estZ) from Pseudomonas putida was introduced into KO11 and the presence of this enzyme reduced the ethyl acetate level to less than 20 mg L-1, a level comparable to that of yeast fermen­tation [38].

2.2