Formulating cost-effective media for the recombinant microorganisms developed for broad-spectrum pentose and hexose utilization (chapter 3, sections 3.2 and 3.3) commenced in the 1990s. For pentose-utilizing E. coli, for example, the benchmark was a nutrient-rich laboratory medium suitable for the generation of high-cell-density cultures.111 Media were then assessed using the criteria that the final ethanol concen­tration should be at least 25 g/l, the xylose-to-ethanol conversion efficiency would be high (90%), and a volumetric productivity of 0.52 g/l/hr was to be attained; in a defined minimal salts medium, growth was poor, only 15% of that observed in the laboratory medium; supplementation with vitamins and amino acids improved growth but could only match approximately half of the volumetric productivity. The use of corn steep liquor as a complex nitrogen source was (as predicted from its wide industrial use in fermentations) the best compromise between the provision of a complete nutritional package with plausible cost implications for a large-scale process. As an example of the different class of compromise inherent in the use of lignocellulosic substrates, the requirement to have a carbon source with a high content of monomeric xylose and low hemicellulose polymers implied the formation of high concentrations of acetic acid as a breakdown product of acetylated sugar residues; to minimize the associated growth inhibition, one straightforward strategy was that of operating the fermentation at a relatively high pH (7.0) to reduce the uptake of the weak acid inhibitor.

In a study conducted by the National Center for Agricultural Utilization Research, Peoria, Illinois, some surprising interactions were discovered between nitrogen nutrition and ethanol production by the yeast P. stipitis.112 When the cells had ceased active growth in a chemically defined medium, they were unable to ferment either xylose or glucose to ethanol unless a nitrogen source was also provided. Ethanol pro­duction was increased by the amino acids alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, leucine, and tyrosine (although isoleucine was inhibitory); a more practical nitrogen supply for industrial fermentations consisted of a mixture of urea (up to 80% of the nitrogen) and hydrolyzed milk protein supplemented with tryptophan and cysteine (up to 60%); the use of either urea or the protein hydrolysate was less effective than the combination of both. Adding small amounts of minerals, in particular, iron, manganese, magnesium, calcium, and zinc salts as well as amino acids could more than double the final ethanol concentration to 54 g/l.

Returning to recombinant E. coli, attempts to define the minimum salts concen­tration (to avoid stress imposed by osmotically active solutes) resulted in the formu­lation of a medium with low levels of sodium and other alkali metal ions (4.5 mM) and total salts (4.2 g/l).113 Although this medium was devised during optimization of lactic acid production, it proved equally effective for ethanol production from xylose. Because many bacteria biosynthesize and accumulate internally high concentrations of osmoprotective solutes when challenged with high exogenous levels of salts, sugars, and others, modulating known osmoprotectants was tested and shown to improve the growth of E. coli in the presence of high concentrations of glucose, lactate, sodium lactate, and sodium chloride.114 The minimum inhibitory concentrations of these sol­utes was increased by either adding the well-known osmoprotectant betaine, increas­ing the synthesis of the disaccharide trehalose (a dimer of glucose), or both, and the combination of the two was more effective than either alone. Although the cells’ tol­erance to ethanol was not enhanced, the use of the combination strategy would be expected to improve growth in the presence of the high sugar concentrations that are becoming ever more frequently encountered in media for ethanol fermentations.

Accurately measuring the potential for ethanol formation represented by a cel — lulosic biomass substrate for fermentation (or fraction derived from such a material) is complex because any individual fermentable sugar (glucose, xylose, arabinose, galactose, mannose, etc.) may be present in a large array of different chemical forms: monomers, disaccharides, oligosaccharides, even residual polysaccharides. Precise chemical assays may require considerable time and analytical effort. Bioassay of the material using ethanologens in a set medium and under defined, reproducible condi­tions is preferable and more cost effective — and broadly analogous to the use of shake flask tests to assess potency of new strains and isolates and the suitability of batches of protein and other “complex” nutrients in conventional fermentation laboratories.115