By 1983, in experimental laboratory programs, selected Bacillus strains had achieved ethanol formation to 20 g/l (from 50 g/l sucrose as the carbon substrate) at 60°C, with ethanol as the major fermentation product; acetic and formic acids remained serious by-products, however, and evidence from laboratory studies suggested that ethanol accumulation followed (and depended on) the formation of those growth- inhibiting acids.46 The ability to run ethanol fermentations at 70-80°C with ther­mophilic microbes remains both a fascination and a conscious attempt to accelerate bioprocesses, despite the low ethanol tolerance and poor hexose-converting abilities of anaerobic thermophilic bacteria. In 2004, exploratory work at the Technical Uni­versity of Denmark tested isolates from novel sources (hot springs, paper pulp mills, and brewery wastewater), using three main criteria for suitable organisms:236

1. The ability to ferment D-xylose to ethanol

2. High viability and ethanol productivity with pretreated wheat straw

3. Tolerance to high sugar concentrations

Five good (but unidentified) strains were identified by this screening program, all from hot springs in Iceland,[29] the best isolate could grow in xylose solutions of up to 60 g/l.

Thermophilic and mesophilic clostridia also have their advocates, especially with reference to the direct fermentation of cellulosic polymers by the cellulosome multien­zyme complexes, as discussed in chapter 2, section 2.4.1). Bypassing the cellulosome is possible if cellulose degradation products (rather than polymeric celluloses) are used as carbon sources — this equates to using bacteria with cellulase-treated materi­als, including agricultural residues and paper recyclates. Laboratory studies with C. cellulolyticum tested cellobiose in this fashion but with chemostat culture so as to more closely control growth rates and metabolism.237 The results demonstrated that a more efficient partitioning of carbon flow to ethanol was possible than with cellulose as the substrate but that the fermentation remained complex, with acids being the major products (figure 3.11). Nevertheless, clostridia are open to metabolic engineering to reduce the waste of carbohydrates as acids and polymeric products or as vehicles for “consolidated bioprocessing” where cellulase production, cellulose hydrolysis, and fer­mentation all occur in one step — this is covered in chapter 4 (section 4.5).