Bacteria are traditionally unwelcome to wine producers and merchants because they are causative spoiling agents; for fuel ethanol production, they are frequent contami­nants in nonsterile mashes where they produce lactic and acetic acids, which, in high concentration, inhibit growth and ethanol production by yeasts.3435 In a pilot plant constructed and operated to demonstrate ethanol production from corn fiber-derived sugars, for example, Lactobacilli were contaminants that could utilize arabinose, accumulating acids that impaired the performance of the ethanologenic yeast; the unwanted bacteria could be controlled with expensive antibiotics, but this experience shows the importance of constructing ethanologens to consume all the major carbon

sources in lignocellulosic substrates so as to maximize the competitive advantage of being the dominant microbial life form at the outset of the fementation.36

Bacteria are much less widely known as ethanol producers than are yeasts but Escherichia, Klebsiella, Erwinia, and Zymomonas species have all received serious and detailed consideration for industrial use and have all been the hosts for recom­binant DNA technologies within the last 25 years (table 3.3).37-43 With time, and perhaps partly as a result of the renewed interest in their fermentative capabilities, some bacteria considered to be strictly aerobic have been reassessed; for example, the common and much-studied soil bacterium Bacillus subtilis changed profoundly in its acknowledged ability to live anaerobically between the 1993 and 2002 editions of the American Society for Microbiology’s monograph on the species and its rela­tives; B. subtilis can indeed ferment glucose to ethanol, 2,3-butanediol, and lactic acid, and its sequenced genome contains two ADH genes.44 The ability of bacteria to grow at much higher temperatures than is possible with most yeast ethanologens led to proposals early in the history of the application of modern technology to fuel etha­nol production that being able to run high-yielding alcohol fermentations at 70°C or above (to accelerate the process and reduce the economic cost of ethanol recovery) could have far-reaching industrial implications.45,46

Bacteria can mostly accept pentose sugars and a variety of other carbon sub­strates as inputs for ethanol production (table 3.3). Unusually, Zymomonas mobilis can only use glucose, fructose, and sucrose but can be easily engineered to utilize pentoses by gene transfer from other organisms.47 This lack of pentose use by the

wild-type organism probably restricted its early commercialization because other­wise Z. mobilis has extremely desirable features as an ethanologen:

• It is a GRAS organism.

• It accumulates ethanol in high concentration as the major fermentation product with a 5-10% higher ethanol yield per unit of glucose used and with a 2.5-fold higher specific productivity than S. cerevisiae.48

• The major pathway for glucose is the Entner-Doudoroff pathway (figure 3.4); the inferior bioenergetics of this pathway in comparison with glycolysis means that more glucose is channeled to ethanol production than to growth, and the enzymes required comprise up to 50% of the total cellular protein.48

• No Pasteur effect on glucose consumption rate is detectable, although inter­actions between energy and growth are important.49

Escherichia coli and other bacteria are, as discussed in chapter 2 (section 2.2), prone to incompletely metabolizing glucose and accumulating large amounts of carboxylic acids, notably acetic acid; with some authors, this has been included under the heading of the “Crabtree effect.”50,51 For E. coli as a vehicle for the production of recombinant proteins, acetate accumulation is an acknowledged inhibitory factor; in ethanol production, it is simply a metabolic waste of glucose carbon. Other than this (avoidable) diversion of resources, enteric and other simple bacteria are easily genetically manipulated, grow well in both complex and defined media, can use a wide variety of nitrogen sources for growth, and have been the subjects of decades of experience and expertise for industrial-scale fermentations — Z. mobilis also was developed for ethanol production more than 20 years ago, including its pilot-scale use in a high-productivity continuous process using hollow fiber membranes for cell retention and recycling.52