Fermentation of the sugars generated from enzymatic hydrolysis of biomass is another important step where a lot of technical advances are needed to make lignocellulosic ethanol technology feasible. What is desired in an ideal organism for biomass-ethanol technology would be a high yield of ethanol, broad substrate utilization range, resistance to inhibitory compounds generated during the course of lignocellulose hydrolysis and ethanol fermentation, ability to withstand high sugar and alcohol concentrations, higher temperatures and lower pH, and minimal by-product formation [143]. Unfortunately, all these features seldom exist together in any wild organism and the need of the industry would be to develop an organism which will at least partially satisfy these requirements [208].

The ability to use the hemicellulose component in biomass feedstock is critical for any bioethanol project. S. cerevisiae and Z. mobilis, the commonly employed organisms used in alcohol fermentation, lack the ability to ferment hemicellulose and derived pentose (C5) sugars. While there are organisms that can ferment C5 sugars (e. g., Pichia stipitis, Pachysolen tannophilus, Candida shehatae), the effi­ciencies are low. These organisms also need microaerophilic conditions and are sensitive to inhibitors, higher concentrations of ethanol, and lower pH [26]. Worldwide, a lot of R&D efforts are being directed to engineer organisms for fermenting both hexose (C6) and pentose (C5 sugars) with considerable amount of success [4]. There are a large number of microorganisms including bacteria and fungi that are capable of breaking down cellulose into monosaccharides either aerobically or anaerobically. The anaerobic bacteria include Bacteroids cellulo — solvents, Bacillus spp. Clostridium cellulolyticum, Clostridium cellulovorans, Cellvibrio gilvus, Candida lusitance, etc. The fermentation of cellulose yields a variety of products, e. g., ethanol, lactate, acetate, butyrate, H2, CO2, etc.

Introduction of bacteria has been the greatest microbiological innovation because they produce less biomass, low concentration of by-products, and high productivity. The bacterium Z. mobilis ferments glucose to ethanol by with a typical yield of 5-10% higher than that of most of the yeasts though it is lesser ethanol tolerant than industrial yeast strains [151]. However, the small bacterium is difficult to centrifuge. Zymomonas being a simple prokaryote, an important possibility for the future is development of genetically modified organisms especially tuned to more ethanol tolerance and improved centrifugability [109].

Clostridium thermosaccharolyticum, Thermoanaerobacter ethanolicus, and other thermophillic bacteria as well as Pachysolen tannophilus yeast [177] are employed in fermenting pentose sugars which are nonfermentable by other organisms usually employed in ethanol production. These bacteria also convert hexose sugars. They have minimum end-product inhibition because very high temperature reactions would allow simple continuous stripping of ethanol from the active fermenting mixture. The yield of alcohol was further improved by cocul­turing C. thermocellum with C. thermosaccharolyticum or C. thermomophydro- sulphuricum [156]. However, the organisms so far studied produce excessive quantities of undesirable by-products and require strict anaerobic conditions which would be difficult to maintain on an industrial scale [53, 154].

Several microorganisms, including bacteria, yeasts, and filamentous fungi, have capacity to ferment lignocellulosic hydrolysates generating ethanol. Among them, Escherichia coli, Z. mobilis, S. cerevisiae, and P. stipitis are the most relevant in the context of lignocellulosic ethanol bioprocesses. These microorganisms have different natural characteristics that can be regarded as either advantageous or disadvantageous in processes of ethanol production from hemicelluloses (Table 9.8).

Pure and mixed cultures of Z. mobilis and Saccharomyces sp. were tested for the production of ethanol by fermentation of medium containing sucrose (200 g/l) at 30°C. The best results were obtained using fermentation for 63 h by a mixed culture and the average hourly ethanol productivity was 1.5 g/l [2, 161]. Ethanol fermentation from culled apple juice was compared by using Sacharomyces and Zymomonas spp. Ethanol production from culled apple juice showed that fer — mentability of the juice could be enhanced by addition of Di-ammonium hydrogen phosphate (DAPH) or ammonium sulfate in Saccharomyces and DAHP in Zymomonas. Trace elements however, inhibited the fermentation in both the cases. Physicochemical characteristics of the fermented apple juices were also analyzed.

Overall, S. cerevisiae proved better than Zymomonas for fermentation of apple juice [161].