Inasmuch as xylose accounts for 30-60% of the fermentable sugars in hard­wood and herbaceous biomass, the fermentation of xylose to ethanol becomes an important issue. The efficient fermentation of xylose and other hemicel- lulose constituents is essential for the development of an economically viable process to produce ethanol from lignocellulosic biomass. Needless to say, co-fermentation of both glucose and xylose with comparably high efficiency would be most ideally desirable. As discussed earlier, xylose fermentation
using pentose yeasts has proven to be difficult due to several factors includ­ing the requirement for O2 during ethanol production, the acetate toxicity, and the production of xylitol as by-product. Xylitol (or, xyletol) is a naturally occurring low-calorie sugar substitute with anticariogenic (preventing pro­duction of dental caries) properties.

Other approaches to xylose fermentation include the conversion of xylose to xylulose (a pentose sugar, part of carbohydrate metabolism, that is found in the urine of individuals with the condition pentosuria [78]) using xylose isomerase prior to fermentation by Saccharomyces cerevisiae, and the develop­ment of genetically engineered strains [79].

A method for integrating xylose fermentation into the overall process is illustrated in Figure 4.12. In this example, dilute acid hydrolysis was adopted as a pretreatment step. The liquid stream is neutralized to remove any mineral acids or organic acids liberated in the pretreatment process, and is then sent to xylose fermentation. Water is added before the fermentation, if necessary, so that organisms can make full use of the substrate without having the yield limited by end-product inhibition. The dilute ethanol stream from xylose fer­mentation is then used to provide the dilution water for the cellulose-lignin mixture entering SSF. Thus, the water that enters during the pretreatment process is used in both the xylose fermentation and the SSF process.

The conversion of xylose to ethanol by recombinant E. coli has been inves­tigated in pH-controlled batch fermentations [80]. Relatively high concentra­tions of ethanol (56 g/L) were produced from xylose with good efficiencies.

In addition to xylose, all other sugar constituents of biomass, including glucose, mannose, arabinose, and galactose, can be efficiently converted to ethanol by recombinant E. coli. Neither oxygen nor strict maintenance of anaerobic conditions is required for ethanol production by E. coli. However, the addition of base to prevent excessive acidification is essential. Although less base was needed to maintain low pH conditions, poor ethanol yields and slower fermentations were observed below the pH of 6. Also the addi­tion of metal ions, such as calcium, magnesium, and ferrous ions, stimulated ethanol production [80].

In general, xylose fermentation does not require precise temperature con­trol, provided the broth temperature is maintained between 25 and 40°C. Xylose concentrations as high as 140 g/L have been positively tested to evaluate the extent to which this sugar inhibits growth and fermentation. Higher concentrations slow down growth and fermentation considerably. Ingram and coworkers [80-83] demonstrated that recombinant Escherichia coli expressing plasmid-borne Zymomonas mobilis genes for pyruvate decar­boxylase (PDC) and alcohol dehydrogenase II (ADHII; adhB) can efficiently convert both hexose and pentose sugars to ethanol. Ethanologenic E. coli strains require simpler fermentation conditions, produce higher concentra­tions of ethanol, and are more efficient than pentose-fermenting yeasts for ethanol production from xylose and arabinose [84].

A study by Sedlak, Edenberg, and Ho [28] successfully developed geneti­cally engineered Saccharomyces yeasts that can ferment both glucose and xylose simultaneously to ethanol. According to their experimental results, following rapid consumption of glucose in less than 10 hours, xylose was metabolized more slowly and less completely. Although the xylose conver­sion was quite significant by this genetically engineered yeast strain, xylose was still not totally consumed even after 30 hours. Ideally, xylose should be consumed simultaneously [26] with glucose at a similar efficiency and speed; however, this newly added capability of co-fermentation of both glucose and xylose has given new promise in the lignocellulosic ethanol technology leading to technological breakthroughs. They also found that ethanol was the most abundant product from glucose and xylose metabolism, but small amounts of the metabolic byproducts glycerol and xylitol also were obtained [28]. Certainly, later studies will be focused on the development or refine­ment of more efficient engineered strains, ethanol production with higher selectivity and speed, and optimized process engineering and flowsheeting.