Ethanol is being hailed as the fuel of the future. Interest in the production of fuel ethanol from renewable sources has increased significantly. In order for fuel ethanol production to become a practical reality, cheaper substrates and more efficient production processes are needed [1, 2]. Biomass, which includes all plant and plant derived material, forms a potential renewable source of sugars that can be fermented to produce fuel ethanol and a variety of other fuels and chemicals. In addition to the many benefits common to renewable energy, biomass is particularly attractive because it is currently the only renewable sustainable energy source for liquid transportation fuel.
Lignocellulosic biomass is an attractive energy feed-stock because it is an abundant, domestic, renewable source that can be converted to liquid transportation fuels (From Biomass to Biofuels: A Roadmap to the Energy Future; Office of Science, US Dept. of Energy, December 2005).
Lignocellulosic biomass consists of three major components: cellulose (˜40-50%), hemicellulose (˜25-35%), and lignin (˜15-20%) [3]. Of these, cellulose and hemicellulose constitute the polysaccharides that can be hydrolyzed to sugars that could be fermented to ethanol. In biomass, the majority of cellulose is a crystalline polymer of glucose that is relatively difficult to hydrolyze into its monomeric sugar residues. Hemicellulose is a short branched polymer of pentose and some hexose sugars that surround the cellulose fibrils and is much less organized [4]. The pentose sugars consist primarily of xylose and to a smaller extent arabinose, while the hexose sugars are usually galactose and mannose. Due to its relatively open structure, the hemicellulose fraction is easier to convert to its sugar monomers by various pretreatment techniques than the cellulose fraction.
For the conversion of lignocellulosic biomass to bioethanol to be economically feasible, it is imperative that the hemicellulose-derived monomeric sugars be fermentable along with the glucose derived from cellulose. Unfortunately, no known native (or wild type) microorganisms are able to efficiently ferment both glucose and xylose to ethanol. Wild type Saccharomyces cerevisiae strains can readily ferment glucose as well as other sugar components of biomass like mannose, fructose and galactose [5]. Xylose, which forms a major portion of hemicellulose, cannot be fermented by the same native strains of yeast. Several non-Saccharomyces strains of yeast, such as Pichia stipitis and Candida shehatae, are known to ferment pentose sugars more efficiently than other yeasts [6]. In such yeasts, the xylose metabolism pathway goes from xylose to xylitol to xylulose [7, 8]. In other yeast strains as well as bacteria and fungi, xylose can also be converted to xylulose via a single enzyme, xylose isomerase (XI). Several yeasts, including S. cerevisiae, that cannot ferment xylose are able to ferment xylulose, the ketose isomer of xylose [9-12] to ethanol. Considerable effort has been focused on the genetic modification of microorganisms so that both xylose and glucose can be efficiently metabolized using the same organism [13-25].
While genetically modified organisms (GMOs) have potential for fermentation of pentose and hexose sugars, their genetic stability, overall ethanol yield, and ability to survive under the conditions of industrial fermentation are unproven [26, 27]. Hence, an alternative approach to fermentation of xylose to ethanol involves using native yeast strains with the addition of exogenous enzymes for the isomerization of xylose. In this approach, the production of xylulose is accomplished using immobilized glucose/xylose isomerase [11, 28-30]. The appeal for this approach is that XI, along with amylase and protease, is among the most widely and cheaply available commercial enzymes [31]. Hydrolysate from lignocellulosic biomass will contain both xylose and glucose. The affinity of XI for xylose is typically 1 to 2 orders of magnitude greater than its affinity for glucose; hence, isomerization of xylose to xylulose will dominate over isomerization of glucose to fructose [31]. However, any fructose formed is readily fermentable by Saccharomyces to produce ethanol, so fructose formation is not a cause for concern.
Although XI is capable of converting xylose to xylulose, under conditions where XI has significant activity, the equilibrium ratio of xylose:xylulose is typically high (on the order of 5:1) [32-34]. Hence, xylose isomerization does not have a favorable forward equilibrium. One way to increase xylose conversion is to drive the isomerization forward by removal of the product xylulose. Simultaneous isomerization and fermentation (SIF), where the isomerization of xylose and the fermentation of xylulose to ethanol occur simultaneously in the same vessel, is one method for increasing xylose utilization. However, SIF does have inherent limitations due to the pH range over which XI is active. All commercially available XI's have optimal activity at pH 7 to 8, and the XI activity drops sharply as the pH decreases. In contrast, the optimal pH for the fermentation is in the range of 4 to 5. The large pH difference associated with these two steps poses a problem for conducting SIF efficiently. The SIF can be carried out at a compromised pH between 4 and 7, but the results are less than optimal for both reactions [11]. Efforts to isolate a XI with optimal activity at significantly lower pH for SIF were also noted in the literature [30]. However, it does not appear that this enzyme has the same level of activity as displayed by the commercially available enzymes.
The instant invention provides a further improvement over one of the co-inventor's prior inventions disclosed in the Fournier et al. U.S. Pat. No. 5,254,468 and the Fournier et al. U.S. Pat. No. 5,397,700, the disclosures of each of which are incorporated herein by reference in their entireties.
Considering the above-mentioned concerns, it is clear that there remains a need in the art for a method of developing a process that enables efficient fermentation of xylose and hexose sugars.