Comparison of Alternatives for the Fermentation of Pentoses to Ethanol by Yeasts

231 Jeffries, Thomas W. Comparison alternatives for the fermentation of pentoses to ethanol by yeasts. In: Lowenstein, Michael Z., ed. Energy applications of biomass: Proceedings of the National Meeting on Biomass R & D for Energy Applications; 1984 October 1-3; Arlington, VA. New York, NY: Elsevier Applied Science Publishers; 1985: 231-252. COMPARISON OF ALTERNATIVES FOR THE FERMENTATION OF PENTOSES TO ETHANOL BY YEASTS T. W. JEFFRIES* *U.S.D.A., Forest Service, Forest Products Laboratory, Madison, Wisconsin SYNPOSIS Hemicelluloses are major components of plant biomass. In hardwoods and agricultural residues, xylose is the principal hemicellulosic sugar. Xylose and other hemicellulosic sugars are recovered from lignocellulose more readily but are fermented with greater difficulty than is glucose. Xylose metabolism employs pathways distinctly different from those involved in the utilization of glucose. With most yeasts, xylose metabolism requires air. Aeration results in cellular respiration (as opposed to fermentation) and low ethanol yields. It is possible, however, to suppress respiration by feeding small amounts of glucose during the xylose fermentation. Some yeasts, such as Pachysolen tannophilus, will metabolize xylose anaerobically. Alternately, other yeasts will anaerobically ferment the keto isomer of xylose, xylulose, after it is formed from xylose by the action of xylose isomerase. In both instances, the fermentation rates are low. Improved strains of P .tannophilus have been obtained by W mutagenesis followed by enrichment for faster growth in nitrate-xylitol broth or by selecting for yeast strains incapable of using ethanol as a carbon source. Several yeasts have been described as superior xylose fermenters, including (in approximate ascending order): Candida tropicalis , Kluyveromyces marxianus , P . tannophilus, the mutant Candida sp. XF 217, and Candida shehatae (and its sex- ually perfect form, Pichia stipitis). The xylose fermentation rate of C . shehatae is 3 to 5 times higher than that obtained with P. tannophilus, but the yields of ethanol from xylose are. similar with the two organisms. The glucose fermentation rate and ethanol yield are lower with C. shehatae) than 232 with P. tannophilus . Unstable petite and grande strains of C. shehatae have bean obtained on urea + xylitol agar, and some show markedly different fermentation rates and products. Further strain improvement and process development should soon provide commercially practicable technology for the fermentation of xylose. 1 INTRODUCTION Within the realm of liquid fuel production from biomass, utilization of lignocellulose has focused largely on the problem of cellulose saccharification. Various approaches have been tried including hydrolysis by extra- cellular streptomycete and fungal cellulases, simultaneous saccharification and fermentation by cellulolytic bacteria, and acid hydrolysis followed by fermentation. To a certain extent, the attention given to cellulose is jus- tifiable. Cellulose comprises about half of the total weight of lignocellulose, and its fundamental constituent, glucose, is an excellent fermentation substrate. In a larger context, however, consideration of cellulose to the exclusion of the two other major constituents, hemicellulose and lignin, is futile. One reason is that it is uneconomical to throw away almost half of the feedstock. Another is that cellulose has appreciable commercial value as fiber. Converted to pulp, a ton of cellulose is worth $400 to $700; converted to ethanol, it is worth less than $300. In the kraft pulping process, lignin and hemicellulose are extracted under alkaline conditions and then burned to recover chemicals and energy. In some instances, the lignin is recovered for other applications. The hemicellulose is largely degraded to organic acids prior to combustion and has no current commercial value. Other technologies are being developed that will enable the efficient fractionation of lignocellulose into pulp-grade cellulose, useful lignin derivatives, and useful hemicellulosic sugars including xylose. The objective of the research described in this paper is to improve our knowledge of pentose metabolism in yeasts and to thereby provide the means for more efficient utilization of xylose. 1.1 Sources and Recovery Hemicellulosic sugars are major constituents of wood and agricultural residues. Table 1 shows the average proximate composition of seven commonly 233 occurring hardwood (angiosperm) and softwood (gymnosperm) species (Ref. 1-3) along with a few major U.S. agricultural residues (Refs. 4-6). Generally, hardwoods have slightly more neutral hemicellulosic sugars and cellulose but less lignin than softwoods. The composition of the hemicellulose differs in hardwoods and softwoods. In hardwoods, the predominant hemicellulosic sugar is the pentose xylose; in softwoods, the predominant hemicellulosic sugar is the hexose mannose. The xylose content of hardwoods is greater than softwoods. Most agricultural crops are angio- sperms and, like hardwoods, have xylose as the predominant hemicellulosic sugar. As described elsewhere in this symposium, low-grade hardwoods are available in relative abundance in the United States, but low-grade softwoods are in relatively short supply. The combination of a greater angiosperm resource and a higher proportion of xylose in that resource make xylose utilization a major concern in production of fuel from biomass. In addition to being relatively abundant, xylose is more readily recovered from hemicellulose than glucose is from cellulose. Dilute acid hydrolysis of hemicellulose yields about 85% to 90% of the xylose present in red oak; dilute acid hydrolysis of cellulose yields only about 50% to 60% of the glucose present. The difference between the xylose and glucose yields can be attributed directly to physical and chemical properties of the two polymers and hence is not readily amenable to process changes. The situa- tion is similar with regard to enzymatic hydrolysis. Although up to 90% of the glucose can be recovered from steam-exploded wood if sufficient cellu- 234 lase is added, at economical enzyme loadings glucose yields are substan- tially lower. Taking the differences in yields of xylose and glucose into account, roughly equivalent amounts of sugar can be recovered from the hemicellulosic and cellulosic fractions. 1.2 Biochemical Pathways and Fermentative Capacities Xylose can be assimilated by many bacteria, yeasts, and filamentous fungi, but initial steps of assimilation in yeasts and fungi are signifi- cantly different from those in bacteria. In yeasts and fungi, xylose is first reduced to xylitol and then oxidized to xylulose. In bacteria, the conversion from xylose to xylulose is catalyzed by xylulose isomerase in a single step (Ref. 7). This paper considers only the activities of naturally occurring yeasts. Most yeasts use a xylose reductase with a specific requirement for NADPH as a cofactor to reduce xylose to xylitol. Next,xylitol dehydro- genase specific for NAD oxidizes xylitol to xylulose. Consequently, assimilation of xylose converts NADPH into NADH. In Candida utilis, the organism best studied in this regard (Ref. 8),NADPH is supplied by the oxidative phase of the pentose phosphate pathway (PPP) in a closed cycle (Fig. 1). Under oxidative conditions, the only mode of fungal xylose assimilation known until 1981, NADH is recycled to NAD by respiration. Under anoxic conditions, NAD cannot be regenerated, and xylose assimilation ceases (Ref. 9). NADPH is used primarily in metabolic syntheses, and is generated mainly by the oxidative PPP (Ref. 10). Thus, production of NADPH by the oxidative PPP is thought to provide the means to assimilate xylose for aerobic production of ethanol by Candida tropicalis and other yeasts (Ref. 12). More recently, certain yeasts capable of fermenting xylose to ethanol in the absence of oxygen (anoxically) (Ref.13) have been shown to possess xylose reductase(s) capable of using either NADH or NADPH as a cofactor (Ref. 14). If NAD(H) can be used for both the reductive and oxidative steps of xylose metabolism, the balance between NAD and NADH can be maintained under anoxic conditions (Fig. 2) and xylose utilization is not dependent on aeration. The observation that P . tannophilus will ferment but not grow anaerobically on D-xylose could be attributed to the insufficient production of metabolic reductant or energy for growth. 235 2 PROCESS ALTERNATIVES 2.1 Coupled Isomerization and Fermentation In 1980, Wang, Shopsis, and Schneider (Ref. 15) showed that yeasts are able to ferment xylulose to ethanol under anoxic conditions. This find- ing had immediate implications because the conversion could be carried out readily by using commercial xylose (glucose) isomerase. The discovery was immediately seized upon and became the basis for considerable research and development in this field. As proposed for commercial practice, the technology would employ exogenous, immobilized xylose isomerase (already commercially derived from bacteria) to convert xylose to an equilibrium mixture of xylose and xylulose. The xylulose would then be fermented to ethanol and the residual xylose recycled over the xylose isomerase. The process would be continued until all xylose was consumed. Several variations on the basic process are possible and most have been attempted, but the principal remains the same. Xylose isomerase could be incorporated directly into the fermentation vessel or the xylulose

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