Repression of xylose utilization by glucose in xylose-fermenting yeasts

Repression of xylose utilization by glucose in xylose-fermenting yeasts CHANDRA J. PANCHAL,LYNDABAST,' INGE RUSSELL,AND GRAHAMG. STEWART Research Department, Lubatt Brewing Company Limited, London, Ont., Canada N6A 4M3

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Received February 29, 1988 Accepted May 4, 1988 I., and STEWART, G. 1988. Repression of xylose utilization by glucose in xylose-fermentPANCHAL, C. J., BAST,L., RUSSELL, ing yeasts. Can. J. Microbiol. 34: 1316- 1320. The xylose-fermenting yeasts Pichia stipitis, Candida steatolytica, and Candida shehatae were subjected to fermentations in synthetic media containing mixtures of glucose and xylose. In all cases, repression of xylose uptake by glucose was observed, although the extent of repression was different with each yeast. While Candida shehatae was found to utilize xylose effectively in the presence of approximately 5% (wlv) glucose, Candida steatolytica could only utilize xylose when the glucose concentration was below 3 % (wlv), and Pichia stipitis required the glucose concentration in the medium to be below 2 % (wlv) before significant xylose utilization occurred. I., et STEWART, G. 1988. Repression of xylose utilization by glucose in xylose-fermentPANCHAL, C. J., BAST,L., RUSSELL, ing yeasts. Can. J. Microbiol. 34 : 1316- 1320. Les levures Pichia stipitis, Candida steatolytica et Candida shehatae, qui fermentent le xylose, ont CtC assujetties B des fermentations dans des milieux synthktiques contenant des mklanges de glucose et de xylose. Dans tous les cas, la rCpression de l'absorption du xylose par le glucose a CtC observke, bien que le niveau de rkpression fut diffkrent pour chaque levure. Tandis que le Candida shehatae s'est avCre un utilisateur efficace du xylose en presence d'approximativement 5 % @/v) de glucose, le Candida steatolytica n'a utilisC le xylose que lorsque la concentration du glucose Ctait infkrieure B 3 % (plv), alors que le Pichia stipitis a requis une concentration infkrieure i 2% (plv) avant que servienne une utilisation significative du xylose. [Traduit par la revue]

Introduction Significant global interest still persists for the use of fermentation ethanol as a fuel or fuel supplement. In North America and Europe a considerable research effort has been devoted to the cost-effective utilization of cellulose-based substrates for the production of ethanol. The major problem areas, however, have been the hydrolysis of cellulose and the fermentation of some of the sugars produced during hydrolysis. The hydrolysis of lignocellulosic materials, either chemically or enzymatically, results in the production of a mixture of compounds, the major components being sugars and lignin, while appearance of minor products such as acetic acid and furfural is dependent upon the type of process employed (Tran and Chambers 1986; Wayman et al. 1987; Wilke et al. 1983). The sugar component is mainly comprised of glucose (from cellulose) and xylose (from hemicellulose) with the former usually being 2 to 4 times more abundant than the latter (Jeffries et al. 1985; Wayman et al. 1987), although variations in this ratio can occur depending on the wood type. For efficient fermentation of a cellulose hydrolysate to ethanol, the major prerequisites of a suitable microorganism are as follows: (i) ability to ferment both glucose and xylose, (ii) ability to overcome glucose repression of xylose utilization, and (iii) resistance to products of cellulose hydrolysis such as acetic acid and furfural (Tran and Chambers 1986). While the commercially used yeast, Saccharomyces cerevisiae, ferments glucose, maltose, and sucrose very efficiently, it lacks the ability to ferment xylose (Batt et al. 1986). In the past few years, some yeasts have been identified that have the capability to ferment both hexoses and pentoses into ethanol (du Preez and Prior 1985; Jeffries 1985; Parekh et al. 1986). These 'Present address: Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ont., Canada N6A 5B9. Printed in Canada I ImprimC au Canada

yeasts, however, have been found to be slow fermenters and are susceptible to varying degrees of glucose repression of xylose utilization (Beck 1986). Since these yeasts possess the genetic capacity to ferment wood hydrolysis sugars, it is of interest to optimize fermentation conditions and to determine the degree of glucose repression encountered by some of these yeasts when utilizing glucose - xylose mixtures. The three xylose-fermenting yeasts Pichia stipitis, Candida steatolytica, and Candida shehatae were subjected to fermentations in synthetic media containing various concentrations of glucose and xylose in an attempt to compare the degree of glucose repression effected, with respect to xylose uptake, on each yeast.

Materials and methods Yeast cultures

The yeast strains investigated, with their Labatt culture collection numbers (and National Research Council Collection Ottawa or CBS numbers), are as follows: P. stipitis strain 1558 (NRC 2548), C. steatolytica strain 1563 (NRC 2959), and C. shehatae strain 1586 (CBS 5813). These were chosen from an initial screening of 15 yeasts that were found to be xylose fermenting (Aitken et al. 1985; G . Calleja, personal communications). Growth -fermentation medium

The nutrient medium (PYN) used for the studies consisted of the following ingredients per litre of distilled water: KH2P04, 2.0 g; (NH4),HP04, 2.0 g; MgSO, . 7H20, 1.13 g; peptone (Oxoid), 3.5 g; yeast extract (Oxoid), 3.75 g; and glucose and xylose at required concentrations; final medium pH 5.6. The minimal medium was prepared as described by Aitken et al. (1985) and consisted of the following ingredients per litre of distilled water: NaH2P04. H20, 69 g; yeast nitrogen base (without amino acids, Difco), 6.7 g; glucose, 120 g; and xylose, 30 g; final medium pH 5.5. A high concentration of phosphate buffer was used to maintain cell viability (Aitken et al. 1985). Inoculation concentrations were 1.0 or 5.0% (wlw) wet weight of cells pregrown in 10% xylose-containing medium (with the above nutrient composition) for 24 - 30 h. Fermentations were carried out in 300-mL shake flasks agitated at 150 rpm and incubated at

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30°C. Fed-batch fermentations were performed with 1.0% (wlw) inocula under conditions as described in the figures. Sampling Ten-millilitre samples were taken periodically and centrifuged at 4 000 X g for 10 min. The cell pellet was dried in a preweighed aluminum dish in an oven at 105OC for 24 h (biomass) and the supernatant was subjected to HPLC analysis to determine substrate and product concentrations. A Spectra-Physics model SP8100 highperformance liquid chromatograph incorporating a BioRad Aminex HPX-87P column operated at 85°C was used in conjunction with a Spectra-Physics model SP6040 XR refractive index detector and a Spectra-Physics SP4270 computing integrator.

Results and discussion The phenomenon of glucose repression in yeast has been the subject of many reports (e.g., Boucherie 1985; Entian 1986; Frohlich et al. 1985; Herrero et al. 1985; Mormeneo and Sentandreau 1986). In Saccharomyces strains, utilization of sugars such as maltose, galactose, sucrose, and dextrin is repressed in the presence of glucose at levels of 1% and higher. In large-scale fermentations, such as in brewing, glucose repression can have a significant impact on time and monetary constraints since a major component of wort (malt extract) is maltose (Stewart et al. 1983). During wort fermentations, the sugars glucose, fructose, and sucrose are first metabolized by the brewer's yeast, and only when these sugars have been almost completely utilized does maltose metabolism commence. In many fermentations this occurs 18-20 h after inoculating the wort with the yeast. Hence, substantial time, fermentation capacity, and monetary savings could be realized if simultaneous fermentation of the sugars could occur (Stewart et al. 1983). In fermentations involving cellulose hydrolysates, it has been reported that the utilization of xylose is also repressed in the presence of glucose (Enari and Suihko 1984; Jefferies et al. 1985; Lucas and van Uden 1986). When the yeasts P. stipitis, C. steatolytica, and C. shehatae were inoculated into synthetic minimal media containing 12% glucose plus 3 % xylose, varying degrees of repression of xylose utilization were observed with the three yeasts. As can be seen in Fig. lA, the uptake of glucose by P. stipitis was fairly rapid, with all of it being utilized within 75 h. The utilization of'xylose, however, was slow until the glucose concentration in the broth was below 2 % . Thus most of the glucose was utilized 50 h after the fermentation had begun, and after 75 h about 30% of the xylose had been consumed. The production of ethanol, glycerol, and arabitol (Fig. 1B) reflected the consumption of glucose, as seen in Fig. 1A. With C. steatolytica, the utilization of glucose was found to be very rapid and all of it was utilized within 48 h (Fig. 1C). However, as seen above with P. stipitis, significant uptake of xylose did not commence until glucose concentration in the medium was under 2 % . This yeast was also found to produce much higher levels of ethanol (Fig. ID), reaching a maximum of about 5.4% (wlv) after 48 h of fermentation. The glycerol plus arabitol concentration levelled off around 1.2 % , while less than 0.2% xylitol was formed, reflecting the low utilization of xylose by the yeast. Candida shehatae was also found to utilize glucose rapidly, with most of it being consumed in 48 h (Fig. 1E). In contrast to the other two yeasts, however, the uptake of xylose commenced almost simultaneously with that of glucose, although initially at a much slower rate. The rate of xylose uptake was found to be much faster after the glucose concentration in the medium had decreased to approximately

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FIG. 1. Fermentation of 12% glucose and 3% xylose minimal media by (A, B) Pichia stipitis strain 1558 (C, D) Candiah steatolytica strain 1563, and (E, F) Candida shehatae strain 1586. (Inoculation concentration, 5 % wet wt. of cells.) (A, C, E) Substrate uptake: 0 , glucose; A , xylose; (B, D, F) Product formation: 0, ethanol; M, biomass; 0 , glycerol plus arabitol; A , xylitol.

3.5 % . At the end of the fermentation (75 h), over 75 % of the xylose had been utilized. As with C. steatolytica, ethanol production in C. shehatae was much higher than with P. stipitis, reaching a level of about 5.1 % (wlv) after 48 h of fermentation (Fig. IF). The levels of the polyols, glycerol, and arabitol were, however, much higher in C. shehatae with the combined concentration reaching 2.5 % after 72 h of fermentation, while xylitol concentrations were generally below 0.3% . In fermentations employing only xylose as the substrate, C. shehatae has been found to produce much higher levels of xylitol (du Preez et al. 1986; Slininger et al. 1985; C. J. Panchal, manuscript in preparation). It was interesting to observe in the above results that the degree of glucose repression of xylose uptake was different for each yeast and that C. shehatae, which is con-


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FIG. 2. Xylose uptake by (A) P. stipitis strain 1558, (B) C. steatolytica strain 1563, and (C) C. shehatae strain 1586 in nutrient media containing 5 % xylose and 0 to 5 % glucose. (Inoculation concentration, 1% wet wt. of cells.) M, 0% glucose; A , 1% glucose; 0, 2% glucose; 0,3% glucose; a, 4% glucose; A, 5% glucose.

sidered to be an asexual form of P. stipitis (Barnett et al. 1983), showed the lowest extent of glucose repression. It was of further interest to determine the level of glucose repression when the cells were subjected to fermentations in synthetic media containing a fixed amount of xylose substrate and varying concentrations of glucose. As can be seen in Fig. 2A, the presence of glucose at levels of 2% or higher caused repression of xylose utilization by P. stipitis. Thus, at 24 h into fermentation, 1 % glucose caused about 5 % repression of xylose utilization, while 2 % glucose caused about 20 % repression of xylose utilization. With 3 % glucose, only 65 %

FIG. 3. Fermentation of xylose and glucose by P. stipitis strain 1558 in fed-batch operation. (A) Sugar uptake. (B) Ethanol and glycerol production. The initial fermentation medium was comprised of 7.3% xylose PYN. After 25 h of fermentation in shake flasks at 150 rpm and 30°C, the medium was diluted with the addition of an equal volume of 14.4%glucose, 2 X-PYN medium. Glucose; 0, xylose; 0, ethanol; W, glycerol. (The drop in xylose and products concentration at 25 h was due to dilution of the medium.)

of the xylose was utilized after 24 h, while during the same time with 4 and 5% glucose, only 50 and 33% of the xylose was used up, respectively. With C. steatolytica, the utilization of xylose in media lacking glucose was initially slow (Fig. 2B), since after 50 h of fermentation only 62% of the xylose was utilized. The repressive effect of glucose was, however, observed when glucose concentrations of 3 % or greater were present in the media. Interestingly, with 3 , 4 , and 5% glucose supplementation, the degrees of repression observed were very similar, being about 20%at 24 h into fermentation. Thus, C. steatolytica was less severely affected by glucose repression than P. stipitis, although the extent of xylose uptake was generally low. The yeast C. shehatae demonstrated much more resilience to glucose repression of xylose utilization than the other two yeasts. Thus in the presence of 1 and 2% glucose, there was a negligible effect on xylose utilization (Fig. 2C), while in the presence of 3 % glucose, only about 10%repression of xylose utilization was seen after 24 h of fermentation. With 4 % glucose in the medium, however, about 22 % repression of xylose utilization was seen during the same time interval, while 5% glucose caused almost a 40 % repression of xylose utilization. The two yeasts P. stipitis strain 1558 and C. shehatae strain 1586 were further subjected to a fed-batch type fermentation where xylose was used as the initial substrate and after 25 h of incubation, glucose was added. It is seen from the results in Fig. 3A that with P. stipitis strain 1558 over 50% of the xylose had been utilized after 25 h of fermentation with the

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production of 1.93% (wlv) ethanol (Fig. 3B). When glucose was added to the medium, however, fermentation of xylose was immediately halted, and as seen from the results, little or no utilization of xylose was observed for the following 25 h. (The sudden drop in xylose concentration seen in the figure was due to dilution of the broth and not utilization of the sugar by the yeast.) Only when glucose concentration in the medium dropped below 3.5 % (wlv) did any significant utilization of xylose recommence. These results are thus in agreement with batch fermentation results mentioned above and indicate that glucose repression of xylose utilization in P. stipitis strain 1558 occurs despite its initial growth on xylose. After 96 h of fermentation, less than 60% of the initial xylose had been utilized while practically all of the glucose had been fermented (Fig. 3A), and 4.52% (wlv) ethanol had been produced (Fig. 3B). When C. shehatae strain 1586 was subjected to a similar fed-batch type fermentation, it was observed that initial xylose utilization was more rapid than was the case with P. stipitis strain 1558. Thus after 25 h of incubation, about 70% of the xylose had been utilized (Fig. 4A), and 2.13 % (wlv) ethanol had been produced (Fig. 4B). When glucose was added to the medium, however, xylose utilization was slowed down for the following 9 h, after which significant utilization occurred. (As in Fig. 3A, the sudden drop in xylose concentration seen in Fig. 4A was due to dilution of the medium.) The glucose concentration in the medium was approximately 5.40% (wlv) when significant xylose utilization recommenced. After 96 h of fermentation, practically all the glucose and over 80% of the xylose in the original medium had been utilized (Fig. 4A), and 3.93 % (wlv) ethanol had been produced (Fig. 4B). These results also corroborate batch fermentation results with C. shehatae described above and show that the extent of glucose repression of xylose utilization encountered by this yeast is considerably less than that encountered by P. stipitis strain 1558. In contrast, however, C. shehatae produced less ethanol (3.93 % (wlv) as compared with 4.52 % (wlv)) but more glycerol (1.11 % (wlv) as compared with 0.31 % (wlv)) than P. stipitis after 100 h of fermentation, although the rate of ethanol production was faster by C. shehatae during the first 50 h of fermentation. In terms of overall ethanol fermentation, therefore, C. shehatae performed less efficiently than P. stipitis under the culture conditions employed. The results obtained above were averages of three separate trials and illustrate the differences among the three yeasts with respect to glucose repression. It would also appear that C. shehatae is more osmotolerant than the other two yeasts, since it could ferment both glucose and xylose readily at high concentrations (also confirmed by other experiments (C. J. Panchal, manuscript in preparation)). In spite of these traits, however, it is clear that at levels of 3 % and higher, glucose had a repressing effect on the utilization of xylose by C. shehatae during the early stages of fermentation. The above results (Fig. 2C) with this yeast, however, illustrate that after 50 h, all the xylose (and glucose) was completely utilized, whereas with P. stipitis only 80% of the xylose was utilized when the medium had been supplemented with 5% glucose (Fig. 2A). (Owing to the very slow utilization of xylose by C. steatolytica, this yeast was not subjected to fed-batch fermentation.) It is unclear what effect glucose has on the xylose metabolic pathway, although repression of xylose permease or xylose reductase cannot be ruled out. As mentioned before, the utilization of sucrose by yeast is also repressible by elevated con-

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FIG.4. Fermentation of xylose and glucose by C. shehatae strain 1586 in fed-batch operation. (A) Sugar uptake. (B) Ethanol and glycerol production. The initial fermentation medium was comprised of 7.3% xylose PYN. After 25 h of fermentation in shake flasks at 150 rpm and 30°C, the medium was diluted with the addition of equal volume of 14.4% glucose, 2 X-PYN medium. a, Glucose; 0, xylose; 0, ethanol; M, glycerol. (The drop in xylose and products concentration at 25 h was due to dilution of the medium.)

centrations of glucose. Sucrose is hydrolysed extracellularly by the enzyme invertase that is secreted into the periplasmic space by yeast (Perlman and Halvorson 1981). It has been shown that control of invertase synthesis in S. cerevisiae occurs at the SUC gene locus (Carlson and Botstein 1982). There are two independently regulated transcripts from the SUC gene coding for an intracellular nonglycosylated invertase and an extracellular glycosylated invertase. It is believed that the regulation of the extracellular invertase possibly occurs by glucose repression (Mormeneo and Sentandreau 1986). While the mode of action of glucose is not known definitively, it has been proposed that glucose repression occurs at both the transcriptional level as well as at the posttranscriptional levels such as translation, glycosylation, and secretion. While it is unlikely that glucose repression of maltose and xylose uptake is affected at the secretion stage (no extracellular synthesis of enzymes is required for uptake of these sugars (Lucas and van Uden 1986)), it has been shown that in S. cerevisiae, the gene SNFl (for sucrose nonfermenting) is mandatory for expression of various glucose repressible genes in response to glucose deprivation (Celenza and Carlson 1986). It is not known, however, if SNFl (which codes for a protein kinase) or a similar gene is required for xylose utilization as is the case with sucrose, galactose, maltose, and melibiose utilization by S. cerevisiae (Celenza and Carlson 1986). Attempts to isolate derepressed mutants of C. shehatae and


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P. stipitis using the glucose analogue, 2-deoxyglucose, have been successful to some degree in this laboratory. It is thus conceivable that glucose repression in these yeasts occurs in a manner similar to that in S. cerevisiae, namely inhibition of hexokinase enzymes (Frohlich et a l . 1985). It has been shown previously that the hexokinase isoenzyme 2 is involved in glucose repression and that its synthesis is regulated in coordination with glucose repression (Frohlich et a l . 1985). The isolation of HXK2 mutants in S. cerevisiae, which are defective in glucose repression, lends credibility to the hypothesis that HXK2 acts as a "recognition site" triggering glucose repression. Whether this is the case in xylose-fermenting yeasts is not known, although the kinetics of xylose utilization in the presence of glucose suggest that this could be possible. Further elucidation of this phenomenon would be required to have the pentose-fermenting yeasts be seriously considered for commercial-scale fermentation of cellulose hydrolysates for the production of fuel ethanol.

Acknowledgements The authors wish to thank their colleagues C. Bilinski, T. D'Amore, Y. HaJ-Ahmad, and K. Mussar for helpful discussions of the manuscript. Technical assistance by C. McLaughlin, I. Hancock, J. Sobczak, and R. Crumplen is also acknowledged. The authors are indebied to Dr. G . B. Calleja, National Research Council, Ottawa, for the yeast strains and communicating unpublished results. The work was carried out under the auspices of the Canadian Bioenergy Development Program, Department of Energy, Mines, and Resources Contract no. 23216-6-6104101-52, and the help of Dr. W. E. Lowe, scientific authority, is gratefully acknowledged. C. M., CALLEJA, G. B., AITKEN, J. C., ROBINSON, R. V., OSTROVSKI, and LEVY-RICK, S. R. 1985. Hydrogen fluoride solvolysis of cellulose. Report for Ministry of Energy, Mines and Resources, Renewable Energy Division, Ottawa. BARNETT, J. A,, PAYNE,R. W., and YARROW, D. 1983. Yeasts: characteristics and identification. Cambridge University Press, Cambridge. BATT, C. A., CARVALLO, S., EASSON,D. D., AKEDO,M., and SINSKEY,A. J. 1986. Direct evidence for a xylose metabolic pathway in Saccharomyces cerevisiae. Biotechnol. Bioeng. 28: 549-553. BECK,M. J. 1986. Effect of intermittent feeding of cellulose hydrolysate to hemicellulose hydrolysate on ethanol yield by Pachysolen tannophilus. Biotechnol. Lett. 8: 5 13-5 16. BOUCHERIE, H. 1985. A study on the control of carbon cataboliterepressed proteins in Saccharomyces cerevisiae. Biochim. Biophys. Acta, 825: 360-364. CARISON,M., and BOTSTEIN, D. 1982. Two differentially regulated mRNAs with different 5' ends encode secreted and intracellular forms of yeast invertase. Cell, 28: 145-154. CELENZA, J. L., and CARLSON, M. 1986. A yeast gene that is essen-

tial for release from glucose repression encodes a protein kinase. Science (Washington, D.C.), 233: 1175- 1180. DU PREEZ,J. C., and PRIOR,B. A. 1985. A quantitative screening of some xylose-fermenting yeast isolates. Biotechnol. Lett. 7: 241 -246. DU PREEZ,J. C., BOSCH, M., and PRIOR,B. A. 1986. Xylose fermentation by Candida shehatae and Pichia stipitis: effects of pH, temperature and substrate concentration. Enzyme Microb. Technol. 8: 360-364. ENARI,T.-M., and SUIHKO, M. L. 1984. Ethanol production by fermentation of pentoses and hexoses from cellulosic materials. CRC Crit. Rev. Biotechnol. 1: 229 -240. ENTIAN,K.-D. 1986. Glucose repression: a complex regulatory system in yeast. Microbiol. Sci. 3: 366-371. K.-U., ENTIAN, K.-D., and MECKE,D. 1985. The primary FROHLICH, structure of the yeast hexokinase PI1 gene (HXK2) which is responsible for glucose repression. Gene, 36: 105- 111. HERRERO, P., FERNANDEZ, R., and MORENO, F. 1985. Differential sensitivities to glucose and galactose repression of gluconeogenic and respiratory enzymes from Saccharomyces cerevisiae. Arch. Microbiol. 143: 216-219. JEFFRIES, T. W. 1985. Effects of culture conditions on the fermentation of xylose to ethanol by Candida shehatae. Biotechnol. Bioeng. Symp. 149-166. E. N. 1985. Effects of JEFFRIES, T. W., FADY,J. H., and LIGHTFOOT, glucose supplements on the fermentation of xylose by Pachysolen tannophilus. Biotechnol. Bioeng. 27: 171- 176. and VAN UDEN,N. 1986. Transport of hemicellulose L u c ~ s c., , monomers in the xylose fermenting yeast Candida shehatae. Appl. Microbiol. Biotechnol. 23: 491 -495. MORMENEO, S., and SENTANDREAU, R. 1986. Molecular events associated with glucose repression of invertase in Saccharomyces cerevisiae. Antonie van Leeuwenhoek, 52: 15-24. PAREKH,S. R., Yu, S., and WAYMAN, M. 1986. Adaptation of Candida shehatae and Pichia stipitis to wood hydrolysates for increased ethanol production. Appl. Microbiol. Biotechnol. 25: 300-304. D., and HALVORSON, H. 0. 1981. Distinct repressible PERLMAN, mRNAs for cytoplasmic and secreted yeast invertase are encoded by a single gene. Cell, 25: 525-536. SLININGER, P. J., BOTHAST, R. J., OKOS,M. R., and LADISCH, M. R. 1985. Comparative evaluation of ethanol production by xylosefermenting yeasts presented with high xylose concentrations. Biotechnol. Lett. 7: 431 -436. STEWART, G. G., PANCHAL, C. J., RUSSELL, I, and SILLS,A. M. 1983. Advances in ethanol from sugars and starch. In Ethanol from biomass. Edited by H. E. Duckworth and E. A. Thompson. Royal Society of Canada, Ottawa. pp. 4-52. R. P. 1986. Ethanol fermentation of red TRAN,A. V., and CHAMBERS, oak acid prehydrolysate by the yeast Pichia stipitis CBS 5776. Enzyme Microb. Technol. 8: 439 -444. WAYMAN, M., SEAGRAVE, C., and PAREKH, S. R. 1987. Ethanol fermentation by Pichia stipitis of combined pentose and hexose sugars from lignocellulosics prehydrolysed by SO2 and enzymatically saccharified. Process Biochem. 22: 55 -59. WILKE,C. R., MAIORELLA, B., SCIAMANNA, A,, TANGU, K., WILEY, D., and WONG,H. 1983. Enzymatic hydrolysis of cellulose. Noyes Data Corporation, Park Ridge, NJ. pp. 64- 102.