231

J e f f r i e s , T h o m a s W . C o m p a r i s o n a l t e r n a t i v e s f o r t h ef e r m e n t a t i o n o f p e n t o s e s t o e t h a n o l b y y e a s t s .I n : L o w e n s t e i n , M i c h a e l Z . , e d . E n e r g y a p p l i c a t i o n so f b i o m a s s : P r o c e e d i n g s o f t h e N a t i o n a l M e e t i n g o nBiomass R & D fo r Energy Appl ica t ions ; 1984October 1 -3 ; Ar l ing ton , VA. New York , NY: Elsev ie rA p p l i e d S c i e n c e P u b l i s h e r s ; 1 9 8 5 : 231-252 .

COMPARISON OF ALTERNATIVES FOR THE FERMENTATION OF PENTOSES TO ETHANOL BYYEASTS

T. W. JEFFRIES*

*U.S.D.A., Forest Service, Forest Products Laboratory, Madison, Wisconsin

SYNPOSISHemicelluloses are major components of plant biomass. In hardwoods

and agricul tural residues, xylose is the pr incipal hemicel lulosic 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

a i r . 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 Pachyso l en t a n n o p h i l u s , will metabolize xylose anaerobi-

cally. 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): Cand ida tropicalis , Kluyveromyces marxianus , P . tanno-

phi lus , the mutant Cand ida sp. XF 217, and Candida shehatae (and its sex-

ual ly perfect form, P i c h i a s t ip i t i s). The xylose fermentation rate of C .

sheha t ae is 3 to 5 times higher than that obtained with P . tannophi lus , 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 . tannophi lus . Unstable petite and grande strains of C . shehatae have

bean obtained on urea + xylitol agar, and some show markedly different fer-

mentation rates and products. Further strain improvement and process devel-

opment should soon provide commercially practicable technology for the fer-

mentation of xylose.

1 INTRODUCTIONWithin the realm of liquid fuel production from biomass, utilization

of lignocellulose has focused largely on the problem of cellulose saccharif-

icat ion. 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-

t i f i ab le . Cellulose comprises about half of the total weight of lignocellu-

lose, 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; con-

verted 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 hem-

icellulose is largely degraded to organic acids prior to combustion and has

no current commercial value. Other technologies are being developed that

wil l enable the eff ic ient f ract ionat ion of l ignocel lulose into pulp-grade

cellulose, u se fu l l i gn in de r iva t i ve s , and useful hemicel lulosic 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

and cellulose but less lignin than softwoods.

icellulose differs in hardwoods and softwoods.

ant hemicellulosic s u g a r i s t h e p e n t o s e

predominant hemicellulosic sugar is the hexose

neutral hemicellulosic sugars

The composition of the hem-

In hardwoods, the predomin-

xylose; in softwoods, the

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 soft-

woods are in relatively short supply. The combination of a greater angio-

sperm resource and a higher proportion of xylose in that resource make

xylose utilization a major concern in production of fuel from biomass.

In addi t ion to being relat ively abundant ,

recovered from hemicellulose than glucose is from

hydrolysis of hemicellulose yields about 85% to 90%

red oak; dilute acid hydrolysis of cellulose yields

xylose is more readily

cel lulose. Dilute acid

of the xylose present in

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-

t ial ly lower. Taking the differences in yields of xylose and glucose into

account, roughly equivalent amounts of sugar can be recovered from the hem-

icellulosic and cellulosic fractions.

1.2 Biochemical Pathways and Fermentative Capacities

Xylose can be assimilated by many bacteria, yeasts, and filamentous

fungi , but ini t ial s teps of assimilat ion in yeasts and fungi are s ignif i-

cantly different from those in bacteria. In yeasts and fungi, xylose is

first reduced to xylitol and then oxidized to xylulose. In bacter ia , 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, assimi-

lation of xylose converts NADPH into NADH. In Candida utilis , the organism

best s tudied 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 con-

ditions, 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 produc-

tion of ethanol by C a n d i d a t rop ica l i s and other yeasts (Ref. 12). More

recent ly, cer tain 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 . tannophi lus 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 tech-

nology would employ exogenous, immobilized xylose isomerase (already com-

mercially 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 fermen-

tation vessel or the xylulose could be produced exogenously and separated

from the xylose prior to fermentation.

The process of sequential isomarization and fermentation is affected

by several factors . At equilibrium in aqueous solution, xylose isomerase

catalyzes the formation of about 17% xylulose from xylose. In comparison

47% fructose is formed from glucose. The lower equilibrium obtained with

xylose is offset somewhat by the higher turnover rate of xylose isomerase

acting on its native substrate. Other reaction conditions, such as tempera-

ture or the inclusion of borate to chelate the xylulose as it is formed, can

affect the equilibrium. The literature on xylose isomerase and the xylulose

fermentation has been covered in earlier reviews (Refs. 16-19).

Cost estimates for the isomerization of xylose have been based on

information from the isomerization of glucose to high fructose corn syrup

(HFCS). Xylose isomerase is used in both instances. But whereas HFCS pro-

duction employs high sugar concentrations and optimal conditions for isomer-

izing glucose, these factors must be compromised with those optimal for fer-

mentation. Reliable values for the cost of HFCS production are difficult to

obtain outside the industry; however, one figure published in 1978 placed

the cost of isomerization a t 2 . 3 t o 3 . 7 ¢ / k g o f f r u c t o s e p r o d u c e d

(Ref. 20). Given that roughly 5.9 kg of sugar are required to produce 1 gal

of ethanol, the isomerization reaction would add about 15¢ to 25¢/gal to the

cost of ethanol production as compared to an equivalent fermentation using

glucose as the feedstock.

236

Although sequential xylose isomerization and fermentation is tech-

nically feasible, it is hampered by several factors: the cost of the enzy-

matic isomerization, the formation of xylitol as a by-product, inhibition of

xylose isomerase by xylitol, the use of separate optimal pile and tempera-

tures for isomerization and fermentation, and the low rate of the xylulose

fermentation. Alternatively, new yeasts might be constructed by recombinant

DNA techniques to possess xylose isomerase. The approach of employing

recombined yeasts suffers from many of the difficulties listed above plus

the basic problem of obtaining adequate expression of enzymatic activity.

Moreover, there are few inherent advantages in carrying out a multistep pro-

cess in a single reactor (or with a single organism) If the process steps

have different optimal conditions or if separate organisms are capable of

carrying out each of the steps more efficiently.

2.2 Fermentation Rates with Different Sugars

The specific xylulose fermentation rate, even with the best strains

of yeasts, is appreciably lower than the rate attained with glucose, and in

some instances, it is lower than the rate attained with the direct fermenta-

tion of xylose. About 60 yeast strains have been screened for their abil-

ities to ferment either xylulose or a mixture of xylose and xylulose under

equilibrium conditions (Refs. 21-23). Results with some of the best strains

are summarized in Table 2. In general, C . tropicalis and Schizosaccharo-

myces pombe ferment xylulose most rapidly, but strains of Saccha romyces

cerevisiae also give better-than-average rates.

Volumetric fermentation rates (g ethanol/L•h) are subject to a great

dea l o f va r i a t i on because cell growth varies under the condi t ions

employed. Indeed, Immobilization of cells can lead to very high volumetric

rates because of high cell densities. Note, however, that specific fermen-

tation rates (g ethanol/g dry wt of cells•h) generally decrease after cells

are immobilized. Although immobilization has been attempted with both the

xylulose and xylose fermentations, the volumetric fermentat ion rates

obtained do not approach those commonly observed in the fermentation of

glucose by free cells of S. cerevisiae or Zymomonas mobilis .

It is better to use specific rates when comparing fermentation of

different sugars. The highest reported specific Xylulose fermentation rate

is about 1/18 of the specific glucose fermentation rate obtained with S .

cerevisiae . On the other hand, the highest reported xylose fermentation

rate is about 1/6 the specific glucose fermentation rate. For both of these

237

pentoses, llttle is known about the regulatory biochemical steps or the con-

ditions optimal for fermentation.

Yeas t s f e rmen t g lucose , xy lose , and xy lu lose a t cha rac t e r i s t i c

r a t e s . In a survey of several different xylose- and Xyluloee-fermenting

yeasts, Maleszka and Schneider (Ref. 22) showed that yeasts capable of fer-

menting xylose were typically poor xylulose fermenters, and vice versa. Two

primary exanples are S. pombe and P. tannophilus . S . pombe is a very good

fermenter of glucose, but it does not metabolize xylose at all; on the other

hand, it is a good fermenter of xylulose. Pachysolen tannophilus ferments

glucose more readily than it does xylose, but it ferments xylulose poorly.

The fermentation rate obtained on glucose is still much lower than that

attained with S. cerevisiae , S. p o m b e , and other yeasts used for commercial

alcoholic fermentations. In this work from our laboratory, C . shehatae has

been shown to ferment glucose at a lower specific rate than P . tannophi lus ,

238

even though it ferments xylose much more rapidly (Table 2 and Figs. 3, 4,

and 5).

2.3 Incidence of Xyose-Fermenting YeastsSixty-four percent of the species listed in Ref. 32 are cited as cap-

able of assimilating xylose and 7% are cited as variable) but none is listed

as capable of fermenting this sugar. A separate taxonomic treatment by

Barnette, Payne, and Yarrow lists P . tannophi lus and P . stipitis as capable

of fermenting xylose (Ref. 35). This discrepancy stems in part from the

inability of these yeasts to grow under anaerobic conditions. Even though

P . tannophi lus will ferment xylose, no cell growth occurs anaerobically, and

because the specific fermentation rate is very low, negative results appear

unless high cell densities are employed as the inoculum. In a study specif-

ically designed to identify xylose-fermenting yeasts (Ref. 36), 200 species

able to ferment glucose anaerobically and to grow on xylose aerobically were

tested for their abilities to ferment D-xylose. In most of these species,

ethanol production on xylose was negligible. Only 19 species produced

between 0.1 and 0.1 g/L of ethanol. Strains of Brettanomyces naardenensis ,

Candida shehatae , Candida tenuis , Pachysolen tannophilus , Pichia segobien-

sis, and Pichia stipitis produced more than 1 g/L ethanol from 2% xylose.

3 PROCESS VARIABLES

3.1 Effects of AerationAeration stimlates cell growth and occasionally stimulates fermenta-

t i on a s we l l . Only a few yeasts are capable of ( l imited) anaerobic

growth. This inability stems in part from a biochemical requirement for

molecular oxygen in the synthesis of membrane steroids. However, Schneider

and co-workers (Refs. 7,8) have shown that the inability of P . tannophi lus

to grow anaerobically cannot be overcome by the addition of ergosterol or

unsaturated fatty acids. Maleszka and Schneider have also shown that oxygen

and mitochondrial function are also required for S . cerevisiae to grow on

xylulose (Ref. 39). These observations suggest that the anaerobic metabo-

lism of pentoses supplies metabolic energy (ATP) fast enough to satisfy only

the basal metabolic demand but does not provide enough ATP to allow cell

growth.

Aside from affecting growth, aeration strongly affects the specific

fermentation of glucose by P. tannophilus . This was first shown by Schef-

fers and Wiken (Ref. 40). Unexpectedly, the stimulation does not extend to

239

xylose (Table 3). Aeration does increase the volumetric fermentation rate,

but this stimulation can be attributed to increase in cell mass.

Pachyso l en tannophi lus is not alone in showing a stimulation of the

glucose fermentation by aeration. The genus Brettanomyces shows this trait

among most of its species (Ref. 40). Aeration is also known to play a role

in the fermentation of glucose by S a c c h a r o m y c e s (Ref. 41), but in this

in s t ance , i t i s p r imar i ly impor t an t in maintaining cell viabil i ty and

ethanol tolerance (Ref. 42).

Aeration decreases the yield of ethanol from xylose by P . tannophi-

lus. It is hypothesized that the reduction occurs by virtue of increased

ethanol respiration (Ref. 41). Under strictly anaerobic conditions, P . tan-

n o p h i l u s produces essentially the same net yield of ethanol from xylose--

after correcting for the amount of xylose going into xylitol--as it does

from glucose (Table 4). The amount of carbon going into xylitol is deducted

from the calculation, because it accumulates early in the fermentation path-

way and essentially represents sugar that is not metabolized. The yield of

xylitol decreases under aerobic conditions and increases under anaerobic

conditions (Ref. 28).

3.2 Effect of Glucose on Ethanol Yields from Xylose

The aerobic ethanol yield from xylose can be improved by adding small

amounts of glucose during the fermentation (Table 5). By using this

approach, a high rate of ethanol production can be achieved with relatively

little ethanol loss. The improvement in yield is not observed under anaero-

bic conditions, and control experiments show that adding glucose at the low

concentrations employed does not affect the rate of xylose assimilation. So

240

the observed improvement in yield is attributed to a decrease in the rate of

ethanol respiration (Ref. 28).

3.3 Effects of Nitrate on Ethanol Production

Nitrate lncreases the levels of PPP enzymes in yeasts, fungi, and

plant cells (Refs. 10,45-57). The enhancement occurs because the PPP is the

primary source of NADPH and because nitrate reductase requires large amounts

of NADPH for nitrogen assimilation. It was for this reason that we examined

the ability of nitrate to stimulate the rate of xylose fermentation in P .

t a n n o p h i l u s (Ref. 48). Although nitrate stimulated the specific aerobic

xylose fermentation rate, cells grew slower on nitrate, and under anaerobic

conditions, the specific rate of ethanol production of nitrate-grown cells

was appreciably lower. The anaerobic effect was dependent on both pregrowth

on nitrate and the presence of nitrate in the medium.

241

3.4 Effects of Nitrate and Xylitol on Strain Selection in P. tannophilus

Xylitol and nitrate were used in an indirect enrichment and selection

method to obtain improved xylose fermenters. These restrictive carbon and

nitrogen sources were used to help select vigorous rather than crippled

mutants. P . tannophilus tends to accumulate xylitol during growth on

xylose, so it was used as a sole carbon source on the assumption that

xyli tol ut i l izat ion is a rate-l imit ing step. P. tannophilus grows slower on

nitrate than on other more readily assimilated nitrogen sources, and

nitrate-grown cells exhibit higher specific aerobic fermentation rates than

ammonia-grown cells. Moreover, nitrate is knowm to induce higher levels of

PPP enzymes; therefore, by using i t as a ni t rogen source, the cel ls are

fully induced for PPP enzymes. Any faster-growing mutant would have meta-

bolic capacities beyond the normal adaptative range of the parent. For

these reasons, cells able to grow well on nitrate should be capable of gen-

erating NADPH at an elevated rate. Hence, nitrate was chosen as the sole

nitrogen source. Taken together, these restrictive conditions slowed growth

so that a minimum of 7 to 10 days was required for significant growth to

occur in liquid or on solid media (Ref. 49).

Strains capable of relatively rapid growth on nitrate + xylitol media

were generally much better xylose fermenters than the parent s train or

mutants obtained under less restrictive conditions (Fig. 6). The strains

derived from nitrate + xylitol enrichment produce ethanol twice as fast and

in 30% better yield than the parent strain under aerobic conditions. More-

over, they have a specific fermentation rate 50% greater under anaerobic

conditions (Fig. 7). These strains are stable under repeated subculture,

and the enrichment and selection method has been successfully employed

several times with P. tannophilus .

Other approaches to obtaining Improved mutants of xylose-fermenting

yeasts have been attempted, including selecting strains of C a n d i d a sp. for

relative growth rates on xylose and xylitol media (Ref. 50) and selecting

strains of P . t a n n o p h i l u s for low rates of ethanol assimilation (Ref. 51).

Both of these methods have led to improved xylose-fermenting strains.

3.5 Candida shehatae as a Rapid Xylose Fermenter

Although mutation and selection methods have been successful in

obtaining incremental improvements in the xylose fermentation rates of

laboratory strains, enrichment and screening of yeast strains from natural

sources has led to the identification of C . sheha t ae as a species capable of

fermenting xylose at four to five times the specific rate of P . t a n n o p h i l u s

242

(Ref . 31) . C a n d i d a sheha t ae produces up to 3.8% (w/w) ethanol from

16% D-xylose (Fig. 8) and about 5% ethanol from 16% D-glucose (Fig. 9). In

comparison to P. tannophilus , which forms much more ethanol on D-glucose

than on D-xylose, with C . shehatae the final ethanol concentrations on these

two sugars and the ethanol yields (after deducting xylitol production) are

about the same (Table 6).

P i c h i a s t i p i t i s is the sexual ly perfect s tage of C . s h e h a t a e .

Although no published study has yet made a detailed comparison of the fer-

mentative capacities of various strains of these two forms, work in this

laboratory has shown that for the most part, they are very similar. As much

variation exists among strains of each form as between the anomorph and the

teleomorph. In other research, separate studies have compared fermentation

characteristics of P . t a n n o p h i l u s with either C . sheha t ae (Ref. 50) or P .

s t ip i t i s (Ref. 53) and found C . s h e h a t a e or P . s t ip i t i s to be the better

fermenter in each case.

Strain improvement Is proceeding with C. shehatae . One of the first

approached tried was to apply the same enrichment and selection method used

successfully with P . tannophilus . According to conventional taxonomic

tests, C . s h e h a t a e is unable to use nitrate as a nitrogen source (nitrate

negative). We have found that some strains will grow to a limited extent In

nitrate + xylitol medium, but this approach has not been successful with

this organism. The fastest xylose-fermenting strain we have obtained to

date is an unstable petite-like variant derived from C. shehatae ATCC 22984

by selection on urea + xylitol medium (Ref. 54).

Certain strains of C . sheha t ae exhibit marked small colonies remin-

iscent of the petite mutation in Saccharomyces when they are grown on urea +

xyli tol agar . Conventionally, the pet i te designation refers to s trains

243

showing small colonies on glucose and deficiencies in respiratory metabo-

lism. Many of the petite-like colonies of C . shehatae ATCC 22984 on xylitol

agar show diminished respiratory capacity as judged by the tetrazolium over-

lay method (Ref. 55). The petite-like colonial morphology of C . shehatae is

expressed on both xylose and glucose. However, strains designated grande on

xylitol exhibit slightly smaller colonial diameters when growing on glu-

cose. Conversely, strains designated petite on xylitol show larger colonial

diameters when growing on glucose. The transitions between small and large

colonial sizes occur in both directions (Table 7).

The xylose fermentation characteristics of petite and grande strains

are related to respiratory act ivi t ies . When a tetrazolium agar overlay is

applied to colonies growing on urea + xylitol agar, five different colony

types can be distinguished (Table 8). Some of these strains, occurring in

low frequency, exhibit a small colony diameter and a strong tetrazolium

react ion on xyl i tol . These strains are poor ethanol producers, but they

form other products. The petite colonies showing a weak tetrazolium reac-

tion on xylitol tend to produce less xylitol and glycerol, but the higher

overall yield of all products formed by grande tetrazolium-positive strains

tends to suggest that these strains have lower endogenous respiratory activ-

ity when growing fermentatively. Neither grande nor petite strains show

signif icant te trazol ium react ions on xylose. Preliminary studies in my

laboratory have shown that a similar petite-like variation occurs in strains

of P . s t i p i t i s . An improved understanding of this petite-like variation

should eventually contribute to the isolation of yeast strains capable of

fermenting xylose economically under practical conditions.

244

3.6 Comparison of Various Xylose-Fermenting Yeast StrainsVarious researchers have used many different media and cultural con-

ditions in studying different yeast strains for their abilities to ferment

xylose. While these strains doubtless possess different optima for ethanol

production, it is useful to compare them under a single set of fermentation

conditions. My lab has recently done such a comparison. Results show that

although the mutant strains of P. tanophi lus and Canida sp. performed better

than their parent strains, all strains of C . shehatae were better than any

other s t rain tested. These results show that further strain development

with C . shehatae as well as enrichment and selection of new isolates should

continue.

4 CONCLUSIONS

1. Xylose is widely avai lable in angiosperm residues and more

readily recoverable than glucose from lignocellulosic materials.

2 . Although a two-stage isomerization and fermentation of xylose is

feasible, direct fermentation of xylose to ethanol can proceed at a higher

specific rate and is more likely to have a lower overall cost.

3. Xylose fermentation rates and ethanol yields are still much lowerthan commercial glucose fermentation, but they are improving. For special-

ized situations where waste xylose streams constitute a disposal problem,

fermentation to ethanol may be economical.

245

4 . Biochemical, genetic, and strain selection studies

recently been undertaken, and i t is expected that they should

better strains and fermentation conditions.

5 ACKNOWLEDGEMENT

have only

resu l t i n

The author wishes to thank Henry Schneider and his coworkers at the

National Research Council, Ottawa, Ontario for useful discussions and for

sharing references and preprints of unpublished data.

6 REFERENCES

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247

248

249

250

251

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