Trends in Biotechnology, Vol. 3, No. 8, 1985

stimulated under aerobic conditions, Emerging technology for fermenting and the classical methods do not pro- vide for aeration during screens for fer- D-xylose mentative activity13. Current efforts to ferment D-xylose Thomas W. Jeffries largely began with the discovery by Wang, Shopsis and Schneider in In the past four years, numerous which convert D-xylose to 198014 that Schizosaccharomyces pombe ethanol have been reported. The conversion occurs most readily under and various other yeasts would’ aerobic conditions. Various aspects of this conversion have provided ferment D-, the keto-isomer of new insight into the mechanisms and metabolic regulation of ethanol D-xylose, to ethanol. This finding was fermentation in yeasts. Although specific fermentation rates, product significant because D-xylose can be yields and product concentrations are significantly lower with D-xylose readily converted to D-xylulose by than with D-, technology is emerging which may prove to be D-xylose (= glucose iso- feasible for commercial fermentation of D-xylose-containing waste merase), Since this is so readily streams. available and produced on such a large scale, technology for the conversion of D-Xylose is the second most abundant by Karczewska4 in 1951. This D-xylose to ethanol by a two-stage in nature, comprising up to 25% observation, however, went largely un- isomerization and fermentation was of the total dry weight of woody angio- recognized - a major review in 19765 rapidly developed 15 18. Interest in the sperms1 and an even larger fraction of recorded that roughly half of all two-stage process waned, however, some agricultural residues2 where it species listed would assimilate when the direct fermentation exists as the polymer . Even D-xylose for aerobic growth but none of D-xylose to ethanol by though it is not yet cheap nor com- would ferment it anaerobically. More- Pa. tannophilus19,20 and other mercially abundant, D-xylose, along over, in a recent taxonomic treatise on yeasts 9,21,23 was discovered. Of these with other hemicellulosic can yeasts6, 64% of the species listed are species only a few, most notably be obtained in good yield (80-90% or cited as capable of assimilating xylose Pa. tannophilus and C. shehatae23, will more) through acid or enzymatic aerobically, but none are cited as carry out the fermentation at rates of hydrolysis of the hemicellulosic frac- capable of fermenting this sugar. practical interest (Table 1). tion. Moreover, D-xylose (or oligo- Another recent taxonomic synopsis7, Several conclusions are immediately meric xylan) is present in many waste however, notes that a few yeast evident from Table 1. First con- streams from sulfite and dissolving species - most conspicuously Pichia ventional D-glucose fermentations by pulp mills, fiberboard and hardboard stipitis, Candida shehatae, Pachysolen S. cerevisiae are 6-35 times faster than manufacturing plants3. Combined use tannophilus and Brettanomyces naar- even the best D-xylose fermentations of D-xylose and D-glucose during denensis - ferment D-xylose to ethanol by C. shehatae or Pa. tannophilus. In production of chemicals or fuel at various rates. In the last four years at this same regard, the D-glucose specific (ethanol) from angiosperm feedstocks least 41 yeast species, including 23 of fermentation rate (g ethanol (g cell dry could improve the overall process the genus Candida and eight of the weight) -1 h-1) of S. cerevisiae is 8-10 economics and using D-xylose from genus Pichia, have been shown times faster than the D-glucose specific waste streams could reduce disposal to produce some ethanol from fermentation rate of these two other costs and provide alternative by- D-xylose8 12. In most instances, the species. Second, reported ethanol product credits for existing processes. conversion to ethanol occurs yields (g product produced/g substrate aerobically. consumed) with D-xylose are only Xylose-fermenting organisms The discrepancy between results of about 56-82% of those reported for The anaerobic production of ethanol classical taxonomic methods for deter- D-glucose. Third, maximum attainable from D-xylose was first demonstrated mining fermentative activity and ethanol concentrations from D-xylose current findings stems largely from the are only about 23-46% of those attain- fact that in many instances, production able from D-glucose (Table 1). T. W. Jeffries is at the USDA, Forest Products Laboratory, 1 Gifford Pinchot of ethanol from D-xylose by yeasts is Taken together, these facts present a Drive, Madison, WI 53705, USA. either obligately aerobic or is greatly dismal picture for the D-xylose fer- Trends in Biotechnology, Vol. 3, No. 8, 1985 209

mentation. Ultimately, D-xylose must anaerobic conditions. Anaerobically, colonies on agar plates as a con- compete economically with D-glucose the cells do not grow; hence the sequence of their diminished res- as a feedstock. In view of the relative apparent increase in the rate is lower piratory capacity. Since they do not ages of the two technologies, however, than when observed aerobically31. possess a functional electron transport there is room for some optimism. There is general agreement that, pathway for ATP generation, they Moreover, it is possible that by using at least under aerobic conditions, must derive all their ATP from D-xylose from waste streams, a plant C. shehatae and Pi. stipitis strains are substrate-level phosphorylation and operator could avoid disposal charges superior to all other known yeast the cell yield is consequently smaller. and thereby offset some of the higher species in their rates of D-xylose fer- Grande strains can respire and hence process costs incurred in the D-xylose mentation. Similarities observed form large colonies. fermentation. between Pi. stipitis and C. shehatae Respiration-deficient mutants might have led to the suggestion that these be particularly useful in the D-xylose Selection of improved strains might be the teleomorphic and ana- fermentation where aeration plays a Improvements in the D-xylose morphic forms (the sexually perfect conspicuousrole in reducing the specific fermentation rate and final and asexual stages) of the same ethanol yield. To date, however, no ethanol concentration have been organism6. However, significant dif- petite mutants have been described for obtained principally by isolating better ferences exist among the various Pa. tannophilus, and it appears to be a strains from nature, and by mutating named strains25,and some strains petite-negative yeast (i. e. petite mutants and selecting strains in the laboratory. exhibit considerable instability in both do not survive more than a few genera- Genetics and strain selection are just their morphological and fermentative tions). In the case of C. shehatae, beginning with D-xylose-fermenting activities 32.Strains of C. shehatae however, unstable petite and grande yeasts. More progress has been made exhibiting high respiratory- and low strains have been demonstrated and with Pa.tarrnophilusthan with fermentative-activities have been iso- their fermentative abilities assessed32. C. sheharae or Pi. stipitis. Methods for lated on agar. As might be expected, strains with crossing strains have been developed A recent quantitative screening of 56 diminished respiratory capacity for Pa. tannophilus28; various yeast isolates identified as Candida (determined by the tetrazolium agar aneuploid and polyploid strains have species, C. tenuis, C. shehatae and Pi. overlay method39) generally show. been constructed29 and their capacities stipitis showed Pi. stipitis CSIR Y633 greater fermentative activity than for ethanol production have been to give the greatest yield of ethanol those exhibiting higher respiratory assessed30. Increasing the chromosome (0.45 g ethanol (g xylose)-1 consumed), capacity. It is worth noting that on number from the haploid to the diploid at the highest volumetric rate (0.92 g yeast malt agar, the strain exhibiting level resulted in a significant increase ethanol (g cells)-1 h-1 )33. most conspicuous petite/grande transi- in the ethanol yield. Further increases tions, C. shehatae ATCC 22984, in ploidy enhanced the ethanol yield Respiration deficiency reverts to a heterogeneous mixture of and D-xylose specific fermentation rate Selection for respiration deficiency, phenotypes with petite-like cells pre- to a lesser extent. Selection of has been proposed as a method to dominating”. The relationships Pa. tannophilus strains capable of rapid improve the efficiency of ethanol pro- between respiration capacity and growth on xylitol-plus-nitrate medium duction 34 37. Normal, respiration- ability to grow on a particular also results in stable isolates of sufficient cells can oxidize ethanol source are complex and incompletely Pa. tannophilus exhibiting up to a two- after fermentation. Ethanol oxidation understood, and because these strains fold increase in the volumetric rates can also occur during fermentation if of C. shehatae are unstable, analysis is (g ethanol 1-1 h-1) of D-xylose (and respiration is not repressed by the difficult. D-glucose) fermentations under aerobic carbon source used38.Respiration- If a yeast strain does not possess the conditions, and a 1.5-fold increase in deficient (petite, rho-) mutants are biochemical machinery enabling it to the specific fermentation rate under strains of yeasts which form small carry out a balanced fermentation of 210 Trends in Biotechnology, Vol. 3, No. 8, 1985 xylose (see later), then the net accumu- mechanisms seems to have relevance to nitrate as a nitrogen source did not lation of NAD(P)H under anaerobic the D-xylose fermentation. show anaerobic fermentation in the conditions will stop metabolism. This The aerobic fermentation of presence of nitrate, whereas cells effect is particularly apparent when a D-xylose by C. tropicalis is similar in grown on ammonium did52. Our reduced carbon source such as xylitol is some ways to the Custers or Kluyver current studies show that shifting used. effects. As mentioned previously, this Pa. tannophilus cells from aerobic Respiration deficiency has been pre- organism (along with several less well growth at pH 3.5 to anaerobiosis at viously reported to affect the use of documented Candida species) will pro- pH 6.5 greatly inhibits fermentative other sugars by yeasts. For example, duce small amounts of ethanol from activity (Dax and Jeffries, unpub- when respiratory competent strains of xylose under aerobic (or micro- lished). Taken together, these findings S. cerevisiae able to use D-, aerobic) conditions, but it will not suggest similar but separate roles for , , a-methyl glucoside when completely deprived of nitrate and pH in regulating the and were converted into their oxygen 47,48.Unlike the Custers effect in anaerobic fermentative mechanism. It petite forms, half of them lost the which the cells adapt to anaerobic fer- is possible that two different xylose ability to ferment D-galactose and two- mentation of D-glucose after 7-8 h45, reductases are present or active under thirds of the petite strains would not complete anaerobic inhibition of the the different conditions. Other factors use raffinose40. An earlier report by D-xylosefermentation has been may function as well. Mahler and Wilkie41 had shown that observed for at least 20 days. Recently, Bruinenberg et al.53 pre- conversion of wild-type S. cerevisiae to Production of ethanol from D-xylose sented a biochemical explanation for the petite form was accompanied by by Pa.tannophilus19,47,49 and by the inhibition of fermentation under loss of the ability to grow on C. shehatae 50,51 in the complete absence anaerobic conditions. It is based on the D-galactose, maltose or a-methyl of oxygen has been demonstrated in necessity of maintaining a balance glucoside. This phenomenon seems in several laboratories. The specific rates between intracellular NAD and some ways similar to effects observed of ethanol production by Pa. tanno- NADH. Assimilation of D-xylose by on D-, and it is likely philus under aerobic and anaerobic yeasts and fungi commonly proceeds that NAD(P) and NAD(P)H concen- conditions are not very different20’24 first through reduction by NADPH to trations play important generalized but anaerobically, significant amounts form xylitol, then through oxidation roles in regulating sugar use by yeasts. of xylitol accumulate in the medium. by NAD to form D-xyhdose54. The oxi- With both organisms, aeration greatly dative portion of the phos- stimulates the volumetric rate of phate pathway provides the NADPH, Process considerations 49 Not all process variables have been ethanol production . Some research- resulting in the oxidation of D-xylose fully evaluated, but the effects of ers have reported that Pa. tannophilus and transfer of reducing power from exhibits only minimal ability to fer- the NADPH pool to the NADH pool. oxygen, D-glucose, nitrogen and pH 49 51 are discussed in the following sections. ment anaerobically . Others report C. utilis, a yeast which can only that the fermentation proceeds well produce ethanol from D-xylose under anaerobically 19,20,52 . The procedural aerobic conditions, possesses the Effect of oxygen basis for discrepancies among the var- common D-xylose reductase which Oxygen (aeration) has a profound iousreports isnot completely uses only NADPH as a cofactor, hence effect on ethanolic fermentations by apparent, but it might result from the its assimilation of D-xylose is depen- yeasts; as has long been recognized - medium or the pH used for cell dent on the oxidative pentose phos- Pasteur described the inhibition of growth. As an example, cells grown on phate pathway. The D-xylose reduc- ethanol production by aeration in 188142. One hundred years later researchers realized that the Pasteur effect is only observed in washed (starved) cell suspensions43,44 . Less familiar but of equal or greater impor- tance are the Custers, Kluyver and Crabtree effects. The Custers effect (also known as the negative Pasteur effect) is the transient inhibition of D-glucose fermentation following transfer of some yeasts (particularly Brettanmyces) to anaerobic condi- tions13’45.The Kluyver effect is similar except that certain yeasts which will ferment D-glucose anaerobically, require oxygen to use other sugars46. The Crabtree effect is the inhibition of respiration by low concentrations of D-glucose 43. With the possible exception of the Pasteur effect, each of these regulatory Trends in Biotechnology, Vol. 3, No. 8, 1985 211 tase(s) of yeasts which ferment little changed under aerobic and D-xyloseanaerobically (such as anaerobic conditions, even though the Pa, tannophilus or Pi. stipitis) accept product yields may shift dramatically. either NADPH or NADH, allowing the cells to recycle anaerobically the NADH generated during the oxida- Effect of D-glucose tion of xylitol to D-xylulose, and thus to The relatively low yield of ethanol bypass the oxidative phase of the obtained during the fermentation of pentose phosphate pathway (Fig. 1). D-xylose seems to result, at least in The explanation presented by part, from the concomitant fermenta- Bruinenberg et al.53 has not been tion of D-xylose and respiration of ethanol that occurs in Pa. tanno- consistently supported by subsequent 38 biochemical studies. For example, philus . It has been shown that the Ditzelmüller et al. 55 purified a xylose addition of small amounts (O. 5%) of reductase of Pa. tannophilus to 95% D-glucose to an active D-xylose fermen- homogeneity and found that it was spe- tation has essentially no effect on the cific for NADP(H). By comparison, rate of D-xylose utilization, but Verduyn et al. 56 purified the xylose markedly increases the aerobic yield of reductase from Pi stipitis and found ethanol. The anaerobic yield of ethanol that the activity with NADH was 70% is not affected, leading to the conclu- that with NADPH. Both organisms sion that small amounts of D-glucose are capable of xylose fermentations. cansuppress ethanol oxidation. Other enter into the regula- Pa. tannophilus, therefore, appears to be glucose-sensitive and exhibits the tion of xylose metabolism under 24 aerobic and anaerobic conditions. For Crabtree effect . C. shehatae differs in example, in examining purified xylitol this regard in that its yield of ethanol, dehydrogenase from Pa. tannophilus, even when growing on pure D-glucose, Ditzelmüller et al. 57 found that at phy- does not generally exceed approxi- Other factors siological pH (7), the equilibrium of mately two-thirds of the theoretical The fermentation of crude acid or the reaction catalysed by this enzyme maximum. Moreover, its volumetric enzymatic hydrolysates of wood pre- favored the accumulation of xylitol and D-glucose fermentation rate is only sents problems over and above those about 50% greater than that observed that the oxidation of xylitol was 25 encountered in the fermentation of strongly inhibited by NADH and with D-xylose . pure D-xylose. Acid hydrolysis pro- ATP. Given these findings, it is easier duces various incompletely charac- to understand why Pa. tannophilus Effects of nitrogen and pH terized inhibitory compounds from the accumulates xylitol under anaerobic The nitrogen source is important for and fractions. conditions, both Pa. tannophilus and C. shehatae. Enzymatic hydrolysates are easier to The stimulation of D-xylose In the case of Pa. tannophilus, growth ferment but are presently more dif- fermentation by Pa. tannophilus by on nitrate can induce cells to higher ficult to obtain: it is generally neces- aeration can be attributed largely to the specific fermentation rates when they sary to treat the lignocellulose with fact that Pa. tannophilus requires are transferred to a less restrictive steam or mild solvent pulping to dis- oxygen for growth58. Mahmourides et nitrogen source52; C. shehatae does not rupt the lignified material before enzy- al. 59 have identified a transition from use nitrate.Peptone,casein and matic depolymerization. Pretreatment oxidative growth to fermentation especially yeastextract stimulate releases the in a during the cultivation of ethanol production in Pa. tanno- polymeric form along with some inhi- Pa. tannophilus. During the philus 19, and an amino nitrogen source bitory compounds such as acetic acid fermentative phase, ethanol accumu- is very important for good ethanol or lignin degradation products. lates and dissolved oxygen production by C. shehatad 22 (Fig. 2). One of the motives for considering concentration in the medium Studies in our laboratory have shown the fermentation of D-xylose is to use increases. Schvester, Robinson and that the maximum specific fermen- hemicellulosic sugars generated in pro- Moo-Young examined the oxygen tation rate is attained with C. shehatae cesses converting lignocellulose to requirement of Pa. tannophilus in a at pH 3.2-3.4, but that somewhat ethanol. A few yeasts, most notably quantitative manner, and found that a higher yields can be obtained at pH C. lusitaniae ATCC 34449, C. blankii significant level of aeration was neces- 2.6. The aeration provided by shaking ATCC 18735, Pi. wickerhamii ATCC sary to stimulate growth and enhance 50 ml of medium in a 125 ml 24215 and C. tenuis CBS 4435 (Ref. 10) the rate of ethanol production60. These Erlenmeyer (equivalent to a sulfite can ferment both D-xylose and cello- -1 -1 authors also observed that ethanol oxidation rate of about 9 mM O2 1 h ) biose. is produced during production lagged behind growth and appears to be near the optimum for enzymatic hydrolysis and inhibits the did not start until the dissolved oxygen growth with fermentation. Product activity of exocellobiohydrolase, an concentration was near zero. As noted yields and fermentation rates are also enzyme which is very important in the earlier, several researchers found that affected by the age and history of the degradation of crystalline . An the specific rate of D-xylose inoculum - cells from 24-hour-old, ability to ferment cellobiose therefore fermentation by Pa. tannophilus is xylose-grown cultures perform best. has obvious implications for simul- 212 taneous sacchariftcation and fermen- tation. It should also be noted that Pa. tannophilus can ferment D-galactose and D- to ethanol; it is, therefore, able to ferment most of the predominant plant mono- saccharides 61. Future directions The use of D-xylose as a feedstock for the production of ethanol has passed the initial flush of discovery and enter- ed into the phase of process improvement and development. One should look for bench marks: (1) The production of at least 5-6% (w/v) ethanol with a yield of greater than 0.4 g/g in 36 h should be achieved before contemplating scale-up and commer- cialization. (2) Recovery and use of by- products such as acetic acid, glycerol and xylitol should be evaluated for their effects on process economics. (3) The possible production of novel chemicals from the pentose phosphate pathway along with traditional pro- ducts such as citric acid, amino acids, vitamins and antibiotics should be con- sidered. (4)An improved knowledge of the biochemical pathways, par- ticularly of the regulatory and rate- limiting steps, is essential before one can expect progress through the application of contemporary genetic techniques. Finally, it is worth remem- bering that fermenting D-xylose is practicable only where it is a by- product of lignocellulose utilization, and that adapting yeasts and fer- mentation processes to particular waste streams or hydrolysates may present unique and difficult problems. Acknowledgement The author gratefully acknowledges Henry Schneider (NRC, Ottawa), Nancy Alexander (USDA, Peoria) and Kent Kirk (USDA, Madison) for use- ful discussions and critical readings of this manuscript. References