Comparative study of the fermentation of D-glucose/D-xylose mixtures with Pachysolen tannophilus and Candida shehatae

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1 Comparative study of the fermentation of D-glucose/D-xylose mixtures with Pachysolen tannophilus and Candida shehatae S. SaÂnchez, V. Bravo, E. Castro, A.J. Moya, F. Camacho Bioprocess Engineering 21 (1999) 525±532 Ó Springer-Verlag 1999 Abstract We have performed a comparative analysis of the fermentation of the solutions of the mixtures of D-glucose and D-xylose with the yeasts Pachysolen tannophilus (ATCC 32691) and Candida shehatae (ATCC 34887), with the aim of producing bioethanol. All the experiments were performed in a batch bioreactor, with a constant aeration level, temperature of 30 C, and a culture medium with an initial ph of 4.5. For both yeasts, the comparison was established on the basis of the following parameters: maximum speci c growth rate, biomass productivity, speci c rate of substrate consumption (q s ) and of ethanol production (q E ), and overall ethanol and xylitol yields. For the calculation of the speci c rates of substrate consumption and ethanol production, differential and integral methods were applied to the kinetic data. From the experimental results, it is deduced that both Candida and Pachysolen sequentially consume the two substrates, rst D-glucose and then D-xylose. In both yeasts, the speci c substrateconsumption rate diminished over each culture. The values q s and q E proved higher in Candida, although the higher ethanol yield was of the same order for both yeasts, close to 0.4 kg kg )1. 1 Introduction The characterization, in the early 1980s, of yeasts capable of directly producing substantial concentrations of ethanol from D-xylose [1, 2], has attracted growing interest in the lignocellulose wastes that could be used for industrial ethanol production. These wastes, after acidic or enzymatic hydrolysis, can be transformed by fermentation into ethanol. In the fermentation of hydrolyzed lignocellulose to ethanol, two primary problems emerge: the fermentation of pentose and the presence of inhibitory compounds from the yeasts [3]. The yeasts traditionally used in the fermentation processes, principally of the genera Saccharomyces and Schizosaccharomyces, can ferment a wide range of sugars, Received: 11 January 1999 S. SaÂnchez (&), E. Castro, A.J. Moya Department of Chemical Engineering, Faculty of Experimental Science, University of JaeÂn, JaeÂn, Spain V. Bravo, F. Camacho Department of Chemical Engineering, Faculty of Science, University of Granada, Granada, Spain although not D-xylose. Therefore, the integral use of lignocellulose waste, among which D-xylose is the major pentose of the hemicellulose fraction, must involve the transformation of this sugar into ethanol. The hemicellulose fraction may represent from 15 to 35% of the total [4], and thus the conversion of the hemicellulose fraction together with the cellulose fraction raises the overall production and transformation. One of the rst yeasts recommended for this transformation was Pachysolen tannophilus [5], although more recently others have been identi ed, such as Candida shehatae, Pichia stipitis, Candida tropicalis, Kluyveromyces marxianus, Kluyveromyces cellobiovorus, Candida butyrii and Candida tenuis. These yeasts can ferment D-xylose and provide ethanol yields exceeding 0.2 kg kg )1 in synthetic culture mediums containing D-xylose as the carbon source [6, 8]. In the present study, a comparative analysis is made of the ethanol fermentation of different mixtures of the two main monosaccharides (D-glucose and D-xylose) in any lignocellulose hydrolysate, using the yeasts Pachysolen tannophilus and Candida shehatae under operating conditions tested for each yeast in previous works [9, 11]. 2 Materials and methods Yeast strain We used the yeasts Pachysolen tannophilus and Candida shehatae 34887, supplied by the American Type Culture Collection. Experimental device All the experiments were carried out at laboratory scale in a batch-culture reactor described elsewhere [12] consisting basically of three temperature-controlled, magnetically stirred fermenters with a usable volume of 2 l. The volume of culture medium used was 0.5 l; the stirring speed was 500 rpm, and the stirring rod was 4 cm long and 0.8 cm in diameter. Culture medium The composition of the culture medium in g l )1 was: MgSO 4,1;KH 2 PO 4, 2; (NH 4 ) 2 SO 4, 3; peptone, 3.6; and yeast extract 4. The initial concentrations of D-xylose (s) and D-glucose (g) ingl )1 were: 525

2 Bioprocess Engineering 21 (1999) P: tannophilus s=g 25=0 24=1 20=5 15=10 10=15 5=20 1=24 0=25 C: shehatae s=g 25=0 20=5 15=10 5=20 0= Maintenance medium and inoculum preparation The yeasts were stored between 5±10 C in 100 ml-test tubes on a sterilized solid culture medium with a composition in g l )1 of: yeast extract 3; malt extract 3; peptone 5; D-xylose 10; agar-agar 20. Before the start of each experiment the microorganisms were inoculated under sterile conditions into glass test tubes containing the solid culture medium described above. These tubes were then kept in an oven at 30 C for 60 h in order to obtain cells at the same growth stage for every experiment. The concentration of the inoculum at the beginning of each experiment was around 0.01 g l )1. Procedure Favourable operating conditions were determined in previous studies [10, 13]: temperature of 30 C and initial ph 4.5. The complete culture medium was sterilized using a pre- lter of glasswool and cellulose nitrate lters with 0.2 lm pore size. Analytical technique Dry-weight calibration was determined by the absorbance of the suspension at a wavelength of 620 nm. The residual concentration of D-xylose was calculated via Miller's reducing-sugar method [14]. Ethanol and xylitol concentrations were quanti ed according to the methods described by Beutler [15] and Beutler and Michal [16], based on the enzymes alcohol dehydrogenase and polyol dehydrogenase. 3 Results and discussion Over the course of the cultures analysed, the concentrations of biomass, residual D-glucose, total residual sugar, ethanol and xylitol formed, were determined for each experiment. In addition, the evolution of the ph was recorded in the culture for each experiment. With respect to ph, no signi cant in uence was detected in the different proportions of D-xylose and D-glucose assayed. In the case of Candida shehatae, only slight decreases were appreciated in experimental time, these coinciding with a notable consumption of the mixture of substrates and with the formation of bioproducts. In the case of Pachysolen tannophilus, these decreases were also only slightly signi cant in the cultures having the greatest proportion of D-xylose. A slight decline in the value of this variable was also detected at around 25 h, at which time there was already an appreciable fall in substrate concentration, as well as a considerable ethanol formation; in the Pachysolen experiments having a greater proportion of D-glucose, values were reached almost independently of the initial proportion of sugars, although the fall in ph exceeded that detected for the cultures in which the initial concentration of D-xylose was higher (Fig. 1). These lower ph values may be due to the excretion of acidic bioproducts, such as acetic acid, glycerol and other alcohols [17]. Fig. 1. Growth curves (open symbols) and ph (solid symbols) versus time for experiments: (h j) Pachysolen tannophilus, s o = 24, g o = 1 kg m )3 ; (s d) Pachysolen tannophilus, s o = 1, g o = 24 kg m )3 ; (m n) Candida shehatae, s o = 0, g o = 25 kg m )3 From the experimental results for both of the yeasts, calculations were made of the speci c growth rates, the speci c rates of substrate consumption (q s ) and ethanol production (q E ), as well as the overall biomass (Y G x=s ), xylitol (Y G Xy=s ) and ethanol (YG E=s ) yields. 3.1 Specific growth rates Figure 1 shows the growth curves of some of the experiments performed with Candida and Pachysolen. These curves resulted from the representation of the values of ln(x/x o ) against time over the course of the cultures. As in these cases, the growth curves for the other experiments show that the lag phase was strongly reduced (approximately 2 h maximum) in both yeasts. Next came an exponential growth phase, the duration of which depended on the initial D-glucose in the culture medium. Thus, in the experiments with the greatest initial concentrations of D-glucose (Fig. 1), the stationary phase immediately superseded the exponential. Nevertheless, in the rest of the cultures of both yeasts, the exponential phase was followed by a period in which growth continued, although at a lower speci c rate during a long time interval, which, in the case of Pachysolen, varied between 40 and 60 h, this interval being shorter in Candida. Finally, the stationary phase was reached, practically coinciding with the total consumption of the substrate.

3 S. SaÂnchez et al.: Comparative study of the fermentation of D-glucose/D-xylose mixtures From the experimental results, the duration of the exponential growth phase was established for each culture (marked between arrows in Fig. 1) and the respective values of maximum speci c growth rate (l m ) were calculated according to the equation ln x=x o ˆ a l m t ; 1 values which are listed in Table 1. In general, in Pachysolen, the presence of D-glucose in the culture medium raised the l m values, though there was no signi cant relationship between these and the initial D-glucose concentration; therefore it appears that these values re ect growth exclusively on D-glucose in this exponential phase. In Candida, however, the presence of D-glucose in the culture medium (even a small proportion) lowered the l m values, though without a signi cant relationship between these and the initial D-glucose concentration; thus, as with Pachysolen, the maximum speci c growth rates appear to correspond to growth only on D-glucose. In addition, in the experiments without either sugar, Pachysolen registered a speci c growth rate comparable to that reached when only D-xylose was present as the substrate. In the case of Candida, the experiment without substrate presented a lower l m value. During the period following exponential growth, in the experiments with a higher initial proportion of D-xylose, the biomass grew lineally with time. The representations of the experimental results of biomass-time enabled the determination of the duration of this period, as well as the formulation, by a least-squares t, of Eq. (2): x ˆ c b t ; 2 with b representing the values of biomass productivity. These results are shown together with those of l m in Table 1. The proportion of sugars used did not signi cantly in uence the b values, these being comparable to those obtained with the same microorganism when only D-xylose was used as the substrate. In both yeasts, the biomass-productivity values were very similar. Nevertheless, in the Candida cultures, a linear phase was not detected when g o ³ 15 kg m )3, while in Pachysolen, this fact was evident for greater D-glucose concentrations (g o ³ 24 kg m )3 ). In addition, for the experiments with equal concentrations of D-glucose and D-xylose, Pachysolen presented a linear time period (t 1 ) substantially greater than that of Candida. The t 1 values are presented in Table 1, in parenthesis together with the value of biomass productivity. This linear-growth period, subsequent to the exponential-growth period, has also been reported by other authors [18, 19]. This period is characteristic of fermentation controlled by physical stages, in which the kinetic control of the bioprocess resides in the transference of oxygen in the cell suspension. This result agrees with the work of Slininger et al. [20], in which dissolved oxygen was found to be almost absent in the culture medium during the exponential phase, even in well-aerated cultures with Pachysolen. 3.2 Specific rate of substrate consumption The experimental results revealed that both yeasts sequentially consume the two substrates used. Firstly, they consume D-glucose for the rst 20 hours of the culture. Then, there is a period in which the biomass production, the substrate consumption and ethanol formation stop or 527 Table 1. Maximum speci c growth rates (l m ), biomass productivities (b), overall biomass yields (Yx=s G ) and speci c xilose-uptake rates (q s ) with P. tannophilus (P) and C. shehatae (C) s o (kg m )3 ) g o (kg m )3 ) l m (h )1 ) b (kg m )3 h )1 ) Y G x=s (kg kg)1 ) t (h) q D s (kg kg )1 h )1 ) q s (kg kg )1 h )1 ) P C P C P C P C P C (t 1 = 99) (36) ± ± 0.12 ± (60) (62) (25) (80) (35) ± ± 0.12 ± (72) ± (74) ± ± ± ± 0.12 ± ± ± (4) (41) t 1 : lineal time period (h)

4 Bioprocess Engineering 21 (1999) 528 increase very slightly. The duration of this interval varies with the initial substrate concentrations, and is especially signi cant in the cultures with similar proportions of the two sugars. Afterwards, the consumption of D-xylose began and again the net formation of biomass and of ethanol. In this sense, du Preez et al. [21] also observed the diauxic phenomenon in the utilization of D-glucose and D-xylose by Candida. These authors, using mixtures of sugars at 1% in the culture medium, found that rst D-glucose was consumed and then D-xylose. For the determination of the speci c substrate-consumption rate, differential and integral methods were applied in the treatment of the kinetic data. Among the equations tested on the two yeasts, the one that gave acceptable reproducibility of the substrate-concentration data for greater time intervals has the form: s ˆ s o a tb : 3 Linearization of this equation gives the expression: ln ln s o =s Š ˆ ln ln a b ln t ; 4 which enables the determination, by least-squares t of the rst member against the ln t, of the values of a and b (Fig. 2a), with which the s values are acceptably reproduced (Fig. 2b). Figure 2a shows the two examples of linearization of Eq. (3) and two time intervals can be discerned. These can be characterized by two different equations (solid lines of the gure) which correspond to the preferred consumption periods of each substrate, beginning with D-glucose. Between the two intervals, there was a period in which the microorganism adapted to the new substrate (broken line). After a and b were calculated with Eq. (3), an analytic determination was made of the speci c rates of the total substrate consumption, q D s, as listed in Table 1, which gives the values for the various culture times of each yeast. The values obtained for the shortest times correspond to the period in which D-glucose is preferentially consumed, while the times longer than 25 hours are characteristic of the stage of preferential D-xylose. The speci c rate of substrate consumption were considerably higher in Candida than in Pachysolen ± roughly 10-fold higher for xylose consumption. In both yeasts, q D s diminished over the course of each culture from a maximum value, which was recorded in the interval of 10 to 20 hours after the beginning of the experiment. For the initial culture times (12 to 15 h), as the initial D-glucose concentration increased, the speci c rates of substrate consumption approached that of the experiment in which only this sugar was used as the substrate. For longer culture times (60 h), the values found were highly similar to those of the experiment made with D-xylose. These results coincide partly with the ndings of Jeffries and Sreenath [22] when they fermented both sugars with Candida shehatae. When these researchers used D-xylose and D-glucose, they found that both sugars, separately, were readily assimilated by Candida, with D-glucose being more rapidly consumed than D-xylose. Nevertheless, in their mixtures, these authors found that the utilization or consumption of D-glucose was inhibited by the presence of D-xylose and the consumption of D-xylose was inhibited by D-glucose. In our experiments, as shown in Table 1, a similar phenomenon was noted, which may be due, as indicated by these authors, to direct competition between the two sugars for the same transport system. Fig. 2. Application of Eqs. (4) A) and (3) B) for the experiments: h Pachysolen tannophilus, s o = 24, g o = 1 kg m )3 ; j Candida shehatae, s o = 15, g o = 10 kg m )3 3.3 Overall biomass yield Instantaneous biomass yield, Y x/s, can be de ned as the quotient between the net biomass produced and the net substrate consumed at a given point of the culture: Y x=s ˆ dx d s o s : 5 If this yield remains constant over the culture, a representation at different times of the values for the net biomass formed (x)x o ) against total net substrate consumed s ot s T should give a straight line with a slope Y x/s and a ordinate different from 0. The yield indicated by the slope corresponds to the entire experiment, and can be distinguished from instantaneous yield by the term ``mean'' or ``overall'' (Yx=s G ). For example, Fig. 3 includes these representations for an experiment of Pachysolen and another of Candida. As observed in these experiments, in all cases,

5 S. SaÂnchez et al.: Comparative study of the fermentation of D-glucose/D-xylose mixtures data of the ethanol concentration produced in the longest time intervals. Among those assayed, the best reproduction of the experimental variations proved to be: Fig 3. Biomass formed (x)x o ) versus total substrate consumed (s ot )s T ) for the experiments with s o = 5, g o = 20 kg m )3 : h Pachysolen tannophilus; j Candida shehatae acceptable ts have been achieved, enabling the determination of the values of Yx=s G (Table 1). In all the cultures, biomass yield is clearly higher (two-fold) in Pachysolen than in Candida. In addition, in Candida, the value of Yx=s G increased the higher the initial D-glucose concentration, reaching the maximum yield value in the experiment with only D-glucose as the substrate. In Pachysolen, the in uence of the proportion of D-xylose/D-glucose was not detected in the overall biomass yield, and was re ected only in a slight decrease on using only D-xylose. When the biomass yield remained constant over the entire experiment and when there was no cell maintenance (therefore Y x=s ˆ Yx=s G ), from the de nition itself of the speci c growth rate (l = 1/x dx/dt) and from the de nition of q s and Eq. (5), it can be deduced that: q s ˆ l : 6 Y G x=s Thus, with the values of speci c rate and yield, q s can be calculated (integral method). During the exponentialgrowth phase l = l m ; for culture times corresponding to the period of linear growth, according to Eq. (2), it holds that: l ˆ 1 dx x dt ˆ b x : 7 This equation makes it possible to determine the value of l in the interval where the biomass shows a linear trend, and, with this, the values of the speci c rate of substrate consumption. In this way the values of q s were determined, these appearing together with q D s in Table 1, at equal culture times. In general, the values calculated for both parameters were similar by the two procedures. 3.4 Specific rate of ethanol production For the calculation of the values of speci c ethanol-production rate, differential (q D E ) and integral (q E) methods were used in the treatment of the kinetic data. In the application of the differential method, certain equations were tested for acceptable reproduction of the E T E T E ˆ AtB1 1 ; 8 in the cultures performed with Pachysolen, and: E E T ˆ A 2 e B 2=t ; 9 in the experiments with Candida. In these equations, E T represents the maximum concentration attainable if the production from the transformation of the D-xylose/Dglucose mixture into ethanol were theoretic, and A 1, A 2, B 1 and B 2 were adjustment parameters. For the determination of these parameters, Eqs. (8) and (9) can be linearized in the form: E T ln ln E T E ˆ ln ln A 1 B 1 ln t ; 10 ln E 1 ˆ ln A 2 B 2 ; 11 E T t and by least-squares ts of the rst member against ln t in Eq. (10) and against 1/t in Eq. (11), the values of these parameters were calculated. Afterwards, from Eqs. (8) and (9), the derivative de/dt can be determined, enabling the determination of speci c rates of ethanol formation in Pachysolen and Candida, respectively. As indicated above, the ethanol formation over the course of the cultures occurs in two intervals separated by a period of very little or no cell growth, substrate consumption or bioproduct formation. For the calculation of q E, the empirical Eqs. (8) and (9) were used in both intervals, depending on the yeast. For example, Fig. 4a provides the graphic representation of Eqs. (10) and (11) for the same D-xylose/D-glucose mixture for both yeasts. The reproduction of the experimental data are included in Fig. 4b, where the solid line corresponds to the values deduced and the dotted line to the experimental values. As in this case, the rest of the cultures showed acceptable ts within the validity intervals in which the above equations were applied. The values for q D E for both yeasts are given in Table 2. In contrast to the trend of the speci c rate of substrate consumption, it was con rmed in Pachysolen that q D E remained approximately constant during the period of linear biomass growth. In the experiments with high D-xylose concentrations, a single interval appears when the rst member of Eq. (10) is represented against ln t, a trend which resembles that found when only this substrate is used, with a highly similar speci c formation rate [23]. In the experiments in which the initial proportion of D-glucose was greater, the two formation zones of ethanol can be discerned (Fig. 4), these providing the two q D E values which appear in Table 2. The values for shorter time periods correspond to the speci c rates of ethanol formation due to the consumption of D-glucose, whereas the longer periods correspond to those of D-xylose. The former are around 10-fold greater than in the latter. When 529

6 Bioprocess Engineering 21 (1999) 530 Fig. 4A,B Application of Eqs. (10) and (11) A and application of Eqs. (8) and (9) for the experiments with s o = 20, g o = 5 kg m )3 B: h Pachysolen tannophilus and n Candida shehatae there was a very high concentration of D-glucose, only q D E could be determined for D-glucose consumption, so that it appears that the microorganism does not succeed in adapting to the new substrate. Similarly, the values obtained in this case were one order of magnitude greater than those found with D-xylose alone. Contrary to Pachysolen, Candida registered a speci c rate of ethanol formation that declined over the experiment, fundamentally after about 20 hours. In addition, for the following culture times, the q D E values of Candida were longer than those of Pachysolen. In Candida, the sequential consumption of the substrates used was re ected also in the q D E values obtained; thus, in the cultures having a high concentration of D-glucose, the speci c rate of ethanol formation proved greater, though it diminished slightly as this concentration increased, as occurred throughout the series. In the experiments with similar concentrations of the two substrates, it was possible to determine the q D E values corresponding to the two periods in which D-glucose was preferentially consumed ± rst, D-glucose and then D-xylose. Also, this yeast reached much greater speci c rates in the rst period. In addition, for the experiments in which the two periods could be differentiated, two hypotheses could be tested: either the instantaneous ethanol productivity (Y E/x ) remains constant, or the speci c rate of ethanol formation is constant. In the former case, given that the instantaneous productivity is de ned by Y E=x ˆ q E =l, then, in the exponential growth phase, with l = l m, the speci c rate of ethanol production would also be constant and its value is given by: q E ˆ l m Y E=x ; 12 q E ˆ b x Y E=x ; 13 Table 2. Overall ethanol (Y G E=s ) and xylitol (YG Xy=s ) yields and speci c ethanol production rates (q E) with P. tannophilus (P) and C. shehatae (C) s o (kg m )3 ) g o (kg m )3 ) Y G E=s (kg kg)1 ) Y G Xy=s (kg kg)1 ) t (h) q D E (kg kg)1 h )1 ) q E (kg kg )1 h )1 ) P C P C P C P C L.P ± 0.16 ± L.P L.P ± L.P ± ± ± ± ± ± ± ± L.P.: Linear Phase

7 S. SaÂnchez et al.: Comparative study of the fermentation of D-glucose/D-xylose mixtures while in the linear phase. Thus the speci c rate of ethanol production would decline with the biomass concentration and therefore with time. The latter case, q E constant, coincides with the previous one in the exponential growth phase. Nevertheless, for the period in which the biomass increases lineally over time, the following could be applied: de x dx ˆ de d x 2 =2 ˆ qe b ; 14 deduced from the de nitions of the speci c rates of growth and of ethanol production, so that a representation of E against x 2 /2, restricted to the time interval in which the biomass presents linear growth, should lead to a straight line with a slope from which the value of q E could be calculated when b is known. From the experimental results, the representations corresponding to the two hypotheses were made ± that is, E against (x)x o ) over the entire course of the experiment and E against x 2 /2, in the linear growth phase, implying that it is possible to admit a linear relationship for the former in the case of Candida, while for Pachysolen this linear relationship can be admitted only within the interval in which the ethanol production is due to D-glucose consumption. In this way, for Pachysolen, in the period of linear biomass growth, a good linearity was not found when E versus (x)x o ) was represented, but an acceptable t of E versus x 2 /2 was achieved. By these procedures, the q E values were calculated (integral method), as shown in Table 2 for the same times in which q D E was determined. Acceptable agreement was achieved in the values of the parameter by the two procedures proposed and in both yeasts. 3.5 Bioproduct yields Starting from a concept similar to that used to determine the biomass yield, values for ethanol and xylitol yields have been calculated. In principle, to nd out whether the ethanol and xylitol yields remain constant, were made graphs of their concentrations versus the quantity of the total substrate consumed, revealing in all cases graphs similar to the one shown in Fig. 5. In each culture, the experimental points acceptably t a straight line, indicating that the yields could be considered constant over the course of the experiment. From the slopes of the lines represented, the overall yield values for ethanol (YE=s G ) and xylitol (YXy=s G ), are listed in Table 2. In Pachysolen, in relation to YE=s G, the overall ethanol yield rose with the initial proportion of D-glucose, although when only this sugar was used as a substrate, the value was very similar to that found when only D-xylose was used. In Candida, no signi cant trend was discerned in the YE=s G values, although these were slightly lower when D-glucose was present than when only D-xylose was the substrate, this trend proving analogous to that of Pachysolen. These values of overall ethanol yield with Candida are close to those reported by Wayman and Parekh [24], who, for initial concentration mixtures of between s o = 14.7 and g o = 34.3 kg m )3 to s o = 54.9 Fig. 5. Ethanol (h, j) and xylitol (s, d) concentrations versus substrate consumption for the experiments with s o = 20, g o = 5 kg m )3 : (open symbols) Pachysolen tannophilus and (solid symbols) Candida shehatae and g o = kg m )3, found values of YE=s G in the interval 0.38±0.41 (kg ethanol) (kg substrate) )1. In relation to the overall yield of xylitol, in general, the values fell with the rise in the initial D-glucose proportion, supporting the idea that this bioproduct appears as an intermediate compound in the metabolism of D-xylose but not in D-glucose, and thus went almost undetected in the experiments in which s o = 0 and g o = 25 kg m )3. Nevertheless, it should be pointed out that this general trend did not hold in the experiment made with Pachysolen when s o = 24 and g o = 1 kg m )3, where high xylitol production resulted (YXy=s G =0.16 kg kg)1 ), an anomalous fact that agrees with the result of the lowest ethanol production (YE=s G =0.24 kg kg)1 ), when in this same yeast, for very similar concentration mixtures, the ethanol production is higher while that of xylitol is lower. References 1. du Preez, J.C.; van der Walt, J.P.: Fermentation of D-xylose to ethanol by a strain of Candida shehatae. Biotechnol. 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