the influence of ph and aeration rate on the fermentation of d-xylose by candida shehatae

ELSEVIER

The influence of pH and aeration rate on the fermentation of D-xylose by Candida shehatae Sebastiain SBnchez,* Vicente Bravo,’ Eulogio Castro,* Albert0 J. Maya,* and Fernando Carnacho+

*Department of Chemical Engineering, University of Jae’n, Ja&z, Spain; ‘Department of Chemical Engineering, University of Granada, Granada, Spain

The effects of the initial pH and air supply on the production of ethanol from D-X$OSe using the yeast Candida shehatae in a batch reactor were investigated. The initial pH was altered within the range of 2.5-6.5 and the specific aeration rate from 0.0-0.3 v v-’ min-‘. The results showed that the mostfavorable initial pHfor ethanol production was 4.5 and aeration via the stirring vortex of the bioreactor was suflcient. Under these conditions. the maximum specific growth rate (F,) was 0.329 h- I; biomass production rate (b), 0.024 kg m -.’ h-- ‘: overall biomass yield (YzJ* 0.036 kg kg-‘; the specific uptake rate of D-XylOSt? (q,), 2.0 kg kg-’ hh’; and the specific ethanol production rate (Q), 0.72 kg kg-’ h-’ (both at 20 h culture time). The average xylitol yield (Y.,,) was 0.078 kg kg-’ and the overall ethanol yield (Y&). 0.41 kg kg-‘. Both q, and qE diminished once the exponential growth phase was over. 0 1997 Elsevier Science Inc.

Keywords: o-xylose: ethanolic fermentation; xylitol; yeasts: Candidu shehatae

Introduction

Yeasts capable of producing ethanol directly from D-xylose in significant quantities were first characterized at the beginning of the eighties.‘,’ This has led to a growing interest in the use of lignocellulose residues for the indus- trial production of ethanol since the conversion of both the hemicellulose and cellulose fractions substantially increases the yield of ethanol. Various yeasts are capable of fermenting n-xylose along with D-glucose. These are Pachysolen tannophilus, Pichia stipitis, and Candida shehatae.

Studies on the possible effects of the availability of oxygen on the metabolism of D-xylose by C. shehatae found that, in principle, an extra supply of oxygen was unneces- sary, although ethanol production was indeed enhanced by added oxygen when using either D-xylose or D-glucose as the carbon source.3,4 Delgenes et al.’ have shown that when the oxygen supply was restricted, some growth occurred, but no ethanol was produced. This indicates that for the efficient conversion of n-xylose into ethanol, the aeration rate should be higher than 0.02 v v-’ mm’. These publications suggested

Address reprint requests to Dr. S. Stichez, University of Jakn, Dept. of Chemical Engineering, Paraje Las Lagunillas, E-23071 Jaen, Spain Received 16 May 1996; revised 24 Feb 1997; accepted 4 March 1997

that an optimum aeration rate must be achieved in order to obtain maximum productivity and ethanol yield.

As far as the pH of the culture medium is concerned, it should be borne in mind that this variable affects cell growth and its influence may vary considerably among yeast strains. The cell membranes are not completely permeable to hydrogen ions and so the intracellular pH and that of the culture medium may not be the same. Apart from affecting cell membrane permeability, pH may also determine the solubility of some components of the medium: thus, a modification in the pH might also cause some micronutrient to precipitate and so become impossible to be assimilated. Du Preez et a1.6 report that in their experiments, the most adequate pH for the growth of C. shehatae was between 3.5-4.5.

The principal aim of this study has been to determine the optimum initial pH of the culture medium and the best aeration rate for the conversion of D-xylose into ethanol using the yeast C. shehatae.

Materials and methods

Microorganism

The yeast C. shehatae ATCC 34887 was supplied by the American

Type Culture Collection (Rockville. MD, U.S.).

Enzyme and Microbial Technology 21:355-360, 1997 0 1997 Elsevier Science Inc. All riahts reserved. 655 Avenue of the Americas, NewYork, NY 10010

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Experimental device

All the experiments were performed at laboratory scale in a batch culture reactor which consisted basically of three temperature- controlled, magnetically stirred fermenters with a usable volume of 2 1 (described fully elsewhere’). The volume of the culture medium used was 0.5 1; the stirring speed was 500 rpm, and the stirring rod was 4 cm long and 0.8 cm in diameter.

Maintenance medium and inoculum preparation

The yeasts were stored between 5-10°C in lOO-ml test tubes on a sterilized solidified culture medium with a composition in g 1-l of: yeast extract, 3; malt extract, 3; peptone, 5; o-xylose, 10; and agar-agar, 20. Before the start of each experiment, the microor- ganisms were inoculated under sterile conditions into glass test tubes containing the solidified culture medium described above. These tubes were then incubated 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 approximately 0.01 g 1-l.

Culture medium

The composition of the culture medium in g 1-l was: MgSO,, 1; KH,PO,, 2: (NH&SO,, 3; yeast extract, 4; and peptone, 3.6.

Procedure

Two series of experiments were performed to study the influence of initial pH and aeration rate on fermentation. All experiments were at 30°C with an initial xylose concentration (s,) of 25 g 1-l. In the first series of experiments, the initial pH values assayed were: 2.5, 3.5, 4.0, 4.5, 5.5, and 6.5 while the additional aeration rate was nil (0.0 v v- ’ min- ’ ), i.e., the only air allowed into the fermentation process was that which entered naturally through the stirring vortex. In the second series, the additional aeration rates studied were 0.075 and 0.300 v v- ’ min-’ while the initial pH was always 4.5.

Analytic technique

Dry weight (x, g ll’) was determined by the absorbance of the suspension at a wavelength of 620 nm. The concentration of residual xylose (s, g 1-l) was calculated by Miller’s reducing sugar method.8 Ethanol and xylitol concentrations were quantified ac- cording to the methods described by Beutler’ and Beutler and Michal” based on the enzymes alcohol dehydrogenase and polyol dehydrogenase.

Results and discussion

In each of the cultures in both series of experiments, the evolution of biomass concentration, D-xylose uptake, and the formation of ethanol and xylitol were followed.

Growth curves for two of the experiments are shown in Figure 1 where it can be seen that there is a short lag phase lasting no more than 10 h followed by a fairly short exponential growth phase (indicated by the arrows) at the end of which the D-xylose concentration is still close to that of its initial value. After this, exponential phase cell growth continues but much more slowly until practically all the D-xylose has been used up and the process enters the stationary phase.

In Figure 1, the changes in pH during two of the experiments are also shown. In general, it can be seen that

l I 5 % 2t/ A- A,

01 0 20 40 60 80 100 120 140'

t 09

Figure 1 Growth curves (solid symbols) and pH (open symbols) versus time for experiments: pHi = 4.5, Q = 0 v v-’ min-‘, n 0; and pHi = 2.5, Q = 0 v v-’ min-‘, 0 0

these values remain practically constant when the initial pH is low whereas when the initial pH is higher or the air flow is high, there is a tendency for the pH to drop slightly during the experiment (by about 0.5 at most). This slight fall in pH is concomitant with an appreciable decrease in the concentration of the substrate and an increase in the presence of bioproducts (particularly ethanol).

Biomass production

The exponential growth phase was quantified via the maximum specific growth rate (pm). The duration of the exponential growth phase was calculated in all cases from representations of In (x/x,) versus time such as those in Figure 1 and the values for l.~,,, shown in Table 1. It can be seen that l.~,,, remains almost constant for the lower aeration rates but that it diminishes appreciably when Q = 0.3 v v-’ min- ’ .

The IL,,, values with aeration rates of 0.0 and 0.075 v v- ’ min- ’ coincide very well with those published by Delgenes et aL5 who arrived at a value of p_,,, = 0.347 h-’ with the yeast strain C. shehatue (Y12878) under very similar experimental conditions to ours (batch reactor with a usable

Table 1 Maximum specific growth rates (p,,,), biomass produc- tivities (b), overall biomass yields (b$,), and specific xylose uptake rates (4,) at different initial pH values (pH,) and aeration rates (0)

VG pH, (v v-lQmin-l) (I!‘) (kg rnF3 h-‘)(kg kx;;‘) (;I (kg kg” h-‘1

2.5 0 0.092 0.021 0.040 3.5 0 0.280 0.024 0.042 4.0 0 0.304 0.023 0.038 4.5 0 0.329 0.024 0.036

4.5 0.075 0.338 0.037 0.075 4.5 0.300 0.242 0.025 0.093 5.5 0 0.290 0.039 0.052 6.5 0 0.262 0.044 0.068

18.0 1.2 20.0 1.8 20.0 1.9 20.0 2.0 40.0 0.8 19.0 1.5 19.0 0.8 20.0 2.0 22.0 1.2

356 Enzyme Microb. Technol., 1997, vol. 21, October

pH and aeration effects on xy/ose fermentation: S. Sinchez et al.

volume of 2 1; aerobic conditions; aeration rate = 1 v v-’ min-‘; stirring speed, BOO rpm), although both their aeration rate and stirring speed were higher than ours.

As far as pH is concerned, the highest p,,, value was reached when pH, = 4.5 and the lowest values were obtained at each extreme of pHi. The specific growth rates obtained on fermenting D-XylOSe with C. shehatne (0.33 hP ‘) were considerably higher than when P. tannophiius (0.26 h-‘) was used under the same experimental conditions,7 although the highest k, values were reached with both yeasts at the same pH value of 4.5.

Du Preez et al.,” on the other hand, got lower p,,, values ranging between 0.05-0.18 h-’ with an initial D-xylose concentration of 50 kg m-j, T = 3O”C, and pH, = 5.5; nevertheless, in our experiment, when pH, = 5.5, T = 3O”C, and s, = 25 kg mP3, the value for CL,,, turned out to be 0.29 h- ’ . The differences in these results may possibly be due to different initial substrate concentrations and aeration rates.

-3.0 /

1 -4.0;

0.0 1.0

/‘8 q

2.0 3.0 -4d 5.0

In t

(a)

The growth phase after the exponential one was quantified using parameter b which was determined via the equation

x=c+bt (1)

given that from representations of the experimental results x-t, the evolution of biomass production versus time may be accepted as being linear. The values for b thus obtained for each aeration rate showed no definite tendency (Table I) while it increased concomitantly with higher pH, values. It is also noteworthy that the values for b with C. shehatae are higher than those obtained with P. tannophilus under the same conditions. ‘*

161

o------L---_._ L i-Y, ‘O..._ j

0 20 40 60 so 100

t th)

Substrate Uptake (b)

The uptake of D-xylose was studied using the following parameters: specific substrate-uptake rate, qs, and overall biomass yield, Y$,. Empirical equations including two parameters, OL and p, were tried for calculating qs. Of the various equations assayed, that which best fits the experimental data over the longest time span is

Figure 2 Application of linearized form of Eq. (2) (a); and application of Eq. (2) for the experiments lb): pHi = 4.5, Q = 0 v v-’ min-‘, n ; pHi = 2.5, Q = 0 v v-’ min-‘, 0

s = s,, . &“j (2)

which was used in a previous paper.7 This equation, once linearized, leads to determination of the parameters m and p. In Figure 2a, In (In (s,/s)) versus In t is shown as an example of this linearization for two experiments and the close fitting of the experimental data to Eq. (2) is evident here (Figure 2b).

authors obtained a result of qs = 0.55 kg kg-’ h-’ under aerobic conditions which is considerably less.

In general, as can be seen in the results for one experiment set out in Table I, qs is quite high at the beginning of the culture but diminishes during the course of the experiment. This agrees with the observations of Alexander et al.” who indicates that this decrease in the value of ys mainly after 20 h can be attributed to the accumulation of ethanol which has an inhibitory effect on the specific substrate uptake rate. The highest qs values, obtained at around 20 h, are about 2 kg D-xylose kg-’ biomass h- ’ which coincides fairly well with the result obtained by Ligthelm et a1.4 using C. shehatae under oxygen-limited conditions, T = 3O”C, and pH = 5.2; nevertheless, the same

Values for q6 corresponding to intermediate times ( 18 -22 h) are set out in Table I. It can be seen that at similar times in the experiments, the value for q, is higher in those cultures to which a lower aeration rate wa$ applied, and thus it would appear that the higher the aeration rate, the greater the inhibition of D-xylose uptake. It can also be seen that the values for q, practically coincides in all the experiments between pHi values of 3.5-5.5 but are lower at the extremes of initial pH.

The specific uptake rates for D-xylose obtained in this series of experiments with C. shehatae are higher than those determined for P. rannophilus under the same experimental conditions14 (s, = 25 kg m-j, pH, = 4.5. T = 30°C); thus. in the experiment where Q = 0 v v- ’ min-’ after about 20 h, the values of qs with C. shehatae are about twice as high as those with P. tannophifus. Our values for qs with C. shehatae are also higher than those published by Sreenath et a1.l5 for batch cultures with the same yeast but different experimental conditions. These authors arrived at q< values

Enzyme Microb. Technol., 1997, vol. 21, October 357

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of around 0.91 kg xylose kg-’ biomass hh’ with an initial cell concentration of 0.72 kg mP3 which indicates that the higher the initial concentration, the lower the value of qs. Their experimental conditions were a temperature of 32°C a stirring rate of 300-400 rpm, and an aeration rate of 0.5 v v-l min-‘; they used a different culture medium from ours with an unspecified pH. Their observation that qs diminishes concomitantly with a rise in the initial concentration of biomass agrees with our findings in that the specific uptake rate decreases during the course of the experiment, i.e., as the biomass concentration increases.

Overall biomass yields have been evaluated from representations of (X-X,) versus (s,,-s). The values for I$, for each culture are set out in Table 1. In general, it would seem that overall biomass yield increases concomitantly with an increase in the aeration rate. This agrees with data available in the literature which confirms that biomass yield is a function of the prevailing aeration conditions; thus, Ligth- elm et aL4 working with the same yeast under aerobic conditions, obtained a yield of YxlS = 0.33 kg biomass kg-’ xylose while under oxygen-limited conditions, it was YXIS = 0.01 kg biomass kg- ’ xylose. Alexander et a1.,3 working with a continuous C. shehatae culture under semiaerobic conditions, achieved biomass yields between 0.01-0.41 kg kg-’ depending in this case not just on the aeration rate but also on the dilution rate involved.

It can also be seen that between pH, 2.5-4.5, the yield remains constant at about 0.04 kg biomass kg-’ xylose. Above pH, = 4.5, the value of I”$, increases. These biomass yields are considerably lower than those obtained with P. tannophifus under identical operating conditions. With this latter yeast at pH, = 4.5, Bravo et a1.j6 arrived at a value for Y$, of 0.16 kg kg-’ and thus it is obvious that C. shehatae is capable of converting a greater percentage of the original concentration of D-xylose into ethanol.

Bioproducts

The formation of the major bioproducts, ethanol and xylitol, was studied via the following parameters: the specific ethanol formation rate, qE, and ethanol, YE,s, and xylitol, YXy,,, yields.

To determine the volumetric rate of ethanol production, dEldt, and then calculate qE, an empirical equation was arrived at which would allow us to predict the concentration of ethanol formed, E, as a function of time. Of the various equations tried, that which comes up with the best fit over the widest time range is

E _ = A . eB/t

ET (3)

where A and B are constants and E, is the theoretical maximum ethanol concentration that can be produced ac- cording to the stoichiometry of the reaction

CSH,,O, -+ i C,H,OH + 5 CO;, + Energy (4)

The values of parameters A and B can be arrived at by where x stands for the biomass concentration at the moment linearizing Eq. (3) and representing the experimental values when the specific ethanol production rate is to be calculated. of In (E/E,) versus l/t until the maximum ethanol concen- The resulting values of qE calculated at around 20 h for tration is reached. As an example of this, a graphic repre- all the experiments are set out in Table 2. It should be noted

-6.0 :m .._-__ 0.00 0.04 0.08 0.12

I/t (h-l)

(a)

I / /

?’ /d’ / I’ /

/ b /

J 0 10 24l 30 40 50

t @I

@I

Figure 3 Application of linearized form of Eq. (3) (a); application of Eq. (3) for the experiments (b): pHi = 4.5, Q = 0.075 v v-’ min-‘, n ; and pHj = 6.5, Q = 0 v v-’ min-‘, 0

sentation of two of the experiments is shown in Figure 3a where the solid line represents the progress deduced via the equation and the open squares correspond to the experimental data. Once the values for parameters A and B for each experiment have been ascertained, the values for the ethanol concentration, E, within the time range in Eq. (3) can be calculated. The values thus arrived at for E are shown in Figure 36 by the solid line while the open squares represent the experimental data. It can be seen that within the time range pertaining to the equation, the calculated results match the experimental ones well.

The derivative dEldt was determined analytically from Eq. (3). By a process of replacement in Eq. (5), it was thus possible to obtain the expression for the specific ethanol production rate as a function of time

1 dE E, - A - B . eB”

qE=Xdt=- xt? (5)


pH and aeration effects on xy/ose fermentation: S. Scinchez et al.

Table 2 Overall ethanol yields (Y&J, xylitol yields (V,,,,), and specific ethanol production rates (qE) at different initial pH values (pHi) and aeration rates (0)

Q pH, (v v-’ min-‘)

CS (kg kg-‘) (kg’z._‘) t (h) (kg kg9’ h-‘)

2.5 0 0.30 3.5 0 0.39 4.0 0 0.34 4.5 0 0.41 4.5 0.075 0.41 4.5 0.300 0.29 5.5 0 0.38 6.5 0 0.40

0.097 22.0 0.39 0.082 20.0 0.59 0.080 20.0 0.52 0.078 20.0 0.72 0.094 19.0 0.64 0.11 19.0 0.51 0.076 20.0 0.57 0.063 20.0 0.49

that, in general, the maximum qE values are reached at this time and that afterwards the specific ethanol formation rate gradually diminishes throughout the course of the experiments. This result coincides on the whole with that observed by Du Preez et al.’ ’ in a batch fermentation of o-xylose with this same yeast. These authors also detected a decrease in the specific ethanol production rate concomitant with an increase in the quantity of ethanol present in the culture; nevertheless, their maximum qE values were about 0.4 kg ethanol kg- ’ biomass h- ’ while in our experiments specific rates of up to 0.8 kg kg-’ h-’ were attained.

It can be seen that qE values are higher in those cultures with lower aeration rates. With regard to the pHi, the highest qE value arrived at after 20 h culture was with pH, = 4.5; the lowest results came at the extremes of the pH values assayed. This initial pH was also that chosen by Bravo et ~1.‘~ for the fermentation of o-xylose with P. tannophilus under the same experimental conditions, but the specific ethanol production rate values obtained with C. shehatue are much higher than those with P. funnophilus; thus, at pH, 4.5, P. tunnophilus produces a qE value of around 0.06 kg kg- ’ h- ’ while C. .shehutue results in a qE of about 0.8 kg kg-’ hK’.

Our values are somewhat higher than those reported in the literature for batch cultures of this same yeast where values for the specific ethanol production rates range from 0.07-0.45 kg kg-’ h-’ depending upon the culture medium employed (above all the nitrogen source), the operating conditions (with or without aeration), and the initial concentration of o-xylose. The highest value found in the literatureI (0.45 kg kg-’ h-‘) was obtained under semiaerobic conditions using an ammonium salt as nitrogen soy;ce and with an initial o-xylose concentration of 50 kg m which was the lowest concentration among those employed within this specific ethanol production rate range. In our study, an enriched medium with (NH,),SO, as nitrogen source was also used and, more importantly, a very low initial o-xylose concentration (25 kg me3), very much lower in fact than those used by previous authors which varied between 50-200 kg mp3.

The overall ethanol and xylitol yields for each of the experiments have been determined from graphic representations of the ethanol and xylitol concentrations obtained versus the concentration of xylose used up such as that shown in Figure 4. In general, it can be seen that the value

8 i

61

” /’ I

_,J ’

-f e-1

. I

s,-s (kg/m3)

Figure 4 Ethanol (H, 0) and xylitol (A, A) yields for the experiments: pHi = 5.5,Q = 0 v v-’ min-‘, D, A; and pHi = 4.5, Q = 0.075 v v-l min-‘, 0, A

of pw, (Table 2) decreases for the highest level of Q while the xylitol yield, YXyts increases slightly along with the value of Q. As for ethanol, it would seem that in the more highly aerated cultures, there is some sluggishness and/or evapo- ration attached to the formation of this product and thus its yield diminishes concomitantly with a rise in the value of Q; thus, as far as ethanol production is concerned, the best conditions are those with least aeration in which the highest values for Y& and the lowest for Y are obtained.

The yield of Y& = 0.41 kg kg -“?s about the same as that found in the literature for this yeast. For example, Du Preez et al.17 and Wayman et al.” obtai;ned ethanol yields of 0.38 and 0.39 kg kg- ’ using initial D-XylOSe concentrations of 50 and 31 kg m-j, respectively.

Our xylitol yield of about 0.1 kg kg- * is lower than that obtained by Ligthelm et al.” who, when using the same yeast and an initial o-xylose concentration of 40 kg rnp3 under oxygen-limited conditions, arrived at a YXyls value of 0.3 kg xylitol kg-’ D-XylOSe.

It can also be seen that while YXyls diminishes concomitantly with pHi, the overall ethanol yield remains fairly constant at around 0.4 kg ethanol kg-’ xylitol except in the experiment where pHi = 2.5 where Y& is lower.

The overall xylitol yield is comparable to that obtained by Sreenath et ~1.‘~ on fermenting D-XylQSe with C. shehutue under similar experimental conditions. At pH, = 3.2- 3.5, T = 30-32°C and a stirring speed of 400 rpm, these authors’ value for YXy,s is one of 0.1 kg kg - ’ compared to our value of 0.082 kg kg-’ at pH, = 3.5, T = 30°C and a stirring speed of 500 rpm.

References

I. Du Preez, .I. C. and van der Walt. J. P. Fermentation of D-XylOSe to ethanol by a strain of Candida sheharae. Biofechnol. Left. 1983, 5, 357-362

2. Bruinenberg, P. M., de Bot, P. H. M., van Dijken, .I. P., and Scheffew. W. A. The role of the redox balances in the anaerobic fermentation of xylose by yeasts. Eur. J. Appl. Microbial. Eiorech- nol. 1983, 18, 287-292

3. Alexander, M. A., Young, V. W.. and Jeffries, T. W. Levels of pentose phosphate pathway enzymes from Candida shehafae grown

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in continuous cultures. Appl. Microbial. Biotechnol. 1988, 29, at controlled low dissolved oxygen levels. Appl. Microbial. Biotech- 282-288 nol. 1989.30, 53-58 Ligthelm, M. E., Prior, B. A., and Du Preez, J. C. The oxygen requirements of yeasts for the fermentation of o-xylose and D-

glucose to ethanol. Appl. Microbial. Biotechnol. 1988, 28, 63-68 Delgenes, J. P.. Moletta, R., and Navarro, J. M. The effect of aeration on o-xylose fermentation by Pachysolen tannophilus, Pichia stipitis, Kluyveromyces marxianus, and Candida shehatae. Biotechnck L&t. 1986, 8, 897-900 Du Preez. J. C.. Prior. B. A.. and Moteiro, A. T. M. The effect of aeration on xylose fermentation by Candida shehatae and Pachy- solen tannophilus. A comparative study. Appl. Microbial. Biotech- nol. 1984, 19, 261-266 Bravo, V., Camacho, F., Sanchez, S., and Castro, E. Influence of the concentrations of n-xylose and yeast extract on ethanol production by Pachysolen tannophilus. .I. Ferment. Bioeng. 1995,79,566-571 Miller, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31, 426-428 Beutler, H.-O. Ethanol. In: Methods of Enzymatic Analysis Vol. 6 (Bergmeyer, H. U., Ed.). Verlag Chemie, Weinheim, 1984, 598- 606

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Castro, G. E. Fermentacidn de disoluciones de o-xilosa y n-glucosa con levaduras. Ph.D. thesis. Faculty of Experimental Science (Jaen), University of Granada, Spain, 1993 Alexander, M. A., Chapman, T. W., and Jeffries, T. W. Continuous culture responses of Candida shehatae to shifts in temperature and aeration: Implications for ethanol inhibition. Appl. Environ. Micro- biol. 1989, 55, 2152-2154 Bravo, V.. Camacho, F., Sanchez, S., and Castro. E. The ethanolic fermentation of solutions of D-XylOSe by Pachysolen tannophilus. The influence of aeration rate. Afinidad 1995, 457, 189-196 Sreenath, H. K. and Jeffties, T. W. Batch and membrane-assisted cell recycling in ethanol production by Candida shehatae. Biotech- nol. Len. 1987, 9, 293-298 Bravo, V., Camacho, F., Sanchez, S., and Castro, E. The effect of pH on kinetic and yield parameters during the ethanolic fermentation of o-xylose with Pachysolen tannophilus. Bioprocess Eng. 1993. 9, 159-165 Du Preez, J. C., Bosch, M., and Prior, B. A. Xylose fermentation by Candida shehatae and Pichia stipitis: Effects of pH, temperature, and substrate concentration. Enzyme Microb. Technol. 1986, 8, 360-364 Wayman, M. and Tsuyuki, S. T. Fermentation of xylose to ethanol by Candida shehatae. Biotechnol. Bioeng. Symp. 1985,15,168-177

Beutler, H.-O., and Michal, G. Neue Methode zur enzymatischen Bestimmung von Ethanol in Lebensmitteln. 2. Anal. Chem. 1977, 284, 113-l 17 Du Preez, J. C., van Driessel, B., and Prior, B. A. Ethanol tolerance of Pichia stipitis and Candida shehatae strain in fed-batch cultures

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the influence of ph and aeration rate on the fermentation of d-xylose by candida shehatae

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