Proces.~ Riorhemiwy 29 (1994) 657-662 1994 Elsevier Science Limited

Printed in Great Britain 0032.9S92/94/$7.00

Influence of Oxygen on Ethanol and Xylitol Production by Xylose Fermenting Yeasts

S. A. Furlan, P. Bouilloud & H. F. de Castro*

Faculdade de Engenbaria Quimica de Lorena, Rodovia ItajubB-Lorena Km 74.5, Lorena, .%o Paulo, Brazil

(Received 20 July 1993; revised version received and accepted 8 September 1993)

The behaviour of Pichia stipitis, Pachysolen tannophilus, Candida shehatae and Candida parapsilosis was investigated to select the most suitable yeast to convert xylose either to ethanol or to xylitol, with little or no formation of by-products. The aeration rate was used as a variable parameter. P. stipitis and C. parapsilosis were the most effective producers of ethanol and xylitol, respectively, both reaching productivities at very low levels of oxygenation. With P. stipitis, better ethanol productivity was attained under microaerobic conditions @,a = 4% h - ‘) while with C. parapsilosis high yields and rates of xylitolproduction were detected at K,a values of about 16.3 h-‘. P. tannophilus and C. shehatae showed lower performances under all conditions used while changes in oxygenation modified the ratio of ethanol to xylitolproduced by these yeasts, suggesting that they are more dependent on the oxygen power input than P. stipitis and C. parapsilosis. The injkence of oxygen transfer rates on ethanol and xylitolftirmation with the best producers is discussed.

NOTATION Y x,s Overall yield of biomass on substrate (g g-‘)

C’ Dissolved oxygen concentration (mg litre-‘) p,,,,, Maximum specific growth rate (h- ‘)

K, a Oxygen transfer coefficient (h - ’ ) P Product concentration (g litre- ‘) Subscripts

QP Productivity (g litre-’ h-‘) S Substrate

9P Specific rate of product formation (g g- ’ P Product h-‘) X Biomas

rP Rate of product formation (g litre- ’ h- ‘) 1 Ethanol

rX Rate of growth (g litre- ’ h- ‘) 2 Xylitol s Substrate concentration (g litre- ’ ) X Biomass concentration (g litre- ‘) Y p/s Overall yield of product on substrate INTRODUCTION

(g g-‘)

*To whom correspondence should be addressed. D-Xylose is a five-carbon sugar, derived from hemicellulose hydrolysate, which can be used as a

6.57

658 S. A. Furlan, P. Bouilloud, H. F. de Castro

feedstock in chemical and biological processes. In This work deals with the variation of oxygen chemical processes it is transformed into furanic transfer rate using yeasts suitable for ethanol and polyols, xylitol and furfural,’ while in biological xylitol production. Unlike most of the previous processes xylitol and ethanol may be produced investigations3,‘2,‘s these experiments were car- depending on the microorganism used and the ried out in fermenters instead of shake flasks, in environmental conditions employed. which aeration conditions are poorly defined.

The conversion of n-xylose to n-xylulose is apparently a critical step in xylose metabolism in yeasts’ and involves two reactions. The first is the reduction of n-xylose to xylitol with a NADPH- or NADH-dependent xylose reductase and the second is the oxidation of xylitol to D-xylulose with a NAD + -dependent xylitol dehydroge- nase.‘,” The role and regeneration of cofactors are highly dependent on oxygen transfer rates.

MATERIALS AND METHODS

Organisms

In 1976 Barnett4 suggested that although a large number of microorganisms were able to metabolize b-xylose aerobically none were able to do this anaerobically (Kluyver effect). It is now known that some microorganisms are able to con- vert n-xylose into ethanol or xylitol without oxygen. The inability of some microorganisms to ferment b-xylose anaerobically is probably due to a redox imbalance between NAD+ and NADH.’ Xylitol oxidation to n-xylulose requires NAD’, which is regenerated by the respiratory chain. In anaerobiosis the conversion of n-xylulose into ethanol leads to a lack of NAD’ and without a hydrogen acceptor metabolic activity stops. As yeasts do not have transhydrogenase activity,h excess NADH cannot be oxidized in this reaction.

The fermentations were carried out with P. tanno- philus ATCC 32691, P. stipitis NRIXL Y 7124, C. shehatae NRRL Y 17024 and C. parapsilosis ATCC 28474. Stock cultures were maintained at 4°C on agar slants containing glucose (20 g litre-‘), yeast extract (10 g litre- ‘) and peptone (10glitree’).

Growth and fermentation conditions

Apart from NADPH-linked xylose reductase, some yeast strains also have an NADH activity that prevents the imbalance of the NAD + /NADH redox system, allowing anaerobic fermentation to occur. The NADH/NADPH ratio has been deter- mined’ for Puchysolen tannophilus, Pichia stipitis and Candidu shehatae as O-04, 0.5 and O-4 respectively. For P. tannophilus, which has the smallest ratio value, ethanol production depends on the presence of hydrogen acceptors such as oxygenx while xylitol accumulation is favoured by microaeration” or anaerobic conditions.“’ The high ratio shown by P. stipitis allows the highest ethanol productivity to be obtained.

Yeast inocula were grown aerobically at 30°C for 24-72 h in Erlenmeyer flasks containing (g litre- ’ ): xylose, 20.0; yeast extract, 2-O; KH,PO,, 5.0; MgSO,. 7H,O, 0.4; and (NH,),S04, 2.0. To prevent a reaction with xylose, (NH4)2S04 was sterilized separately. The pH was adjusted to 4.5 with orthophosphoric acid. Fermentation runs were performed in a 2-litre Setric Set 2’ reactor with a working volume of 1.6 litres. Xylose (50 g litre-‘) was used as substrate and other nutrients were kept at the concentrations used for inoculum preparation. Stirring and aeration rates kept at the concentrations used for inoculum pre- paration. Stirring and aeration rates varied with the experiment. The initial cell concentration was adjusted to 3 X 1 Oh viable cells ml- ’ . Temperature was maintained at 30°C and pH initially set to 4-5 was not controlled. Samples were taken periodic- ally for analysis of ethanol, xylitol and biomass concentration.

Analytical procedures

Recently, several research groups have shown interest in evaluating the performance of P. stipitis, “,12 P. tannophilus’*‘3,‘4 and C. sheha- tae ‘2,‘s for ethanol production while Candida parapsilosis, ‘+I 8 Candida tropicalis, Ix Candida guilliermondii I9 and P. tannophilus I” have been investigated for xylitol production.

Biomass concentration was determined by optical density at 620 nm, with a calibration curve of optical density against biomass dry weight. Xylose, ethanol, xylitol and arabitol concentra- tions were analysed by HPLC on an Interaction Ion 300 column using 0.025~ H,SO, as eluent. Oxygen concentration in the liquid phase was measured by an Oximeter ‘PO, NUM’ with an ‘Ingold’ electrode. K,a values were determined by the polarographic method for each pair of condi-

Ethanol and xylitol production by yeasts 659

tions (aeration and stirring rates) used throughout this work with a calibration curve prepared using uninoculated medium.20 The following rela- tionships were found: for K, II = 4.8 h- ’ (O*OS wm and 250 rpm); K,a= 16.4 h-’ (0.3 wm and 250 rpm); K,a = 35.4 h-l (O-9 wm and 250 rpm) and &a = 99.6 h-’ (O-6 wm and 500 rpm).

RESULTS AND DISCUSSION

Yeast strain selection Fermentation runs were carried out initially under microaerobic (0.08 wm and 250 rpm: K,a= 48 h-‘) and aerobic (0.9 wm and 250 rpm: K,a = 35.4 h- ‘) conditions. The kinetic parameters were calculated when the product concentration (either ethanol or xylitol) in the culture medium was maximal (Table 1).

All the yeasts tested except for C. parapsilosis produced ethanol under both aeration conditions. At a K,a of 4.8 h- ‘, the highest ethanol con- centration ( 15 g litreP ‘) was attained with P. stipitis at 104 h, followed by P. tannophilus (9.5 g litrec’) at 165 hand C. shetutue (8-O g litre-‘) at 180 h. Increasing K,a values to 35.4 h- ’ led to an increase in the maximum specific growth rate and growth yield at the expense of ethanol formation.

Only P. stipitis failed to accumulate xylitol. The other yeasts accumulated xylitol at different rates and yields. The best producer was C. parapsilosis attaining concentrations of 30 g litre- ’ within 117 h. P. tannophilus and C. shetatae produced less with maximum xylitol concentrations of 12-5 g litre ’ and 6.0 g litre- ‘, respectively. Here again, better results were achieved under micro- aerobic conditions.

Besides showing superior production rates, P. stipitis and C. parupsifosis were able to form only one product of interest preferentially or exclus- ively. For the two remaining yeasts, a mixture of both products at different concentrations appeared under microaerobic and aerobic condi- tions. In the former conditions, the ratio between ethanol/xylitol concentrations was found to be 0.80 and 1.3 for P. tannophilus and C. shetatae, respectively. Under aerobic conditions, a similar ratio was obtained for P. tannophilus but increased for C. shetatae since less xylitol was accumulated. The differences in the product ratio resulting from changes in the degree of the aera- tion rate may reflect the higher sensitivity of these strains to oxygen. Factors other than oxygen2’ can influence the relative amounts of ethanol and xylitol formed, but this is outside the purpose of this work.

It is important to note that product yields and rates were lower than those reported by other workers.“*‘7 This can be explained by differences of medium composition and in this work no attempt was made to enhance yeast performance by adding nutrient supplement. The influence of oxygen transfer rates was the major concern in these experiments, and particular attention was given to determining the appropriate degree of aeration for efficient production of either ethanol or xylitol with the chosen yeast strains.

Ethanol production by I? slipiris When P. stipitis was tested at low K,a (4.8 h- ‘) and high K,a (35.4 h-l) values, better ethanol yield ( YPlis = 0.35 g g- ‘) and higher ethanol pro- ductivity ( Qpl = O-14 g litre’ h- ’ ) were obtained under the former conditions. A typical fermenta-

Table 1. Kinetic parameters for yeast strains tested under microaerobic (K,a=4.8 h-‘) and aerobic (K,a=35.4 h-‘) conditions

Yeast strain &a Biomass value (h -‘,J yx,, kw*x

(gg-‘) W’)

Ethanol

QPI YPIIS kg-‘) (g litre - ’ h - I)

Xylitol

e (g litreP: h-’ 1

Pichia stipitis 4.8 0.09 0.21 104 0.35 0.14 35.4 0.14 0.5 38 0.25 0.16

Not detected Not detected

Pachysolen 4.8 0.1 @22 165 0.21 0.06 189 0.27 0.06 rannophilus 35.4 0.11 0.25 75 0.13 WO8 0.22 0.13

Candida 4.8 0.06 0.27 180 0.23 0.04 2:: 0.16 0.03 shehtatae 35.4

;:; 0.33 52 o-2 1 0.1 105 O-04 0.1

Candida 4.8 0.25 Not detected 117 0.6 1 0.26 parapsilosis 35.4 0.16 0.23 Not detected 147 0.44 @1.5

“t,: Time required for the maximum product concentration to be reached.

660 S. A. Furlan, P. Bouilloud, H. F. de Castro

tion run carried out under microaerobic condi- tions showing ethanol, biomass and xylose concentrations is presented in Fig. 1. With a lag phase of 10 h and a maximum ethanol concentra- tion of 15 glitree’, this yeast strain showed a closer fit to the reaction type I of Gaden, indicat- ing that growth and product formation were asso- ciated. This type of reaction is evident in Fig. 1, where the concentrations of both biomass and ethanol display a near-linear relationship with fer- mentation time, especially in the active period of fermentation (25-100 h). By applying the Luedeking and Piret equation, the numerical value could be found:

r,, =4.55 rx (I)

To minimize the negative effects of cell growth on ethanol yield, which are accentuated after 70 h of fermentation, an attempt was made to modify the metabolic pathway from growth to product formation by shutting down the air feed supply at this point in the fermentation. A comparison of the batch process running under microaerobic conditions (run 1) and the fermentation carried out with a shift in the operating regime from microaerobic to anaerobic conditions (run 2) is shown in Fig. 2.

When the oxygen supply was cut off, growth stopped and became dissociated from product formation. A better fermentation yield value was attained ( Y,,,s = 0.38 g g- ’ ), although a decrease in ethanol productivity from 0.14 to 0.09 glitree’ h-’ was b o served. Furthermore, by shift- ing the degree of aeration rate, there was a decrease in the xylose uptake rate and about 20%

d Time (hours)

Fig. 1. Concentrations of xylose, ethanol and biomass as a Fig. 3. Concentrations of xylose, xylitol and biomass as a function of time for a fermentation carried out with I’. szipiris function of time for a fermentation carried out with Cl pavq- under microaerobic conditions. silosis under oxygen limitation.

of the initial xylose concentration remained unused.

Although P. sripitis has NADH-linked xylose reductase activity, which prevents the imbalance of NAD + /NADH and allows xylose fermentation to ethanol anaerobically,’ the results obtained in this set of experiments suggest that microaerobic conditions (K,a = 4.8 h- ’ ) were the most suitable operating conditions for xylose fermentation with P. stipitis.

Xylitol production by C. parapsilosh A value of K,a = 16.8 hh ’ was selected for the fermentation of xylose with C. purapsifosis.‘h The concentration profiles of xylose, biomass, xylitol and oxygen as a function of fermentation time are shown in Fig. 3. With initial xylose concentrations

0 60 80 100

Time (hours)

Fig. 2. Comparison of the concentration profiles achieved in the conversion of xylose to ethanol by I? sripitis under microaerobic conditions (run I, solid line) and with a shift in the operating rcgimc from microaerobic to anaerobic condi- tions (run 2, dotted line).

-2 240 3 e .s E 8 20

s

0 0 20 40 no 100 120 I

Time (hours)

Ethanol and xylitolproduction by yeasts 661

of up to 50 g litre-‘, the peak xylitol concentra- tion appeared at 98 h and corresponded to the point where 88% of xylose was used. Complete sugar consumption was observed at 123 h.

The values attained for xylitol and biomass yields ( YPz,s= 0.65 g g-’ and Yxls= 0.11 g g-‘) and for the xylitol productivity ( Qp2 = 0.32 g litre-’ h-‘) we re higher than those achieved under microaerobic conditions (Table 1). The results also indicate that the concentration of dis- solved oxygen in the reactor rapidly decreased in the first 25 h of the process from 6.5 to 0.1 mg oxygen litre - ’ , and was maintained thereafter at this level. As oxygen became limiting, most of the xylitol was produced. From a plot of specific growth rate and specific rate of xylitol formation against fermentation time (Fig. 4) a relationship of about 7-O was found. This suggests that better production of xylitol was attained in oxygen- limited conditions.

To clarify this, two experiments were carried out under extreme conditions of aeration: anaero- biosis and oxygen excess (0.6 wm and 500 rpm: &a= 99.6 h-‘) (Table 2). Like most of yeasts metabolizing o-xylose, C. parapsilosis was not

0.3 0.4

Limitation of mygcn

Fig. 4. Evolution of specific growth rate (p) and specific rate of xylitol formation (qpr) as a function of time for the conversion of xylose by C. parapsilosis under oxygen limitation.

able to ferment this sugar anaerobically (Kluyver effect). On the other hand, oxygen excess condi- tions favoured growth ( Yxls = 0.29 g g- ‘) while the accumulation of xylitol decreased consider-

ably ( Ypz/s =o-10 gg-’ and QP2 = 0.023 g g-’ h-’ ).

As noted earlier, growth and product formation were associated but the value found from eqn (2) indicates that the formation of biomass was pre- ferred to xylitol.

qPZ = 0.46 ,u (2)

In fact, oxygen led to NADH oxidation to NAD- and a high NAD+/NADH ratio led to xylitol oxidation to xylulose; therefore, less xylitol was accumulated. This agreed with several studies, in that oxygen limitation is the main factor in the formation of xylitol. For C. parupsifosis, these conditions were reached after 25 h of the process running at K,n values of about 16.4 h - I.

CONCLUSION

P. stipitis and C. parapsilosis were found to be good producers of ethanol and xylitol, respect- ively from o-xylose. The chosen yeast strains were tested under different oxygen transfer rates and different optimal aeration levels were found.

Low oxygen transfer (K,a = 4.8 h- ’ ) was found to be best for ethanol production by P. stipitis while oxygen-limited conditions (K,a = 16.8 h- ’ ) were suitable for the conversion of xylose to xylitol by C. parapsilosis. Although oxygen supply was the main factor controlling of the conversion of n-xylose into ethanol, xylitol or biomass, other factors imposed by the culture microenvironment, such as inhibitors found in industrial feedstock based on xylose or addition of nutrients, may change the conditions described in this work. Attention should now be focused on adjusting these operational conditions for substrates derived from sugarcane bagasse. Recent work has indicated that minor differences are found when

Table 2. Influence of aeration rates on the conversion of xylose to xylitol with Candida parapsifosis

Stirring rate Aeration rate yx,s (rpm) Ivvm) fgg-‘/

yw,s QP fgg_‘) (g litre _ i h - ‘)

Anaerobic No fermentation was observed 250 0.3 0.1 I 0.65 0.32 500 0.6 0.29 O-10 0.02

662 S. A. Furlan, P. Bouilloud, H. F. de Castro

sorghum bagasse is used as substrate for xylitol production with C. parapsilopsis.22 It is expected that similar results would be achieved using waste material readily available in countries, such as Brazil, with established fuel alcohol production.

9.

10.

ACKNOWLEDGEMENTS 11.

The financial assistance of CNPq (Conselho National de Desenvolvimento Cientifico), Brazil is greatly acknowledged. Thanks are also due to Dr P. Strehaiano and the staff of the Laboratoire de G&tier pour les Bioindustries, Ecole Nationale Supirieure d’Ingenieurs du Genie Chimique, Toulouse, France, where the experimental work was carried out.

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