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Preparation of fermentation media from agricultural wastes and their bioconversion into xylitol
J. M. Cruz , J. M. Domínguez , H. Domínguez & J. C. Parajó
a a a a a

Department of Chemical Engineering, University of Vigo (Campus Ourense), As Lagoas, Ourense, 32004, Spain Available online: 09 Dec 2009

To cite this article: J. M. Cruz, J. M. Domínguez, H. Domínguez & J. C. Parajó (2000): Preparation of fermentation media from agricultural wastes and their bioconversion into xylitol, Food Biotechnology, 14:1-2, 79-97 To link to this article:

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FOOD BIOTECHNOLOGY, 14(1&2), 79-97 (2000)


J. M. Cruz, J. M. Domínguez, H. Domínguez and J. C. Parajó Downloaded by [University of Calcutta ] at 23:47 06 February 2012 Department of Chemical Engineering University of Vigo (Campus Ourense) As Lagoas, 32004 Ourense, Spain

Three agricultural wastes with high xylan content (barley bran, corn cobs and corn leaves) were hydrolysed with dilute sulphuric acid solutions under a variety of operational conditions in order to obtain xylose solutions suitable as fermentation media. The operational conditions for hydrolysis were selected on the basis of both the kinetic models developed for xylose generation and the concentrations of reaction byproducts affecting fermentation. Hydrolysates were supplemented with nutrients and fermented with Debaryomyces hansenii without any previous detoxification stage. Experimenal data concernmg batch fermentations are compared to the results obtained in the fermentation of hydrolysates from Eucalyptus wood, a typical raw material for prehydrolysis processes.


Xylitol, a naturally occurring five carbon polyalcohol, has a growing interest for the food industry due to both its high sweetening power and its technological properties (xylitol is not-cariogenic, is tolerated by diabetics and has a negative heat of solution) (Pepper and Olinger 1988).

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Downloaded by [University of Calcutta ] at 23:47 06 February 2012 Both chemical and biotechnological processes start from xylose solutions generated from lignocellulosic materials (LCM) having xylan as the main component of their hemicellulosic fraction. 1995. which can be converted into xylitol by NAD(P)H-dependent aldehyde reductase (Hofer et al. Dominguez et al. corn cobs. In some cases (for example. 1996 a-c.80 CRUZETAL. three agricultural residues with high xylan content (barley bran. 1996 a-c. 1991. literature has considered the fermentative production of xylitol with yeasts. and the xylose generation was assessed by means of a kinetic model. In this work. 1994. xylitol was produced from Dxylose by catalytic hydrogenation. In the last years. 1990. 1971). Heikkila et al. MATERIALS AND METHODS Raw Materials Samples of barley bran. acetic acid liberated from acetyl groups. In order to allow the selection of operational conditions leading to fermentable hydrolysates. 1997). corn cobs and corn leaves) are studied as alternative raw materials for xylitol production. Xylitol is found in certain fruits and vegetables. 1997 a-b. among them Debatyomyces hansenii (Girio et al. the generation of xylose and reaction byproducts by acid hydrolysis was studied. 1997 a-b). The reaction media obtained under selected conditions were neutralized. but its negligible concentrations make the extraction difficult and expensive. The hydrolysis of xylan leads to xylose-containing solutions which can be used to make fermentation media for xylitol production. Traditionally. Parajo et al. when Eucalyptus globulus wood is used as a raw material). For comparative purposes. 1995. LCM hydrolysates also contain undesired reaction byproducts (such as furfural and hydroxymethylfurfural generated by sugar dehydration. additional experiments were carried out with hydrolysates from a reference raw material {Eucalyptus globulus wood). non structural compounds of wood corresponding to the extractive fraction and lignin-degradation products) which can hinder or even prevent fermentation. However. Xylitol is formed as a metabolic intermediate in the fermentation of D-xylose. vacuum evaporated to increase the concentration of the carbon source and fermented to xylitol (without previous detoxification) using the yeast Debaryomyces hansenii. corn leaves and Eucalyptus globulus wood were . hydrolysates must be detoxified to allow reasonable kinetics and yields in the further fermentation (Parajo et al.

(1995) (3 % H2SO4) 130 °C. Hydrolysates were neutralized with CaCO3 to a final pH of 6. cooled. H2SO4 0. flow rate. homogenized in a single lot to avoid compositional differences and stored until use. Aliquots from the reaction media were taken at time zero and at selected reaction times.PREPARATION OF FERMENTATION MEDIA 81 dried. Hydrolysates were assayed by HPLC using an Interaction ION-300 column (mobile phase. Analysis of LCM Aliquots from the homogenized lots were submitted to moisture determination and to quantitative hydrolysis in two. Acid Hydrolysis Hydrolysates of barley bran. sterilized and used directly as fermentation media. The clarified liquors were supplemented with nutrients. milled to a particle size less than 1 mm. . The time zero in prehydrolysis was taken when the isothermal operation started (the heating period was not considered in the kinetic modelling). and the CaSO4 precipitate was separated from the supernatant by filtration. corn cobs and corn leaves were obtained in autoclave at 130°C with 1-5% H2SO4 solutions during 0 to 3 hours.45 um membranes and analysed by HPLC using the same procedure described above. xylitol. Hydrolysates of Eucalyptus globulus wood were obtained under operational conditions reported by Parajo et al. reaction time= 1 h). and 16 g/g for corn leaves (in this latter case. liquid/wood ratio = 8 g/g. The moisture content of samples was considered as water in the material balances.stage. The absorbance of hydrolysates was measured at 276 nm. acetic acid. and the second one after dilution of the media to 3 weight percent sulphuric acid at 121°C during 1 hour) (Vazquez et al. furfural and hydroxymethylfurfural (HMF). ethanol. 0. The solid Downloaded by [University of Calcutta ] at 23:47 06 February 2012 residue after hydrolysis was considered as Klason lignin. acid treatment (the first step with 72 weight percent sulphuric acid at 30°C during 1 hour. in order to obtain a semiquantitative estimate of the phenolic compounds (Parajo et al. 1997a). This method allowed the direct determination of glucose.01 M. filtered through 0. a higher ratio was necessary to obtain a homogeneous reaction media). IR and UV detection). arabinose. xylose.4 mL/min.5. Results concerning the composition of the raw materials are shown in Table 1.. using liquid/solid ratios of 8 g/g for barley bran and corn cobs. 1991).

8 20.3 30.2 12. 3 g malt extract/L and 5 g peptone/L. At given fermentation times.4 1. FRACTION Barley bran Cellulose Hemicelluloses Downloaded by [University of Calcutta ] at 23:47 06 February 2012 -Xylan -Araban Lignin Acetyl groups 26.45 um membranes and analysed by HPLC using the method described above.4 30.3 3.82 CRUZETAL.54 22.6 23. 3 g yeast extract/L.6 3. filtered through 0.7 37.6 Eucalyptus globulus wood 46.2 16. centrifuged. Table 1. Neutralized hydrolysates were supplemented with nutrients as described by Parajo et aL (1997a) and fermented with adapted yeasts (which were maintained in agar slants made from hydrolysates instead of commercial xylose solutions).9 3.. Culture media and fermentation Freeze-dried cells were grown in a fermentation medium containing 10 g pure xylose/L. Fitting of data The experimental data were fitted to the proposed kinetic models by minimization of .6 6. Microorganisms were maintained in agar slant tubes containing a medium formulated with the same components and concentrations as the previous one plus 20 g agar/L.6 0.Composition of the raw materials used in this study (results expressed as percent of the initial weight of sample. oven-dry basis).1 21.0 RAW MATERIAL Corn cobs Corn leaves 31.6 Microorganism Debaryomyces hansenii NRRL Y-7426 was used in the experiments.9 3.3 4. Fermentation trials were carried out in an orbital shaker (200 rpm) at 30 °C under microaerobic conditions using 250 mL Erlenmeyer flasks containing 50 mL of culture media. samples from the fermentation media were taken.

1997a). make the search for alternative materials with high xylan contents leading to clean hydrolysates interesting.PREPARATION OF FERMENTATION MEDIA 83 the sum of squares using commercial software with a built-in optimisation routine based in the Newton's method.4 g of xylitol/L with Pichia stipitis (volumetric productivity QP = 0.29 g/g with Candida guilliermondii.06 g/L-h and a product yield of 0.04 g/g). (1995) reported a volumetric productivity of 0. furfural .68 g/g when a detoxification stage (charcoal adsorption) was peformed (Parajo et al. On the other hand. but these parameters increased up to 1. (1996) reached a volumetric productivity of 0. For example. the operational conditions leading to directly fermentable hydrolysates of barley bran. 1997b). corn cobs and corn leaves were explored in the present work. In this way. The time courses of the concentrations of xylose (the main reaction product) and reaction byproducts with potential effect on fermentation (other sugars.43 g/g in the bioconversion of raw hydrolysates with Debaryomyces hansenii.16 g/L-h and a product yield of 0. Both yield and productivity can be significantly raised by detoxification of the raw hydrolysates: Parajo et al. The fermentation of Downloaded by [University of Calcutta ] at 23:47 06 February 2012 non-detoxified LCM hydrolysates is characterized by slow kinetics and limited productivities and yields in comparison with studies dealing with fermentation media made from commercial sugars or detoxified hydrolysates. Good results were also achieved when hydrolysates were subjected to other detoxification treatments such as overliming and extraction with organic solvents (Parajo et al.1 g/L-h and 0. product yield: Yp/s = 0.01 g/L-h. Ferrari et al. whereas Felipe et al. The fact that detoxification of hydrolysates is expensive and that the type and concentration of inhibitors depends on the LCM used as a raw material. strong inhibitory effects have been found dealing with fermentation media made from Eucalyptus wood hydrolysates. acetic acid. the toxic effects caused by reaction byproducts are dependent on both the LCM used as a raw material and the operational conditions under which the hydrolysis was carried out. RESULTS AND DISCUSSION Preparation of culture media The presence of fermentation inhibitors in hydrolysates from LCM is one of the major problems concerning the biotechnological production of xylitol. (1992) obtained 1.

Parajo et al. where C is the xylose concentration (g/L). The reaction scheme is: Xn(s) ki ->• Xylose k2 -» Furfural and other degradation products where ki and k2 represent kinetic coefficients. The results show a similar pattern for the three raw materials. The results obtained have been fitted to a kinetic model from literature which has been applied to the kinetic modelling of LCM (Ranganathan et al. 1986. Under the above hypotheses. a is the fraction of susceptible xylan in the raw materials. and hydroxymethylfurfural) were measured in hydrolysis assays carried out at 130 °C under different operational conditions (reaction time and catalyst concentration) for the three LCM. and t is the reaction time (minutes). In the reaction media. it can be inferred that: K2 . The initial xylose concentrations showed that a part of xylan reacted during the heating period. first-order kinetics with the existence of two xylan fractions in the raw materials. 1994). the xylose concentration decreased owing to the participation of degradation reactions. The reaction rate increased Downloaded by [University of Calcutta ] at 23:47 06 February 2012 with the catalyst concentration in all cases. only one of them (denoted Xn(s>) being susceptible to reaction. pseudohomogenous. The model assumes irreversible. with a maximum xylose concentration achieved at intermediate reaction times (15 minutes to 1 hour) depending on the catalyst concentration and raw material. k2 (min"1) is the kinetic coefficient corresponding to the decomposition of xylose reaction. Maloney et al. Figure 1 shows the xylose concentrations achieved in hydrolysis assays.84 CRUZETAL. At prolonged reaction times. Co is the initial xylose concentration (g/L). and xylose is degraded to furfural and other decomposition products. CpX is the potential xylose concentration (g/L) calculated assuming a quantitative conversion of xylan into xylose. .A T . the susceptible xylan is converted into xylose. 1985. kt (min'1) is the kinetic coefficient corresponding to the conversion of susceptible xylan into xylose.

.PREPARATION OF FERMENTATION MEDIA 85 1%H2SO4 40 2%H2SO4 3%H2SO4 4% H2SO4 5% H2SO4 35 " 3 30 - Downloaded by [University of Calcutta ] at 23:47 06 February 2012 a) Barley bran b) Corn cobs c) Corn leaves 50 100 Time (min) FIGURE 1 150 200 Experimental and calculated data of xylose concentration in hydrolysis experiments.

86 CRUZ ET AL. However.6 g/L (corresponding to almost 98 % conversion of the susceptible xylan). k2 and a for experiments performed with the same acid concentration. The values of the kinetic coefficients and the regression parameters are shown in Table 2. For experiments performed with barley bran the model predicted a maximum xylose concentration of about 35 g/L (corresponding to the 96 % conversion of the susceptible xylan). in experiments performed with corn leaves the maximum xylose concentration predicted for the model was 21.4 g/L (corresponding to 93 % conversion of the susceptible xylan). its value remaining below 24% of the correspondent maximum potential value (calculated from material balances). Tables 3a and 3b show the experimental time courses of glucose and arabinose concentrations. Other sugars (mainly arabinose and glucose) were generated during hydrolysis as reaction byproducts. On the contrary. Considering the data of Figure 1. Under the conditions leading to the maximum xylose concentrations. Harsher conditions led to decreased xylose concentration and increased contents in inhibitors. The experimental data showed in Figure 1 were fitted to the above equation to obtain the values of the regression parameters ki. Based on these idea. the manufacture of culture media was accomplished under milder operational conditions when necessary (see below). limited cellulose saccharification . Araban was readily transformed in arabinose with a tendency similar to that observed for xylan. but arabinose had little influence on the bioconversion of the hydrolysates. Figure 1 shows the close agreement found between experimental and calculated values of xylose concentration. The maximum xylose concentration in experiments performed with corn cobs increased up to 41. Finally. The reaction time necessary to reach the maximum Downloaded by [University of Calcutta ] at 23:47 06 February 2012 xylose concentration varied along wide limits depending on the acid concentration used. The model provided reliable information on the conditions leading to optimal xylose concentrations. the optimal conditions for hydrolysis can be considered around 15 minutes and 3 % of catalyst for barley bran and corn leaves and around 60 minutes and 1 % for corn cobs. the final decision selection of operational conditions for making culture media was made considering also the data concerning the concentrations of fermentation inhibitors (acetic acid and furfural). the concentration of glucose continuously increased with the severity of the experimental conditions.

.3 and 5.8 g of acetic acid/L were present during the fermentation of glucose from softwood hydrolysates by S.0779 0. Roberto et al. cell adaptation and the chemical processing of media (Watson et al. Parekh et al.945 1 2. a fact confirming the possibility of reaching a selective separation of xylan from cellulose.4. (min-1) 0. 1989.25-10"3 1 0.989 Downloaded by [University of Calcutta ] at 23:47 06 February 2012 1% 2% 3% 4% 5% k. (1999) reported no inhibitory influence when 4.2 g/L inhibited the growth of Candida utilis. Wilson et al. 1990. Frazer and McCaskey 1989.17-10"3 0.0982 0. The values of the acetic acid concentrations were 2. special attention was paid to study the acetic acid.12-10"3 1 4. Since the final objective of this work was to ferment the raw hydrolysates.03-10"3 1 4.176 0. On the contrary.0395 6.970 0.80-10"3 1 3.2 g/L for barley bran. 1991).7 g acetic acid/L reduced the utilization of pentoses by Pichia stipitis (Wilson et al.PREPARATION OF FERMENTATION MEDIA 87 Table 2. 1984. The acetic acid released from acetyl groups increased gradually with time and catalyst concentration (see Table 3c).971 0.42-10"4 0. and values of R2 measuring the correlation of equations.984 0. cerevisiae. 5. furfural and hydroxymethylfurfural generated. The maximum allowable concentrations of these compounds depend on a variety of factors. Larsson et al.760 0.22-10"3 1 0.990 0. (min"1) 0.171 0. Watson et aL (1984) reported that acetic acid concentrations higher than 1.283 2.0791 0. the kind of microorganism utilized.973 0. whereas 2. Under the harshest conditions assayed.396 4. including the type of bioconversion. These reaction byproducts can cause inhibitory or toxic effects in microorganisms.Results obtained for the regression parameters involved in the kinetic models.192 Com leaves a k2 (min"1) 2. 1986. % H2SO4 Barley bran a k2 ki (min"1) (min"1) 1 0. and 1.7 g . Furfural generated from pentose by dehydration reactions increased with the severity of the experimental conditions (see Table 3d).53-10"3 1 0.240 2.05-10"3 1 R2 0.977 9.45 g/L inhibited completely the growth of Pachysokn tannophylus. 2.82-10"3 1 R2 0.25-10"3 1 2.990 0.995 0. corn leaves and corn cobs respectively under the conditions leading to maxima in xylose concentration.0884 0.160 1.148 RAW MATERIAL Corn Cobs R2 k2 a (min"1) 1.992 0.960 3.0810 0.62-10"4 0.947 k.O8-1O"3 0.75-10"3 0.992 was observed.4H0" 4 0.967 0.0987 0.896 0.983 0. These latter values are near the threshold value leading to inhibitory effects on yeasts.966 2. Delgenes et al. 1989).

2 0.6 5.4 7.1 6.7 2.0 2.8 4.9 7.3 2.3 3.5 2 % H2SO4 3 % H 2 SO 4 5 % H2SO4 BB' 2.2 0.3 0.6 2.9 1.3 6.4 2.3 4.0 5.6 6.0 2.0 3.4 0.4 3.3 2.0 2.9 CL3 0.8 1.5 CL3 0.7 1.1 BB1 0.3 2.5 6.2 0.1 2.3 4.8 2.2 0.5 1.9 4.9 7.0 BB' 1.5 6.5 6.0 4.8 5.1 CL3 2.6 CL3 0.2 0.0 2.4 0.5 5.6 4.6 3.9 5.4 15 2.9 1.6 CC 2 1.4 2.04 0.4 3.7 7.) 0 0.1 4.7 1.0 6.3 0.9 CL3 2.4 1 % H2SO4 2 % H2SO4 3 % H 2 SO 4 4 % H 2 SO 4 5 % H2SO4 BB1 5 10 IS 30 60 120 180 CC 2 0.9 5.5 2.6 3.6 5.4 2.4 2.4 2.02 0.0 3.4 7.4 0.0 7.2 2.1 0.1 4.8 1.4 BB1 5.2 0.5 4.5 2.6 2.4 4.8 1.0 5.8 4.5 1.9 4.7 5.6 4.2 0.3 4.9 7.9 1.5 2.3 5.0 3.2 5.9 5.0 2.6 4.8 BB' 5.5 2.1 0.4 3.9 6.7 2.7 4.04 0.4 6.1 4. 1.2 3.2 2.6 3.2 0.9 3.6 4.01 0.4 1.5 CL3 0.0 2.4 0.7 1.03 0.04 0.5 CL3 1. CL3 = Com Leaves 0.8 6.04 0.8 0.7 2.7 1.3 CC2 0.4 0.8 2.6 6.7 3.6 CC2 2.7 0.4 5.3 7.1 2.6 3.3 0.5 6.6 1.1 0.6 2.6 1.9 7.6 0.01 0.0 6.9 CC2 3.) 0 5 1 % H2SO4 BB' 5.2 2.6 5.2 2.3 2.4 3.3 3.02 0.9 2.1 3.1 2.2 2.2 c) Acetic acid concentration (g/L) Time (min.0 5.6 5.8 2.6 4.1 2.0 2.9 3.4 3.9 4.01 0.0 CC2 2.4 6.3 7.3 0.5 4.5 1.8 CL3 0.3 0.) 0 5 10 15 1 % H2SO4 BB' 2.0 4.6 2.4 2.4 0.7 1.1 0.0 6.1 3.3 5.2 CL3 0.2 3.5 1.7 1.7 4.2 5.7 2.8 CL3 0.4 BB' 0.0 CL3 2.9 6. Table 3.2 2.2 2.1 0.4 3.9 5.9 2.2 3.3 8.5 2.6 0.1 0.1 2.3 3.4 3.8 7.3 4.7 2.4 5.7 2.1 6.4 0.8 CL3 1.1 3.7 2.5 5.8 1.4 2.3 2.7 1.2 6.4 2.1 5.1 0.0 5.7 4.8 CC2 3.8 1.8 6.0 1.4 CC2 CL3 l-9_.7 4.7 6.3 6.1 6.3 2.9 4.7 CLJ 0.7 2.2 0.9 5.2 2.4 5.6 2.4 CC2 2.5 4.7 0.3 2.3 CC2 3.2 5.3 6.0 4.3 2.6 4.1 1.1 7. CC 2 = Com Cobs.8 6.1 0.7 1.0 3.5 3.6 3.8 6.4 1.2 3.2 0.4 1.3 2.3 3.9 BB 1 = BarleyBran.0 3.9 8.0 10 15 30 60 120 180 BB' 5.7 2.3 6.1 2.) 0 5 10 15 30 60 120 180 1%H 2 SO 4 BB' CC2 0.5 b) Arabinose concentration (g/L) Time (min.1 2.0 2.3 2.4 4.7 2.1 0.8 BB' 3.4 2.5 3.-Byproduct concentrations achieved under different operational conditions.9 3.4 1.2 2.3 2 % H 2 SO 4 3 % H 2 SO 4 4 % H 2 SO 4 5 % H2SO4 1.4 0.7 5.5 2.7 4.4 CC 2 0.7 CC2 0.0 7.6 2.2 0.4 CL3 0.4 5.8 1.9 3.4 2.01 0.6 2.0 2.0 3.5 2.4 CC 2 Time (min. a) Glucose concentration (g/L) 4 % H2SO4 BB' 2.3 6.2 2.8 3.6 2.0 5.4 2.2 0.4 2.1 63 CL3 0.6 4.1 2.8 3.01 0.1 30 Downloaded by [University of Calcutta ] at 23:47 06 February 2012 60 120 180 3.5 8.0 7.7 3.0 BB' 1.9 6.6 3.4 5.4 3.1 1.4 2.1 5.7 1.0 CL3 1.6 4.1 6.6 3.6 2.1 0.5 2.1 0.5 2.2 0.02 0.8 7.2 0.6 5.5 2.9 1.0 2.3 CC2 CL3 0.5 2.4 2.3 4.5 CC2 0.7 4.9 5.2 4.1 3.6 0.5 3.8 CL3 0.8 BB1 5.0 2.5 2.5 2.1 BB' 1.3 2.8 1.0 2.5 2.88 CRUZETAL.7 1.6 4.6 6.0 4.8 2.3 2.2 2.5 2.03 0.7 7.4 2.1 .7 BB' 2.6 1.9 5.1 5.5 4.0 3.4 CC2 0.2 2.3 7.9 8.3 6.1 2.1 6.3 2.0 5.6 CC 2 1.7 46 50 4.3 0.4 2.4 2.9 2.7 3.6 3.3 8.3 3.8 7.1 CL3 2 % H2SO4 3 % H 2 SO 4 4 % H2SO4 5 % H2SO4 BB 1 0.9 CC2 0.7 5.1 3.1 d) Furfural concentration (g/L) Time (min.0 1.9 5.0 2.0 2.6 3.0 5.8 3.6 2.8 4.6 4.6 2.4 4.4 2.5 6.5 2.4 2.4 2.01 0.1 3.9 6.5 2.03 0.0 7.7 6.8 3.6 2.7 7.1 0.3 CC2 0.2 BB' 0.1 5.3 6.3 BB' 0.3 2.0 2.0 2.4 5.0 4.8 1.

1988). the growth of Pichia stipitis was reduced by 25 %.32. the operational conditions for making fermentation media from corn cobs were fixed in a H2SO4 concentration of 2% and 15 min reaction time. 47 % and 99 % respectively. On the contrary. (1999) observed the metabolization of 2 g/L of the overall amount corresponding to furfural and hydroxymethylfurfural by S. (1997) working with C.6 g/L from corn cobs and 3. For example. With this modification. Taherzadeh et al.74 to 0. According to reported results. but an almost complete growth inhibition in media containing 2 g/L of higher.PREPARATION OF FERMENTATION MEDIA 89 furfural/L were produced from barley bran. Under the operational conditions selected for making fermentation media. Considering these results and the furfural concentrations achieved in hydrolysis.33 g/L).7 g furfural/L on the cell growth at the expense of xylitol and ethanol.74 and 0.5. (1991) observed an activating effect of furfural on the Pichia stipitis growth at concentrations up to 0. the xylose concentration decreased only slightly. it must be considered that in the present work the furfural concentration in hydrolysates was reduced during vacuum evaporation (see below).34 g/L for barley bran. it was concluded that the furfural content in media from either barley bran or corn leaves are satisfactory. but some problems could happen when corn cobs are used as a raw material. in order to reduce the inhibitor concentration below the threshold reported in literature. 1996) although Preziosi-Belloy et al. 8. (1996) found that in the presence of 0. the furfural concentrations were 0. 0.1 g/L from corn leaves. no inhibitory effects were observed in the presence of 0.08 g of hydroxymethylfurfural/L in the fermentation of sugar cane bagasse hydrolysates with Candida tropicalis (Pessoa et al. and Delgenes et al. On the other hand.1 g/L in all cases. respectively. (1984) reported that the maximum specific growth rate of Pachysokn tannophilus was reduced by 63% at a furfural concentration of 0. The hydroxymethylfurfural concentrations (data not shown) were below 0. cerevisiae. this concentration being lower than the limit causing inhibitory effects on fermentation. Watson et al. the toxic effect of furfural in Pichia stipitis is related to the inhibition in respiration and oxidative phosphorylation (Weigert et al. Roberto et al. parapsilosis also observed an activating effect of 0. corn cobs and corn leaves.2 g/L to 3. In this case the optimal conditions predicted from the model for xylose were modified to milder conditions for the manufacture of fermentation media.35 g/L and completely inhibited at 1. (1997) reported a . 1 and 2 g Downloaded by [University of Calcutta ] at 23:47 06 February 2012 furfural/L. whereas the acetic acid concentration decreased from 5.7 g/L and the furfural concentration decreased markedly (from 0.75 g/L.. Based on this idea. Preziosi-Belloy et al.5 g/L.

Figure 3 shows the time courses of xylose consumption. Felipe et al. This step removed a part of the furfural contained in the media (see Figure 2). 1996. where 40 % of the initial glucose remained unconverted at the end of fermentation. neutralized and vacuum-concentrated to obtain new fermentation media with similar concentration of the carbon source. 1996). 1992. xylitol production and cell growth in media made from the four raw materials considered in this part of the experimentation. Concentrated hydrolysates were supplemented with nutrients. 1996 a-c. fresh fermentation media and exhausted cultures is shown in see Table 4. 1997). Additional information on the composition of hydrolysates. A preliminary analysis of results shows that suitable fermentation media can be obtained from barley bran. Glucose was consumed before 24 hours except in the case of Eucalyptus globulus hydrolysates. In these hydrolysates. The experimental results presented in these fermentations are the average of duplicate assays. Dominguez et al. the average deviation between assays being 4. slightly stimulatory effect on xylitol production by Candida pampsilosis caused by the presence of 1. including corn cobs (Cao et al. whereas strong inhibition was observed in the case of Eucalyptus wood hydrolysates. a well know raw material (Eucalyptus wood) was hydrolysed under reported conditions. For comparative purposes. In order to check the fermentability of hydrolysates made from barley bran.90 CRUZ ET AL. The effects of inhibitors are strongly influenced by the raw material and by the hydrolysis technology. Fermentation of hvdrolysates Several authors have reported on the biotechnological production of xylitol from Downloaded by [University of Calcutta ] at 23:47 06 February 2012 LCM. 1997 a-b. sterilized and used for fermentation with the yeast Debaryomyces hansenii. Figure 2 shows the general idea of the chemical-biotechnological process considered in this study. corn cobs and corn leaves.5 g of furfural/L. Parajo et al. the correspondent liquors were vacuum-evaporated to reach initial xylose concentrations about 45 g/L. Complementary information on the composition of selected process streams is listed Table 4. corn fiber (Saha and Bothast 1996) and Eucalyptus globulus wood (Ferrari et al. . corn cobs and corn leaves.1 %. glucose and xylose are potential carbon sources for microorganisms. 1995.

VOLATILE COMPOUNDS (ACETIC ' ACID. but the percent of xylose consumption was about 80 % in the case of Eucalyptus globulus wood hydrolysates (see Figure 3).. EXTRACTIVES. corn cobs or corn leaves.) CaCO3 NEUTRALIZATION/ CENTRIFUGATION EVAPORATION Downloaded by [University of Calcutta ] at 23:47 06 February 2012 CaSO4 1 B (Sugar solution) NUTRIENTS STERILIZATION Debatyomyces hansenii INOCULATION FERMENTATION EXHAUSTED MEDIUM FIGURE 2 Framework of the chemical-biotechno logical process considered in this study (compositional data shown in Tables 3 and 4). FURFURAL.. Xylitol production was strongly inhibited in media made from Eucalyptus globulus wood: concentrations as low as 1. poor product yield and reduced specific rate of product . A related consumption pattern was observed for xylose: 95 % of the initial amount of this sugar was consumed after 81 hours of fermentation when using hydro Iysates from barley bran.PREPARATION OF FERMENTATION MEDIA H2SO4 .5 g xylitol /L were achieved at the end of fermentation. with low volumetric productivity. ACID HYDROLYSIS SOLID RESIDUE 91 RAW — MATERIAL WATER.

SO 4 15 minutes Corn Leaves 3 % H2SO4 15 minutes Eucalyptus globulus wood 3 % H2SO4 60 minutes N .1 1.0 < 0.5 12.1 48.1 < 0.1 12.6 < 0.2 0.9 5.1 < 0.1 3.6 0.1 STREAMS A g xylose/L g glucose/L g arabinose/L g acetic acid/L g furfural/L gHMF/L g xylose/L g glucose/L g arabinose/L g acetic acid/L g furfural/L gHMF/L g xylose/L g glucose/L g arabinose/L g acetic acid/L g furfural/L gHMF/L g xylose/L g glucose/L g arabinose/L g acetic acid/L g furfural/L gHMF/L Downloaded by [University of Calcutta ] at 23:47 06 February 2012 C 45.2 5.1 0.4 0.0 3.7 2.1 1.2 < 0.1 < 0. RAW MATERIAL HYDROLYSIS CONDITIONS 35.6 4.1 41.1 < 0.9 3.3 < 0.1 4.5 2.1 26.5 5.2 4.1 < 0.3 3.6 0.3 7.1 g xylose/L g glucose/L g arabinose/L g acetic acid/L gfurfural/L gHMF/L g xylose/L g glucose/L g arabinose/L g acetic acid/L g furfural/L gHMF/L g xylose/L g glucose/L g arabinose/L g acetic acid/L gfurfural/L gHMF/L g xylose/L g glucose/L g arabinose/L g acetic acid/L gfurfural/L gHMF/L 1.3 <0.1 35.2 0.1 < 0.9 3.2 < 0.1 29.8 1.7 2.6 2.7 2.2 0.5 < 0.3 < 0..3 < 0.6 3.5 0.1 21.3 2.7 4.1 1.4 3.5 2.Table 4.1 2.1 22. fresh fermentation media and exhausted fermentation media (nomenclature of streams according to Figure 2).5 9.2 2.1 17.6 6.1 42.7 0.1 5.6 0.Data concerning the chemical compostion of hydrolysates.5 < 0.0 2.7 4.3 0.1 0.4 7.5 9.6 5.7 D g xylose/L g glucose/L g arabinose/L g acetic acid/L gfurfural/L gHMF/L g xylitol/L e ethanol/L g xylose/L g glucose/L g arabinose/L g acetic acid/L gfurfural/L gHMF/L gxylitol/L g ethanol/L g xylose/L g glucose/L g arabinose/L g acetic acid/L gfurfural/L gHMF/L gxylitol/L g ethanol/L g xylose/L g glucose/L g arabinose/L g acetic acid/L gfurfural/L gHMF/L gxylitol/L g ethanol/L Barley Bran 3 % H2SO4 15 minutes Corn Cobs 2 % H.0 5.9 7.

PREPARATION OF FERMENTATION MEDIA 93 " Barley " • " Corn leaves * Corn cob —•. .Eucalyptus Downloaded by [University of Calcutta ] at 23:47 06 February 2012 25 50 75 Time (h) FIGURE 3 100 125 Time course of hydro lysate fermentations with Debaryomyces hansenii.

barley bran and corn leaves: after 81 hours. Qp.018 1} YP!S (g/g) 0.Stoichiometric and productivity parameters for bioconversion assays carried out with different raw hydrolysates (results calculated for fermentation times leading to maximum xylitol concentrations).61 0. On the contrary.2 29.0 1.64 0. Raw material Barley bran Corn cobs Corn leaves Eucalyptus Downloaded by [University of Calcutta ] at 23:47 06 February 2012 wood 1) Nomenclature: P. Among the inhibitors.7 g of ethanol/L were detected in media made from the considered raw materials (see Table 4). maximum xylitol concentration. This fact is in agreement with the data reported by Ferrari et al.036 0. specific product formation rate globulus l) P(g/L) 26.6 21. .006 formation (see Table 5). the highest acetic acid concentration leading to the lowest xylitol production rate.0-9. The differences in behaviour observed between wood and agricultural wastes can be ascribed to the different type and amount of inhibitors generated as hydrolysis byproducts. These results are particularly interesting when considering both the absence of detoxification treatments in the processing scheme and the limited initial biomass concentration used in experiments.36 0..044 V(g/g-h) 0.33 0. Table 5.26 0. product yield. Their concentrations in the several fermentation media (see Table 4) are closely correlated to the susceptibility of media to fermentation. volumetric productivity. (1992). A significant part of xylose was used for cell growth (see Figure 3c) or derived towards ethanol production. In this way.025 0. which are dependent on the chemical nature of the raw materials. after 81 hours of fermentation. product yields and specific xylitol production are given in Table 5. this later being a marketable fermentation byproduct. Additional data on volumetric productivities. the xylitol concentrations were in the range 21.94 CRUZ ET AL. qP. 2.52 0.2-29 g/L for the three feedstocks. satisfactory xylitol production was achieved in media made from hydrolysates of corn cobs.032 0.5 " Q P (g/L-h) 0. acetic acid and furfural seem to be responsible for the major effects.

Chen.PREPARATION OF FERMENTATION MEDIA 95 who observed absence of xylitol production by Pichia in media made from Eucalyptus wood hydrolysates containing 10. Elsevier Appl. M. 1997.. J. M. Wood hydrolyzate treatments for improved fermentation of wood sugars to 2. Vol. Moletta. G. N. 1991. I. 1996. Biores... and Tsao. M. Du. and Navarro. Ho.. and McCaskey.. A. S.. J. Biochem. J. ACKNOWLEDGMENTS The authors are grateful to Paula Valevona Corral for her excellent collaboration in the realisation of this work as a part of her degree project. J. Enzyme Microb. J. and Tsao. C . Technol. 40: 753759. Delgenes. C. 18(9): 1013-1018. S. 56: 281-283. 18: 31-42. Hyöky. P. Internacional patent: WO 91/10740. Alves. Biotechnol.. Gong. J. Albornoz. N. Felipe. Technol. Proc. and Navarro. and Saucedo.. J. D. Lett. 1992. Almeida. Sá-Machado. Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism in Debaryomyces hansenii. Moletta. and Viljava. (Grassi. Dominguez. Girio.). T. Techno 1. Enzyme Microb. M. Acid hydrolysis of wheat straw and process considerations for ethanol fermentation by Pichia stipitis Y7124. S. M. L.. Eds. L. Science. T. 1989. Downloaded by [University of Calcutta ] at 23:47 06 February 2012 REFERENCES Cao. A process for the simultaneous production of xylitol and ethanol. Sarkki. G. Gosse.. J. 1996. E. Delgenes. Ethanol production from Eucalyptus wood hemicellulose hydrolysate by Pichia stipitis. Z.5 g acetic acid/L. Cao. . Int. Frazer. and dos Santos. 1990. M D. 61: 85-90. M 1996. A. Mancilha. Fermentation of eucalyptus hemicellulosic hydrolysate to xylitol by Candida guilliermondii.. A. 1994. F. T. and Silva.. N. J.. In: Biomass for Energy and Industry. XUGA 38303A98). S. Roseiro. R. J. A. and Amaral-Collaco. Duarte-Reis. Biores. B.. Dilute acid hemicellulose hydrolysates from corn cob for xylitol production by yeast. Girio. 2.. M. H. 16: 1074-1078. Ferrari. G. F. E. Pichia stipitis and Candida shehatae.. C. R. S. 1990. G. C.. and Amaral-Collaço. I. J. The authors are also grateful to the "Xunta de Galicia" for the financial support of this work (Proj. 8: 132-135. T. F. W. Peito. X. Roberto. C. G.. A. G. Biotechnol.. Ethanol production from corncob pretreated by the ammonia steeping process using genetically engineered yeast. G.. Neirotti. R. Silva. Effects of lignocellulose degradation products on ethanol fermentations of glucose and xylose by Saccharomyces cerevisiae. L.. Krishnan. M. Gong. Biomass. T. 19: 220-225. M.1-10. M. R. Zymomonas mobilis. Technol. Bioeng..3-butanediol.. T. Rahkila. M. M. Heikkilä.

Parajó. 21: 124-129. Vázquez. L. 1991.M. 1997b. J. Prehydrolysis of Eucalyptus wood with dilute sulphuric acid: operation in autoclave. T. Zacchi. M. W.. Parajó. Betz. Domínguez. A. and Olinger. L. S. and Domínguez. Enzyme Microb. S. Yu. 13: 125-131. Technol. H. C. Maloney. and Domínguez.. Prog.. An engineering analysis of the production of xylose by dilute acid hydrolysis of hardwood hemicellulose. J.. I C. 32: 599-604. 1971. and Domínguez. J. 16: 39-43. Pepper. Technol. Domínguez. Biotechnol. 21: 18-24. Biores. J. Biochem. . and Baker. C. Downloaded by [University of Calcutta ] at 23:47 06 February 2012 Parajó. J. M. I. Parekh. C. Biores. Preziosi-Belloy. K. 2: 192-202. N. Proc. M G. 1994. H. J. B. Ranganathan.. Production of xylitol from wood hydrolysates by Debaryomyces hansenii. Bioproc. 1986. J. J.. Charcoal adsorption of wood hydrolysates for improving their fermentability: influence of the operational conditions. 36: 271-275. J. Kinetics studies of wheat hydrolysis using sulphuric acid. Parajó. Mancilha. and Navarro. Hahn-Hägerdal. 24: 151-159. V.. Santos. T. Stenberg. Technol. Höfer. M. Domínguez. 1996.. Eng. M. Fermentation of hemicellulosic sugars and sugar mixtures to xylitol by Candida parapsilosis. G. E.. Chapman. 57: 179-185. Can. Domínguez. S. T. 52: 102-108. The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Palmqvist. M. M. Cultivation of Candida tropicalis in sugar cane hemicellulosic hydrolyzate for microbial protein production. and Domínguez. P.96 CRUZ EX AL. C. A. 1988. Parajó. C . Lett. D. M. Domínguez.. Nolleau.. H. H. M. Metabolism of the obligatory aerobic yeast Rhodotorula gracilis: induction of an enzyme necessary for D-xylose catabolism. V. 18: 593-598. Larsson.. I. and Domínguez.. S. and Nilvebrant. 1996a. 1985. J. Biotechnol.. G. and Wayman. D. Biotechnol. Tengborg. Bioproc. Holz Roh Werk. and Bakhshi. Food Technology 42: 98-106. J. Adaptation oí Candida shehatae and Pichia stipitis to wood hydrolysates for increased ethanol production. C... M. Biophys. E. 1996c. L. 51: 83-88.. J. 252: 1-12. Biotechnol. H. Parajó. J. S. 1999. Utilization of sugar cane bagasse hemicellulosic hydrolyzate by Candida guilliermondii for xylitol production. and Silva. Roberto. Microbiol. 1996b.. Eng. 1997. and Kotyk. Lacis. Study of charcoal adsorption for improving the production of xylitol from wood hydrolysates. Chem. J. Eng. M. Pessoa. Alonso. S. Improved xylitol production with Debaryomyces hansenii Y-7426 from raw or detoxified wood hydrolysates. Appl. Production of xylitol from concentrated wood hydrolysates by Debaryomyces hansenii: Effect of the initial cell concentration. and Domínguez. A. A. 63: 840-844. 1986. 1995. C . S. 25: 300-304.. J. and Domínguez. N. Domínguez. H.. Technol. Acta. and Sato.. C. Xylitol in sugar-free confections. Xylitol production from eucalyptus wood hydrolysates extracted with organic solvents. N. Enzyme Microb. McDonald. H. 1997a. Biochem. A. J. Felipe. J. Parajó. M.

PREPARATION OF FERMENTATION MEDIA 97 Saha. C . Prior. C. C. Rev. Comparative fermentability of enzymatic and acid hydrolysates of steam-pretreated aspenwood hemicellulose by Pichia stipitis CBS 5776. 1999. B. 1984. Biotechnol. Microbiol. H.. and Nishikawa N. and Dellweg. Biotechnol. M. Klein. J. Biosci. C. Taherzadeh. B. J. E. Enzyme Microb.. J. Lett. Microb. 6: 451-456. 45: 299-306. P. Agroquím. fraccionamiento y aprovechamiento. J. Parajó. Watson. Biotechnol. 87 (2): 169-174.. Wilson.. N. M.. Technol. Alimen. 1991. Production of L-arabhol from L-arabinose by Candida entomaea and Pichia guilliermondii. L. 10: 895-900. and Bothast. and Lategan. 1989. Conversion of furfural in aerobic and anaerobic batch fermentation of glucose by Saccharomyces cerevisiae. 31: 592-596. B. Downloaded by [University of Calcutta ] at 23:47 06 February 2012 . Vázquez. C. Weigert. R. 1988. A. G. Niklasson. 31(2): 143-164. 1996. J. Deschatelets. G. Lage. Appl. G. Transformación de materiales lignocelulósicos: Composición. Xylose fermentation by yeasts. Gustafsson. Appl. M. A.. Tecnol. and Vázquez. Lauterbach.. and Lidén.. Bioeng.. Factors in acid treated bagasse inhibiting ethanol production from D-xylose by Pachysolen tannophilus. L. Influence of furfural on the aerobic growth of the yeast Pichia stipitis.