Culture parameters affecting xylitol production by Debaryomyces hansenii immobilized in alginate beads

Process Biochemistry 48 (2013) 387–397

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Culture parameters affecting xylitol production by Debaryomyces hansenii immobilized in alginate beads Belinda Pérez-Bibbins a,b , José Manuel Salgado a,b , Ana Torrado c,b , María Guadalupe Aguilar-Uscanga d , José Manuel Domínguez a,b,∗ a

Department of Chemical Engineering, Faculty of Sciences, University of Vigo (Campus Ourense), As Lagoas s/n, 32004 Ourense, Spain Laboratory of Agro-Food Biotechnology, CITI (University of Vigo)-Tecnópole, Parque Tecnológico de Galicia, San Cibrao das Vi˜ nas, Ourense, Spain c Bromatology Group, Department of Analytical and Food Chemistry, Faculty of Sciences, University of Vigo (Campus Ourense), As Lagoas s/n, 32004 Ourense, Spain d Instituto Tecnológico de Veracruz-UNIDA, Av. Miguel A. de Quevedo 2779, Col. Formando Hogar, CP 91860, Veracruz, Mexico b

a r t i c l e

i n f o

Article history: Received 4 August 2012 Received in revised form 15 January 2013 Accepted 17 January 2013 Available online 26 January 2013 Keywords: Xylitol Debaryomyces hansenii Dimorphism Immobilization Corncob hydrolyzate Vinasses

a b s t r a c t Yeast immobilization offers operational advantages such as high cell concentration, and some drawbacks related to cell leaking and restricted mass transfer inside particles. The influence of bead size, chitosan, bead charge, volume of liquid media, and the use of corncob hydrolyzates and vinasses as culture medium were analyzed on xylitol production by Debaryomyces hansenii immobilized in alginate beads. The results showed a profuse growth of free cells, accounting 75–95% of total biomass, but electron micrographs revealed the generation of a dense biofilm with hyphal morphology at the bead surface and a very low intraparticular growth. Xylitol production was not affected by the size of particle; however chitosan had a negative effect. The use of corn cob as carbon source and twofold diluted vinasses as economic nutrients incremented xylitol concentration to 13.7 g L−1 (YP/S = 0.56 g g−1 ; QP = 0.29 g L−1 h−1 ). The best conditions corresponded to high bead charges and intermediate liquid volumes (44 g Na-alginate and 110 mL liquid medium). These results showed the feasibility of employing these cheap substrates, reflected the importance of the microaerobical conditions, and pointed to the favorable effect of cell immobilization on the metabolism of xylitol production. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction The increasing demand of consumers for natural products has intensified the biotechnological production of natural additives, including xylitol, a naturally occurring sugar alcohol sweetener, discovered in 1891, that has sweetness similar to sucrose but 40% lower energy, negative heat of dissolution, low viscosity in solution, absence of the Maillard reaction, insulin-independent metabolism, higher chemical stability, and several biomedical properties [1]. Xylitol can replace sucrose on a weight-for-weight basis, particularly considering that xylitol has a pleasant taste and no unpleasant aftertaste. With these properties and being a sweetener recognized since the 1960s, xylitol has found increasing use in the food industry, especially in confectionery, and it is also an important sugar substitute for diabetics. Additionally, xylitol has been shown to decrease mutants Streptococci levels, plaque formation, and the incidence of caries [2], being commonly used for oral hygiene as

∗ Corresponding author at: Department of Chemical Engineering, Faculty of Sciences, University of Vigo (Campus Ourense), As Lagoas s/n, 32004 Ourense, Spain. Tel.: +34 988 387416; fax: +34 988 387001. E-mail address: [email protected] (J.M. Domínguez). 1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2013.01.006

well as the preparation of chewing gum, comfits, and pharmaceutical and cosmetic products [3]. In spite of this, the relatively scarce worldwide production of xylitol can be attributed to the high cost of production by chemical means (6.39 D/kg), which is 10 times higher than other traditional sweeteners [4]. Xylitol is nowadays produced commercially by chemical synthesis, after (a) prehydrolysis of lignocellulosic materials, (b) concentration followed by physical–chemical treatments, (c) hydrogenation in presence of nickel, palladium or ruthenium catalysts at 80–140 ◦ C and 50 atm [5], and (d) final xylitol recovery by concentration and crystallization, yielding a product with a purity of 99.7% and a yield of 50–60% with respect to the initial xylose [6]. Alternatively, xylitol can be produced by fermentation, a process which shows certain advantages including milder conditions of pressure and temperature and lower costs of downstream due to the production of lower amounts of by-products [4,7,8]. However, carbon source and nutrients must be economically competitive in order to ensure the feasibility of the process, considering that, particularly, the high cost of yeast extract can impair the economics of fermentation, its cost being estimated in almost 40% of the final cost of some biotechnological processes [9]. Corncob, a major waste obtained in corn production and generally employed as animal feed, recycled fertilizer in soil, or burned as

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a fuel [10], is a largely available source of xylose after acid hydrolysis [11]. On the other hand, vinasses, the main liquid wastes from the distillation process, are acidic effluents with high organic content and high chemical and biological oxygen demand [12], which can be efficiently employed as economic nutrients for larger-scale bioproduction of natural food additives using strains of Lactobacillus rhamnosus, Debaryomyces hansenii or Aspergillus niger [9]. The efficiency of the fermentation can be augmented by using high cell concentrations, which can be achieved with immobilized cell systems. This technology provides higher fermentation rates, permits the reuse of higher cell densities in bioreactors for extended periods of time, facilitates the separation of the biocatalysts from the liquid phase where the desired products are present, improves stability and protection of the cells against inhibitors [13], encourages continuous processes that may lower capital costs, and also precludes the need to separate the cells from the substrate and products following processing. Particularly, immobilization in Caalginate beads stands out because their production method does not require drastic conditions and, in regard to food applications, utilizes ingredients that are accepted as food additives. Alginate also shows low cost as immobilization support. However, although cell immobilization techniques could improve the productivity, the use of immobilized systems often shows lower mass transfer rates from the liquid and gas phases to the immobilized biomass, and leakage of cells into the medium. This work deals with the feasibility of xylitol production by D. hansenii immobilized in alginate beads with the perspective of scaling-up the fermentation process. Different operational variables were studied prior to the substitution of all the components of a standard synthetic medium by xylose-containing hydrolyzates originated from corn cobs and vinasses as the source of nitrogen and mineral salts as a cheap alternative medium. 2. Materials and methods 2.1. Reagents Culture media components: Glycerol (141339.1211), yeast extract (403687.1210), malt extract (403690.1210) and peptone (403695.1210) were supplied by Panreac Química (Barcelona, Spain). d(+) xylose (29013.237) was purchased to BDH Prolabo GPR Rectapur, VWR (Leuven, Belgium). Immobilization reagents: Sodium-alginate (373059.1209), CaCl2 ·2H2 O (191232.1211), acetic acid (122703.1611) and tri-potassium citrate 1-hydrate (141492.1210) were supplied by Panreac Química (Barcelona, Spain). Chitosan (448877) was purchased to Sigma Aldrich (St. Louis, MO, USA). HPLC analytical reagents and standards: Xylitol (S X3375), d(+) xylose (95729), d(+) glucose (49139), d(−) arabinose (A3131), 5-(hydroxymethyl)furfural (H40807) and furfural (48070) were supplied by Sigma Aldrich (St. Louis, MO, USA). Ethanol 96% (v/v) (131085.1611), formic acid 98% (v/v) (131030) and H2 SO4 (131058.1211) were purchased to Panreac Química (Barcelona, Spain). Reagent for hydrolyzates preparation: H2 SO4 (131058.1211), CaCO3 (141212.1211) and activated charcoal (211238.0914) were supplied by Panreac Química (Barcelona, Spain). Field Emission Scanning Electron Microscopy (FE-SEM) reagents: Cacodylic buffer (C0125), glutaraldehyde solution (SG5882) and liquid nitrogen (00474) were supplied by Sigma Aldrich (St. Louis, MO, USA). 2.2. Microorganism and cell immobilization D. hansenii NRRL Y-7426 (CECT 11369) was maintained in cryovials on 30% (v/v) glycerol and growth medium at −80 ◦ C. Monthly, vials were transferred into slants containing the growth medium and agar, and maintained at 5 ◦ C. A loop full of a slant culture was transferred to 125 mL Erlenmeyer flasks containing 50 mL of synthetic medium composed by 30 g L−1 xylose, 3 g L−1 yeast extract, 3 g L−1 malt extract, and 5 g L−1 peptone. The flasks were maintained under agitation at 200 rpm in a constant temperature incubator shaker (Optic Ivymen System, Comecta S.A., distributed by Scharlab, Madrid, Spain) at 30 ◦ C for 24 h. Cells recovered by centrifugation (Ortoalresa, Consul 21, EBA 20, Hettich Zentrifugen, Germany) at 2755 × g for 15 min and 4 ◦ C were rinsed twice with sterile water before entrapment in calcium alginate beads. A cell suspension (4 mL) containing 3 g L−1 cells was added into 46 mL of sodium alginate 4% (w/v) previously sterilized in autoclave (Trade Raypa SL, Terrassa, Barcelona) for 1 h at 100 ◦ C. Alginate-biomass suspension was pumped with a peristaltic pump (Master flex,

Cole Parmer Instrument Co., model 77200-60) and dripped into CaCl2 ·2H2 O–water 2% (w/v) with different gage-needles (Braun, Melsungen AG, Melsungen, Germany): 30 G (˚ 0.3 mm × 12 mm), 27 G (˚ 0.4 mm × 40 mm), 25 G (˚ 0.5 mm × 16 mm), 23 G (˚ 0.6 mm × 25 mm), 22 G (˚ 0.7 mm × 30 mm), 21 G (˚ 0.8 mm × 40 mm), 20 G (˚ 0.9 mm × 25 mm), 19 G (˚ 1.1 mm × 40 mm), 18 G (˚ 1.2 mm × 40 mm), to obtain beads with the following diameters: 1.60, 1.82, 2.27, 2.39, 2.69,2.76, 2.78, 2.83 mm respectively. The beads were maintained in the CaCl2 solution at 4 ◦ C for 24 h and washed with sterile distilled water before being introduced in the culture broth. Some particular experiments were performed using Ca-alginate–chitosan beads obtained by dropping 4% Na-alginate suspension into a hardening solution with 4% (w/v) CaCl2 ·2H2 O, 0.5% (w/v) chitosan and 1% (v/v) acetic acid, with the help of a 27 G (˚ 0.4 mm × 40 mm) gage needle and a peristaltic pump. 2.3. Corn cobs and chemical characterization Corn cobs from the campaign of 2010 were collected in Mondariz (Pontevedra, Spain), dried at room temperature and milled to a particle size suitable for acid hydrolysis (<5 mm). The composition of the raw material was determined after Soxhlet extraction and subsequent quantitative acid hydrolysis in two-stages [14]: 72 wt% sulfuric acid treatment at 30 ◦ C/1 h, followed by 3 wt% sulfuric acid hydrolysis at 121 ◦ C/1 h, and final analysis of the hydrolysis products by HPLC as described in Section 2.8. The solid residue obtained after hydrolysis was considered as Klason lignin. Data indicating the mean values of three replications gave the following composition expressed in percentage (dry basis): cellulose: 31.5 ± 1.5; hemicelluloses: 34.9 ± 0.3; acetyl groups: 4.1 ± 0.6 and Klason lignin: 21.6 ± 1.5. 2.4. Acid hydrolysis (prehydrolysis) and preparation of hydrolyzates by concentration and charcoal detoxification Dried corn cobs (50 g) were hydrolyzed with dilute sulfuric acid in autoclave using 1 L Pyrex bottles, under conditions (2% H2 SO4 , 15 min, 130 ◦ C, liquid:solid ratio of 8:1 g g−1 ) optimized in previous works [15]. After cooled, the liquid phase from the acid hydrolysis was neutralized with CaCO3 to a final pH of 6.0, and the CaSO4 precipitated was separated from the supernatant by filtration. The liquor composition after prehydrolysis was: 34.7 ± 0.69 g L−1 xylose; 2.6 ± 0.08 g L−1 glucose; 3.4 ± 0.15 g L−1 arabinose; 3.2 ± 0.14 g L−1 acetic acid; 0.28 ± 0.05 g L−1 formic acid and 1.1 ± 0.18 g L−1 5-(hydroxymethyl)furfural. In particular experiments, neutralized hydrolyzates were detoxified with activated powdered charcoal at a mass ratio of hydrolyzate:activated charcoal of 10 g g−1 at room temperature under stirring for 1 h. Previously, powdered charcoal was activated with hot water and dried at room temperature. Liquors were recovered by filtration and used for preparing culture media [16]. 2.5. Vinasses Vinasses from the 2010 campaign were kindly supplied by the certified brand of origin of Valdeorras (Ourense), Spain, and stored at 4 ◦ C. Vinasses have the concentration indicated by Salgado et al. [9]: C (60.1 mg g−1 ), N (3.2 mg g−1 ), Fe2+ (61.0 mg kg−1 ), Mn2+ (8.1 mg kg−1 ), Zn2+ (3.5 mg kg−1 ), Ca2+ (945.8 mg kg−1 ), Mg2+ (122.8 mg kg−1 ), Al+3 (128.0 mg kg−1 ) and Cu2+ (12.7 mg kg−1 ). 2.6. Fermentation conditions All fermentations, except for the factorial experiments, were carried out by duplicate placing 30 g beads in 250 mL Erlenmeyer flasks with 100 mL of culture medium. Incubation was done in a rotary shaker at 31 ◦ C and 200 rpm. pH was measured at the beginning and end of the fermentations (pH meter BASIC 20+, CRISOL, Alella, Barcelona Spain). All media were sterilized in autoclave at 100 ◦ C for 1 h. The synthetic culture medium was the same than for inoculum growth. The corn cob hydrolyzates were supplemented with 3 g L−1 yeast extract, 3 g L−1 malt extract and 5 g L−1 peptone except for those experiments where vinasses were added. The effect of vinasses as economic nutrients was assayed at different concentrations using the detoxified hydrolyzate as C source, and the synthetic medium as control. In this experiment the detoxified hydrolyzate and vinasses were vacuum evaporated (Buchi rotavapor R-210/215, Flawil, Switzerland) at temperatures below 40 ◦ C to be concentrated 1.25 and 6 times respectively. The concentrated hydrolyzate and vinasses were mixed at different ratios and sterile distilled water was added to complete the total volume so that the hydrolyzate was finally reconstituted to the initial concentration, while vinasses were diluted to obtain 25, 50 or 75% of their initial concentration. The culture medium used for the experiments corresponding to the factorial design was prepared mixing 1 L of vinasses and 1 L of twofold vacuum concentrated charcoal-treated corn cob hydrolyzate to reach a final concentration of 50% vinasses. 2.7. Experimental design and statistical analysis for xylitol production The simultaneous effect of the volume of liquid medium added (V) and the amount of inoculated alginate beads (C) on the volumetric xylitol production (Pxylitol ), the specific xylitol production (rxylitol /total biomass ), and the consumed xylose

B. Pérez-Bibbins et al. / Process Biochemistry 48 (2013) 387–397 Table 1 Second order experimental design. Independent variables: alginate bead charge (C) and volume of medium (V). Experiment

1 2 3 4 5 6 7 8 9 10 11 12 13

Coded values

Uncoded values

C (g)

V (mL)

C (g)

V (mL)

1 1 −1 −1 1.267 −1.267 0 0 0 0 0 0 0

1 −1 1 −1 0 0 1.267 −1.267 0 0 0 0 0

45.0 45.0 10.0 10.0 49.7 5.3 27.5 27.5 27.5 27.5 27.5 27.5 27.5

140.0 61.0 140.0 61.0 100.5 100.5 150.5 50.5 100.5 100.5 100.5 100.5 100.5

to xylitol yield (Yxylitol/xylose ) was studied by means of an 22 orthogonal central composite design with ˛ = 1.267 and five replicates in the center of the domain, according to Akhnazarova and Kafarov [17] and Box et al. [18]. Experimental domain and coding criteria are given in Table 1. Significance of the coefficients of the models was calculated using Student’s t test (˛ < 0.05) as acceptance criterion. Models consistency was verified by Fisher’s F test (˛ < 0.05) applied to several means squares (QM) ratios (Table 2). The immobilization was done in 2.27 mm diameter alginate beads. The agitation rate (200 rpm) was fixed to the minimum necessary to ensure a good mass transfer between the beads and the bulk medium while limiting the shear stress to avoid biomass damage on the surface of the alginate particles. 2.8. Analytical methods During fermentation, 1 mL of sample was taken at selected times and centrifuged at 3421 × g at 4 ◦ C for 10 min. The liquid phase of the samples was employed for glucose, xylose, arabinose, furfural, 5-(hydroxymethyl)furfural (HMF), acetic acid, formic acid, xylitol, glycerol and ethanol analysis by High Performance Liquid Chromatography (HPLC) (Agilent, model 1200, Palo Alto, CA) using a refractive index detector with an Aminex HPX-87H ion exclusion column (Bio Rad 300 mm × 7.8 mm, 9 ␮ particles) with a guard column, eluted with 0.003 M sulfuric acid at a flow rate of 0.6 mL min−1 at 50 ◦ C. The cells obtained after centrifugation were suspended in water for the determination of free cell concentrations, meanwhile Ca-alginate beads (1 g) were collected at the beginning and the end of fermentation runs, dried with an absorbent paper until no water was released on dry paper, and dissolved with citrate at 4% (w/v) under mild heating (50 ◦ C) and agitation (100 rpm) for 10 min. The resulting suspension was centrifuged (2755 × g during 15 min) and resuspended in water for the determination of immobilized cell concentration. Free and immobilized cell concentrations were determined by absorbance using a UV–VIS Cintra 6 Spectrophotometer (GBC Scientific Equipment Ltd., Braeside, Australia) at 600 nm and correlated with the cell dry weight through the corresponding calibration curve. 2.9. Bead diameter measurement Chemical stability and proliferation of biomass immobilized in Ca-alginate beads was estimated by measuring the diameter at the beginning and the end of fermentation, using an electronic Digital Caliper (Cometa, S.A., Barcelona, Spain). 2.10. Field Emission Scanning Electron Microscopy (FE-SEM) Ca-alginate beads with D. hansenii immobilized were recovered after three repeated batches made with fresh synthetic medium, washed with sterile distilled water and 0.1 M cocadylic buffer, fixed with 2% (v/v) glutaraldehyde in 0.1 M cocadylic buffer (pH 7.4) and dehydrated with ethanol at increasing concentrations (30, 50, 70, 80, 90, and 100% (v/v)). Then beads were dried until the critical point, introduced in liquid nitrogen, fractured in halves, and subsequently the observations were performed by FE-SEM (Model JSM-6700F, Jeol, Japan).

3. Results and discussion 3.1. Influence of bead size on xylose to xylitol bioconversion A preliminary assay was performed to study the influence of the bead size, ranging from 1.60 to 2.83 mm in diameter, on

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the conversion of synthetic xylose into xylitol using D. hansenii cells entrapped in calcium alginate. The same synthetic medium employed for inoculum proliferation was used to eliminate any other variable that could affect yeast growth and xylitol production. Although this medium avoided the need of cell adaptation [3], successive batches were done in this case to eliminate the differences due to the possibility of heterogeneity in the number of viable cells during bead preparation for the different series, and to increase the concentration of the immobilized biomass from the first batch fermentation. Cells were immobilized in 4% Ca-alginate taking into account that Domínguez et al. [19] postulated the suitability of using this high alginate concentration for improving bead strength without affecting xylitol production by D. hansenii during long periods of time. Fig. 1 shows that only the first batch exhibited small differences among the series without a defined pattern in xylose consumption and xylitol production, which could be due to inocula heterogeneity as a consequence of different times of preparation of the beads. These differences disappeared completely in the second and the third batch, thus indicating that the bead diameter had no effect on these parameters by this yeast in the range of particle sizes here studied. Therefore, these results permit the selection of the most adequate bead size for any reactor culture only in terms of hydraulic and economic reasons of bead preparation. The improvement of productivities from batch I to batch II, and the similarity between batches II and III corroborates those data observed previously by Domínguez [16], where reusing D. hansenii immobilized in Ca-alginate allowed increasing the xylitol production rate from 0.31 g g−1 L−1 in the first trial to 0.76 g g−1 after three recycles, remaining at similar levels until the sixth experiment. However, Carvalho et al. [20], using Candida guilliermondii cells immobilized in Ca-alginate beads grown in sugarcane bagasse hydrolyzates, observed almost steady bioconversion rates with average values of xylitol concentration in consecutive trials. Some additional considerations regarding to biomass preferential growth can be pointed out. Fig. 2 shows the final concentration of free and immobilized biomass after the three batchwise operations. From these results the following observations can be highlighted: (a) the immobilized biomass increased gradually from 0.5 g L−1 at the beginning of the first batch until 1.0–1.5 g L−1 at the end of the third incubation. This bead colonization was enough to reduce the lag phase from the first to the second batch (Fig. 1). (b) However, it was remarkable the profuse growth of the free cells (around 5 g L−1 in most cases), which represented 80% of the total biomass at the end of the cultures and showed the prevalence of the free over the immobilized biomass. (c) It can be observed a slight tendency to reach lower values of immobilized biomass as bead size increases. The coexistence of mixed cultures composed by immobilized and free cells was also described by several authors working with different immobilization systems [3,21]. Two mechanisms could be assumed to explain the development of free biomass. The first mechanism explains the presence of free cells on the basis of cell leakage from the support due to shearing forces during agitation. Considering that total (free and immobilized) biomass was approximately the same for all series (around 6.4 g L−1 after three batches), if the complementarity among free and immobilized biomass concentrations was hypothesized to be due to cell leakage, the loss of cells should be stronger for the smaller beads, where a higher number of collisions should happen during fermentation because of the higher number of particles for the same weight of alginate beads. However, the pattern depleted in Fig. 2 turns down this hypothesis, considering that bigger beads presented less immobilized biomass at the end of the fermentation. The second mechanism deals with the preferential growth of the free cells generated from the particles surface due to better

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Table 2 Experimental results and analysis of variance for Models 1–3 describing the effect of the alginate bead charge (C) and volume of liquid medium (V) on xylitol production (Pxylitol ), xylitol/total biomass ratio (rxylitol/total biomass ), and xylitol/xylose yield (Yxylitol/xylose ), at 31 h of incubation. C and V in the models are referred to codified values. Uncoded values

Response values

C

V

45.0 45.0 10.0 10.0 49.7 5.3 27.5 27.5 27.5 27.5 27.5 27.5 27.5

140.0 61.0 140.0 61.0 100.5 100.5 150.5 50.5 100.5 100.5 100.5 100.5 100.5

Pxylitol (g L−1 )

Yxylitol/xylose (g g−1 )

Xexperimental

Xcalculated

Xexperimental

Xcalculated

Xexperimental

Xcalculated

7.72 7.21 3.25 1.32 7.93 1.87 6.69 3.08 6.97 6.93 7.47 7.63 7.48

7.78 6.54 3.48 0.83 8.35 2.01 6.40 3.93 7.25 7.25 7.25 7.25 7.25

1.89 0.48 0.78 0.21 1.47 0.42 1.80 0.18 1.22 1.27 1.43 1.43 1.39

2.00 0.46 0.82 0.13 1.41 0.45 1.68 0.26 1.35 1.35 1.35 1.35 1.35

0.61 0.30 0.31 0.08 0.52 0.19 0.48 0.13 0.40 0.41 0.44 0.46 0.45

0.58 0.31 0.32 0.05 0.53 0.20 0.49 0.14 0.43 0.43 0.43 0.43 0.43

CM Coefficients of the modela (CM); t student 7.25* it C 2.50* V 0.97* −0.35* CV −1.29* C2 −1.30* V2 Significance analysis of the coefficientsb Exp. error variance t (˛ < 0.05; FD = 4) SSc

rxylitol/total biomass (g g−1 )

t

CM

t

CM

t

50.524 20.732 8.069 2.182 9.060 9.124

1.35* 0.38* 0.56* 0.21* −0.26* −0.24*

32.441 10.731 15.977 4.503 6.346 5.691

0.43* 0.13* 0.14* 0.02 −0.04* −0.07*

39.31 14.19 14.99 1.70 3.85 6.63

0.1049 2.132 FDd

0.0088 2.132 QMe

SSc

0.0006 2.1320 FDd

QMe

SSc

Model (M) Error (E) Exp. error (Eexp) Lack of fitting (LF)

69.79 2.17 0.42 1.75

5 7 4 3

13.959 0.310 0.105 0.583

4.09 0.08 0.04 0.05

5 7 4 3

0.819 0.012 0.009 0.015

0.30 0.01 0.0024 0.0026

Total

71.96

12

5.997

4.18

12

0.348

0.30

Significance analysis of the modelf F (QMM/QME) F (QM(M + LF)/QMM F (QME/QMEexp) F (QMLF/QMEexp)

a b c d e f g *

FDd

QMe

4 8 4 4

0.074 0.001 0.001 0.001

12

0.025

45.03 F75 (˛ < 0.05) = 3.84 0.64 F58 (˛ < 0.05) = 6.04 2.95 F47 (␣ < 0.05) = 6.04 5.56 F43 (˛ < 0.05) = 6.39 2 g = 0.970/0.948 r 2 /radj

70.67 F75 (˛ < 0.05) = 3.97 0.63 F58 (˛ < 0.05) = 4.82 1.31 F47 (␣ < 0.05) = 6.09 1.73 F43 (˛ < 0.05) = 6.59 2 g r 2 /radj = 0.981/0.967

117.38 F84 (˛ < 0.05) = 3.84 0.50 F48 (˛ < 0.05) = 6.04 1.03 F48 (␣ < 0.05) = 6.04 1.07 F44 (˛ < 0.05) = 6.39 2 g r 2 /radj = 0.983/0.975

SIGNIFICATIVE

SIGNIFICATIVE

SIGNIFICATIVE

Coefficients for the terms of the model (i.t., independent term; C, alginate beads charge (g); V, volume of liquid medium (mL)). Coefficients significance was calculated using Student’s t test (˛ < 0.05). Sum of squares. Freedom degrees. Mean squares (QM = SS/FD). Models consistency was verified by Fisher’s F test (˛ < 0.05) as indicated in Section 2. Regression coefficients (adj: adjusted). Significative coefficients.

substrate conditions or oxygen availability in the bulk medium [22–24]. Thus, Liouni et al. [25] attributed the ability of cells located on the periphery of the single layer beads to multiply and be released in the suspension, leading to a system of immobilized and free cells. The electronic micrographs (Fig. 3) taken for different cross sections of the incubated beads support the hypothesis since the yeast grew mainly at the surface of the particles. In effect, considering that the yeast diameter is considerably higher (3–5 ␮m) than the alginate porous matrix (10–100 nm) [26,27], yeast growth inside the beads was restricted to those cells located inside the polymer during immobilization, and strongly limited to the space corresponding to the ∼10 ␮m macroporous formed during alginate gelification [26]. On the contrary, the cells initially entrapped at the surface of the beads or very close to it grew preferentially toward the outer surface of the particles, forming

a dense biofilm composed by a large number of cells showing a pseudohyphal morphology, which seems to be more efficient for extending into the liquid medium. The transformation from oval-rounded to pseudohyphal cells or even mycelium has been previously described for different yeasts including Saccharomyces or Candida strains among others [28,29]. Nevertheless, to the best of our knowledge, there are only two references describing the dimorphic behavior of D. hansenii strains, which was related to O2 limitation or lignin derived compounds [30], and to quorum sensing mediated by alcohol-based molecules [31]. Then, the cells growing at the bead surface as a biofilm appear to be exposed to oxygen and steric restrictions, which could be easily overcome by cell elongation as psedudohypae, and gemation and release from the biofilm to the culture medium where the mass transfer processes are more favored, thus explaining the preferential grow of

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Fig. 1. Xylose consumption and xylitol production in a repeated discontinuous fermentation by Debaryomyces hansenii immobilized in Ca-alginate with different bead sizes ranging from 1.60 to 2.83 mm in diameter. 1.60 mm (); 1.82 mm (); 2.27 mm (); 2.39 mm (x); 2.69 mm ( ); 2.76 mm (♦); 2.78 mm (); (+) 2.83 mm.

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Free biomass (gL-1)

6 5

immobilized biomass observed in those cultures made with smaller beads since as the diameter decreases, the surface area also increases for the same weight of alginate. Biofilm formation and, mainly, gel swelling by alginate hydration during fermentation [32], can finally explain the increment of the radius of the spheres in 187 ± 31 ␮m after the three incubations, regardless of the initial particle size. Nevertheless, in order to set up an optimum value for further experiments, it was defined the efficiency of immobilization (εi ) as the ratio of immobilized cells (Xi ) to total (free and immobilized) cells (Xf + Xi ). Table 3 summarizes this information. Beads with lower diameters showed higher efficiencies. In particular, the use of beads with 2.27 mm of diameter (25 G) augmented the efficiency up to 23.4%. This value clearly decreased with the higher diameter (2.83 mm) to only 15.0%, as it was expected from Fig. 2. Considering the similar xylitol production and total levels of biomass for all the bead sizes assayed, the differences in the amount and percentage of immobilized cells between experiments do not appear to be high enough to affect significantly the xylitol production in these conditions of incubation.

4

3.2. Effect of chitosan addition

3

In order to improve beads strength, a new assay was performed comparing the behavior of two experiments employing immobilized cultures made with alginate beads prepared with and without chitosan. Fig. 4 shows the time course for two successive batches. The xylose consumption and xylitol production rates were slightly higher in the experiments conducted in absence of chitosan, although the differences decreased in the second batch. Since the levels of free biomass were similar for both series in both batches, the differences appear to be related to the immobilized biomass in each case. In effect, considering that the mode of preparation of the beads causes that chitosan will be preferably located at the surface of the particles, thus hindering the development of the biofilm, these results support the importance of the cells located on the beads surface on the overall xylitol production. In any case, in view of the small differences between the series, the use of chitosan could be only justified in long-time fermentations (continuous or using several repeated batches) or in systems submitted to shear-stressing conditions. However, in

2 1 0 1.60 1.82 2.27 2.39 2.69 2.76 2.78 2.83 Bead diameter (mm)

Fig. 2. Final concentration of free and immobilized biomass after the third batch with different bead sizes ranging from 1.60 to 2.83 mm in diameter.

free rounded cells. On the other hand, the formation of this superficial biofilm increases the mass transfer restrictions inside the particle, thus hindering the already space-limited yeast development inside the beads. The growth of immobilized cells as a surface biofilm on the alginate particles also agrees with the higher concentration of

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Fig. 3. Micrographs of cells immobilized in Ca-alginate after three reuses with synthetic medium, by Scanning Electron Microscopy (SEM). (A) Ca-alginate bead (50× magnification), (B) biofilm on the surface (350× magnification), (C) yeast at the surface and inside the Ca-alginate particles (750× magnification), and (D) Debaryomyces hansenii cells with gemation scars inside Ca-alginate macroporous (4500× magnification).

two-three repeated discontinuous flask fermentations chitosan is not necessary, being avoided in subsequent experiments. 3.3. Use of corn cob hydrolyzates as substrate The liquid phase obtained after acid hydrolysis of corn cobs was assayed as fermentation medium for xylitol production by D. hansenii immobilized in 2.27 mm alginate beads without the addition of chitosan. These hydrolyzates have been assayed for xylitol production due to the high xylose content of corn cob [33–35], but they also contain some of fermentation inhibitors. According to Leonard and Hajny [36], these inhibitors could be summarized in four groups: (i) minerals and metals present in raw materials or released as a consequence of corrosion of the equipments during the hydrolysis; (ii) hemicellulosic-derived products such as furfural and 5-(hydroxymethyl)furfural generated by degradation of xylose and glucose respectively, and acetic acid from acetyl groups; (iii) lignin-derived products including phenolic compounds, aromatic acids and aldehydes; and (iv) extractives-derived from compounds. Parajó et al. [37] also reported the presence of organic acids with inhibitory ability such as caproic, caprylic, pelargonic and palmitic acids obtained from lignocellulose. To avoid their negative effects on yeast growth and metabolism, detoxification with activated charcoal was efficiently employed as a suitable technology for the biosynthesis of xylitol from different wastes containing inhibitors [38–40]. Considering the protective effect of immobilization on cells resistance to toxics [13], a new experiment was conducted with D. hansenii immobilized in alginate beads and cultured in corn cob hydrolyzates with and without activated charcoal

pretreatment to evaluate the need of detoxification in this case. A third culture was also conducted with comparative purposes using the synthetic medium before described but simulating in this case the sugar composition of hydrolyzates. Fig. 5 shows the time course of xylose consumption and xylitol and ethanol production as the mean value of two independent experiments. From these results it can be concluded that the best conditions for xylitol production corresponded to the charcoal-detoxified medium, generating 13.5 g L−1 xylitol in 56 h of incubation (QP = 0.24 g L−1 h−1 ; YP/S = 0.57 g g−1 ) a value slightly higher than that obtained with crude hydrolyzates (P = 12.9 g L−1 ; QP = 0.23 g L−1 h−1 ; YP/S = 0.53 g g−1 ). Conversely, poor performance was observed using the synthetic medium, reaching only 10.5 g L−1 xylitol (QP = 0.19 g L−1 h−1 ; YP/S = 0.43 g g−1 ). This favorable effect of the media prepared by hydrolysis of wastes can be attributed to the additional effect of some of the compounds present in the hydrolyzates [37]. In spite of the higher xylitol yields obtained with the fermentation broths prepared from hemicellulosic hydrolyzates, these media also produced the highest concentrations of ethanol (3.1–3.2 g L−1 ) and glycerol (0.34–0.52 g L−1 ) (data not shown) compared to only 1.9 g L−1 ethanol and 0.17 g L−1 glycerol with the synthetic medium. This behavior also reflects a positive effect of some of the compounds from the waste media on these metabolic pathways. 3.4. Use of vinasses as economic nutrients In order to extend the process to an industrial scale, it was studied the replacement of commercial nutrients by wine vinasses. Two

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393

Table 3 Immobilized (Xi ) and free (Xf ) biomass concentration, efficiency of biomass immobilization (εi ), xylitol production, xylose concentration, yield of xylitol produced per consumed xylose (YP/S ) and xylitol productivity (QP ) at the time indicated for each experiment. D: diameter. εi (%)

Time (h)

Xylitol (g L−1 )

22.9 ± 0.1 22.9 ± 0.1 23.4 ± 0.2 20.4 ± 0.2 18.8 ± 0.0 17.5 ± 0.1 16.4 ± 0.2 15.0 ± 0.1

30 30 30 30 30 30 30 30

12.4 ± 0.3 12.5 ± 0.2 12.8 ± 0.7 12.1 ± 0.0 12.7 ± 0.4 11.8 ± 0.5 12.2 ± 1.6 12.9 ± 0.4

(2) Effect of chitosan addition in Ca-alginate beads with 2.27 mm of diameterb With chitosan 1.2 ± 0.1 5.2 ± 0.1 6.4 ± 0.2 Without chitosan 1.6 ± 0.0 5.7 ± 0.1 7.2 ± 0.1

19.3 ± 0.1 21.4 ± 0.0

60 60

(3) Use of corn cobs hydrolyzates as substrate + synthetic nutrients 1.9 ± 0.3 5.9 ± 0.2 7.8 ± 0.5 Detoxified 1.7 ± 0.4 5.4 ± 0.2 7.1 ± 0.6 Non detoxified 2.1 ± 0.1 5.1 ± 0.4 7.2 ± 0.5 Synthetic

24.5 ± 0.2 23.7 ± 0.3 29.3 ± 0.1

(4) Use of corn cob hydrolyzates as substrate + vinasses as nutrients 1.7 ± 0.0 6.0 ± 0.5 7.7 ± 0.5 Corn cob + synthetic nutrients 1.2 ± 0.1 4.7 ± 0.5 5.9 ± 0.6 Vinasses 25% Vinasses 50% 1.2 ± 0.0 5.1 ± 0.5 6.2 ± 0.5 1.2 ± 0.1 5.6 ± 0.5 6.8 ± 0.6 Vinasses 75% Synthetic media 1.0 ± 0.2 5.8 ± 0.5 6.8 ± 0.7 (5) Optimization of the operational conditions in Erlenmeyer batch cultures 0.7* 3.4 4.1 1 (45 g, 140 mL) 3.2* 11.9 15.1 2 (45 g, 61 mL) 3 (10 g, 140 mL) 0.1* 4.0 4.2 0.5* 5.8 6.3 4 (10 g, 61 mL) * 1.4 4.0 5.4 5 (49.7 g, 100.5 mL) 0.1* 4.3 4.5 6 (5.3 g, 100.5 mL) 0.5* 3.3 3.7 7 (27.5 g, 150.5 mL) 3.6* 13.7 17.3 8 (27.5 g, 50.5 mL) 0.8* ± 0.0 4.6 ± 0.1 5.4 ± 0.2 9 Central (27.5 g, 100.5 mL)

Experiment

Xi (g L−1 )

Xf (g L−1 )

(1) Influence of beads size on xylose to xylitol bioconversiona 1.3 ± 0.1 4.5 ± 0.2 D = 1.60 mm 1.5 ± 0.1 4.9 ± 0.5 D = 1.82 mm D = 2.27 mm 1.5 ± 0.1 4.9 ± 0.3 D = 2.39 mm 1.2 ± 0.2 4.9 ± 0.2 1.2 ± 0.0 5.0 ± 0.0 D = 2.69 mm 1.2 ± 0.0 5.5 ± 0.5 D = 2.76 mm 1.0 ± 0.2 5.2 ± 0.3 D = 2.78 mm 1.0 ± 0.1 5.4 ± 0.0 D = 2.83 mm

a b *

Xi + Xf (g L−1 ) 5.8 ± 0.3 6.4 ± 0.6 6.4 ± 0.4 6.1 ± 0.4 6.2 ± 0.0 6.7 ± 0.5 6.2 ± 0.5 6.4 ± 0.1

Xylose (g L−1 )

YP/S (g g−1 )

QP (g L−1 h−1 )

0.6 ± 0.1 0.5 ± 0.0 0.4 ± 0.1 0.3 ± 0.1 0.3 ± 0.0 0.3 ± 0.1 0.5 ± 0.1 0.2 ± 0.1

0.54 ± 0.01 0.53 ± 0.01 0.57 ± 0.07 0.52 ± 0.01 0.55 ± 0.00 0.50 ± 0.03 0.52 ± 0.07 0.53 ± 0.00

0.41 ± 0.01 0.42 ± 0.01 0.43 ± 0.03 0.41 ± 0.00 0.42 ± 0.02 0.39 ± 0.02 0.41 ± 0.05 0.43 ± 0.01

11.5 ± 0.3 12.5 ± 0.4

3.3 ± 0.5 2.6 ± 0.1

0.56 ± 0.03 0.60 ± 0.04

0.23 ± 0.01 0.25 ± 0.01

56 56 56

13.5 ± 0.1 12.9 ± 0.2 10.5 ± 0.2

0.4 ± 0.1 0.8 ± 0.3 2.1 ± 0.7

0.57 ± 0.01 0.53 ± 0.01 0.43 ± 0.02

0.24 ± 0.01 0.23 ± 0.00 0.19 ± 0.00

22.1 ± 0.1 19.7 ± 0.2 18.6 ± 0.1 18.1 ± 0.2 26.5 ± 0.3

48 48 48 48 48

6.7 ± 0.1 12.3 ± 0.3 13.7 ± 0.1 12.2 ± 0.0 12.6 ± 0.3

5.9 ± 0.2 0.9 ± 0.0 1.1 ± 0.0 2.6 ± 0.0 0.3 ± 0.0

0.33 ± 0.01 0.51 ± 0.01 0.56 ± 0.01 0.53 ± 0.00 0.50 ± 0.02

0.14 ± 0.00 0.26 ± 0.01 0.29 ± 0.00 0.25 ± 0.00 0.26 ± 0.01

17.2 21.2 3.2 7.5 25.5 2.5 12.8 20.9 14.9 ± 0.0

31 31 31 31 31 31 31 31 31

7.7 7.2 3.3 1.3 7.9 1.9 6.7 3.1 7.3 ± 0.3

14.0 3.6 17.4 12.6 10.8 18.2 13.6 1.8 13.4 ± 0.3

0.61 0.30 0.31 0.08 0.52 0.19 0.48 0.13 0.43 ± 0.02

0.25 0.23 0.11 0.04 0.26 0.06 0.22 0.10 0.24 ± 0.01

Data corresponding to the third batch. Data corresponding to the second batch. Immobilized biomass measured at the end of the culture.

additional series consisting on the detoxified hydrolyzate without any other supplementation and the synthetic medium previously used were performed as control cultures. The results (Fig. 6) clearly showed the need for supplementation when corn cob hydrolyzate was used as the only nutrient source since this series gave the lowest xylitol production. In spite of it, the non-sugar components present in the hydrolyzate allowed the total exhaustion of xylose, although the consumption rate was the lowest. The synthetic medium was the best series in terms of substrate consumption rate, but when xylitol production was considered the difference with the series supplemented with vinasses was smaller. In fact, the series supplemented with vinasses at 50% concentration showed the same xylitol production profile than the series made with synthetic medium, which indicates that wine vinasses provide the necessary nutrients to compensate the deficit in corncob hydrolyzates without showing remarkable inhibitory effects. In this way, Salgado et al. [9] explained that the amino acid profile and protein concentration of vinasses are relevant for the xylitol production by D. hansenii, as well as metals, which also played an important role. Specifically, D. hansenii showed a strong dependence with the initial amount of Mg2+ . Although there were not strong differences among the levels of vinasses supplementation assayed, the best xylitol production corresponded to the intermediate value, which aims to a slight nutritional deficit and toxic effect of the series assayed with the lowest and the highest vinasses addition respectively. These results are confirmed in Table 3.

3.5. Optimization of the operational conditions in Erlenmeyer batch cultures Xylitol production by D. hansenii is strongly dependent on the occurrence of a redox imbalance generated under microaerobic conditions, which lead to a NADH excess that partially inhibits the xylitol to xylulose oxidization [41]. Although oxygen-limited conditions allow the best cell metabolic state for xylitol accumulation, they limit biomass generation, thus reducing the net xylitol production in the system. In consequence, working in batch cultures requires a delicate balance between biomass growth and xylitol generation trough the establishment of adequate aerobic conditions. Thereupon, a new set of experiments was conducted to maximize the xylitol production in this system by optimizing the microaerobic conditions according to the factorial design described in Table 1. Kinetic assays were performed in each experimental condition quantifying xylose, xylitol and free biomass (Fig. 7). Ethanol, glycerol and acetic acid were also measured, but they were produced in negligible amounts. Immobilized biomass was measured at the end of the fermentation. The experimental domain and the conditions assayed are listed in Table 1, meanwhile Tables 2 and 3 collect the experimental results. The experimental data (Fig. 7) confirmed that the volume of the added liquid medium had a strong positive effect on the aerobic conditions. Experiment 8, with the lowest volume of liquid corresponding to the highest gas transfer rate, generated the

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Batch I

8 15 6 10 4 5

2

0

25

Xylose (gL-1)

10

20 Xylose (gL-1)

30

12 Xylitol, free biomass (gL-1)

25

10

20

30

40

50

60

15 10 5

0 0

20

0

70

0

time (h)

24

36

48

60

72

84

60

72

84

60

72

84

time (h)

Batch II

25

12

14

20

Xylose (gL-1)

10

15

8

10

6

4 5 2

16

Xylitol (gL-1)

20

Xylitol, free biomass (gL-1)

12

12

8

4 0

0 0

10

20

30

40

50

60

70

0

time (h)

0

Fig. 4. Xylose consumption, free biomass concentration and xylitol production by Debaryomyces hansenii immobilized in Ca-alginate with and without quitosan in discontinuous fermentations. Free biomass (,䊉); xylose (,), xylitol (,). Filled symbols, with chitosan; void symbols, without quitosan.

Pxylitol = 7.25 + 2.50C + 0.97V − 0.35CV − 1.29C 2 − 1.30V 2

Model 1

As it was expected, V appears in Model 1 with a positive coefficient. But it is remarkable the strong positive effect of high bead charges (C) on xylitol production, which is in agreement with the convenience of using high inocula that provide higher productive

24

36

48

time (h) 5

4

Ethanol (gL-1)

highest substrate consumption and biomass production rates, but yielded very low levels of xylitol (3.1 g L−1 ). On the opposite, the cultures with the highest volume assayed (experiment 7) grew slowly, generating low biomass levels but producing high xylitol concentrations. Since the behavior in the experiments with intermediate situations of volume of liquid and beads charge is not so easily predictable, polynomial second order empirical models were calculated in order to understand better the combined effect of these two operational variables and define favorable conditions for xylitol production (Model 1), total biomass to xylitol ratio (Model 2) and consumed xylose to xylitol yield (Model 3). The experimental data employed corresponded to the longest incubation time before xylose depletion (31 h), which represents well the behavior of all the experiments during all the period of incubation. Model 1 describes xylitol production with a high degree of significance, as Table 2 shows. The corresponding response surface (Fig. 8) also reflects the strong effect of the independent variables on the xylitol production, and predicts values between 0.31 and 8.54 g L−1 xylitol for the experiments with low bead charge and volume of liquid, and with high charge and intermediate volume respectively.

12

3

2

1

0 0

12

24

36

48

time (h) Fig. 5. Discontinuous fermentation with Debaryomyces hansenii in synthetic and hydrolyzate media with and without detoxification. Synthetic medium (); crude hydrolyzate medium (䊉) and charcoal-treated hydrolyzate medium ().

biomass in less time. The relevance of the positive coefficient for variable C could also be related to the higher amount of immobilized biomass measured in the experiments with high bead charges, as it will be discussed later. Anyway, the CV interaction term with negative coefficient indicates that there is a limitation for increasing both volume and charge. To interpret this it must be considered that the oxygen availability depends on the ratio between the biomass oxygen requirements and the rate of oxygen dissolution into the liquid medium. In consequence, different growth rates

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35

30

30 Xylose (gL-1)

25 20

Xylose (gL-1)

395

15

25

20 15 10

10

5 5

0 0

10

20 30 time (h)

40

50

0

10

20 30 time (h)

40

50

0

10

20 30 time (h)

40

50

0

0

10

20

30 40 time (h)

50

60

70

20

20

Xylitol (gL-1)

16

Xylitol (gL-1)

16

12

12 8 4

8

0 4

20 0

10

20

30

40

50

60

70

time (h) Fig. 6. Discontinuous fermentation of Debaryomyces hansenii immobilized in Caalginate using wine vinasses and detoxified corncob hydrolyzate medium: 75% (䊉); 50% (); 25% (); 0% (); synthetic medium ().

can generate different aerobic conditions for similar degrees of aeration depending on the relative rates of oxygen consumption and dissolution from the gaseous space. Taking into account the convenience of working with high charges, this interaction term suggests lowering the volume of liquid so that high growth rates have enough aeration to avoid suboptimal oxygenation. The two negative second order terms for C and V have similar meaning and define the existence of a maximum value for xylitol production of 8.54 g L−1 for C = 0.93 and V = 0.25 in codified values, they corresponding to 44 g and 110 mL respectively. Higher values for C and V would lead to an excessive oxygen limitation caused for a very high oxygen demanding biomass or gas transfer restrictions respectively. Ultimately, the interaction term reflects that kLa is not a single universal criterion to ensure a good xylitol production since the occurrence of different situations of substrate concentration, inoculum size and age, or temperature of incubation generate different oxygen uptake rates thus leading to different aerobic conditions. Therefore, it is necessary to fix the best aeration strategy in accordance with the particular culture conditions in each case, considering both dissolved oxygen and/or oxygen transfer rate [42–44]. It is interesting to analyze the behavior for experiment 2. Although it reached high biomass levels and xylitol production at early times, the culture quickly started to reconsume it after

Free biomass (gL-1)

0

16

12 8 4

0

Fig. 7. Xylose consumption, free biomass concentration and xylitol production by Debaryomyces hasenii immobilized in Ca-alginate with different bead charges and volume of liquid medium, according to Table 1. (1) 45 g and 140 mL (䊉); (2) 45 g and 61 mL (); (3) 10 g and 140 mL (); (4) 10 g and 61 mL (♦); (5) 49.7 g and 100.5 mL (); (6) 5.3 g and 100.5 mL (+); (7) 27.5 g and 150.5 mL (); (8) 27.5 g and 50.5 mL (*); and central point (x).

xylose depletion, which indicates an active respiratory metabolism favored by an aerated system. So, in these aerobic conditions it seems interesting to point out the good results in xylitol production, especially when they are compared to experiment 8, which showed a very close biomass profile and minimal xylitol concentration. Indeed, it is experiment 2 the only case that shows a clearly different pattern when the volumetric or the specific xylitol production is considered. In this case, the biomass to xylitol ratio lowers significantly with regard to the other experiments with high productions. These results indicate that conditions for experiment 2 generated a suboptimal metabolic state for xylitol production. However, it

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the importance of microaerobiosis on the metabolic conditions that favor xylitol accumulation. rxylitol/total biomass = 1.35 + 0.38C + 0.56V + 0.21CV − 0.26C 2 − 0.24V 2

Model 2

The yield xylitol/(consumed xylose) was also modeled (Model 3, Table 2). The main effects of the variables (first and second order terms) showed the same behavior for Models 1 and 2, although small differences can be appreciated. Taking into account that this yield parameter includes both xylitol and specific xylitol productions, it is not surprising in Model 3 the disappearance of the CV interaction term due to the opposite sign of this effect on Models 1 and 2. Considering that bead charge was the independent variable with higher effect on xylitol production (Model 1) but lower effect on specific xylitol production (Model 2), it was also expected that bead charge and liquid volume showed similar coefficients when yields (Model 3) are considered. Yxylitol/xylose = 0.43 + 0.13C + 0.14V − 0.04C 2 − 0.07V 2

Model 3

Finally, the concentration of the immobilized biomass at the end of the fermentation was measured and the immobilization efficiency (εi) was calculated. The levels of the immobilized biomass were in the range 0.1–3.6 g L−1 for experiments 6 and 8, and εi values varied between 2.5 and 25.5% for experiments 6 and 5 (Table 3). It is very interesting to consider these two parameters, together with the concentration of free biomass, for the next pairs of experiments performed in similar conditions of liquid volume (i.e., similar oxygenation rates): 1 with 3, and 5 with 6. As expected, the immobilized biomass was clearly higher for the experiments with higher bead charge (1 and 5), which were also the experiments with the highest εi, and those that gave clearly higher xylitol productions. Since the levels of free biomass were almost the same for experiments 1 and 3, and 5 and 6 independently on the charge, these results point again, as it was previously hypothesized in the experiment with and without chitosan, to a favorable effect of the immobilized biomass on xylitol production, which could be related to the metabolic changes occurring in these conditions as a consequence of oxygen, substrate and space limitations affecting the immobilized cells [45]. This is an interesting issue to be studied in much depth. In any case, biomass immobilization offers, at least, the advantage of allowing the easy recuperation and maintenance of fresh inocula for efficient new cultures. 4. Conclusions

Fig. 8. Response surfaces showing the effect of the bead charge (C) and the volume of liquid phase (V) on xylitol production (P), xylitol/total biomass ratio (r) and xylitol/xylose yield (Y), according to Models 1–3.

was compensated by a profuse growth and a high level of biomass with low xylitol yields but high enough to allow reaching good net productions. To analyze quantitatively the effect of the operational variables on the specific xylitol production a new empirical model was calculated (Model 2, Table 2). The most noticeable difference with Model 1 is the stronger relative effect of the volume and the positive coefficient for the CV interaction term in Model 2, both of them reflecting

High xylitol productions in batch cultures of D. hansenii immobilized in alginate beads can be achieved by optimization of the conditions that lead to microaerobic environments through the selection of adequate combinations of the bead charge and the volume of added liquid medium. The oxygenation rate must be fixed depending on biomass concentration and growth rate to avoid suboptimal microaerobic conditions for cells maintenance. In spite of the profuse growth of free biomass, the results obtained in this work point to an additional favorable effect of yeast immobilization, which grows preferentially on the bead surface as a biofilm with hyphal morphology. The use of a completely waste origin culture medium promoted a xylitol production comparable to that obtained in a synthetic medium. Acknowledgments We are grateful to the Spanish Ministry of Science and Innovation for the financial support of this work (project CTQ2011-28967), which has partial financial support from the FEDER funds of the European Union, to MAEC-AECID (Spanish Government) for the financial support for Pérez-Bibbins, B., to the Galician Government

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Postdoctoral Program “Isidro Parga Pondal” for the financial support for Torrado A., and to Solla E. and Méndez J. (CACTI-University of Vigo) for their excellent technical assistance in microscopy. References [1] Parajó JC, Domínguez H, Domínguez JM. Biotechnological production of xylitol. Part 1. Interest of xylitol and fundamentals of its biosynthesis. Bioresour Technol 1998;65:191–201. [2] Yuen HK, Westwater C, DeGarmo J, Bandyopadhyay D. Immediate effect of xylitol chewing gum and mouth rinse on salivary levels of mutans streptococci in adults with systemic sclerosis: a pilot study. J Exp Integr Med 2012;2: 89–92. [3] Cunha MAA, Rodrigues RCB, Santos JC, Converti A, Silva SSD. Repeated-batch xylitol bioproduction using yeast cells entrapped in polyvinyl alcohol-hydrogel. Curr Microbiol 2007;54:91–6. [4] Tran LH, Yogo M, Ojima H, Idota O, Kawai K, Suzuki T, et al. The production of xylitol by enzymatic hydrolysis of agricultural wastes. Biotechnol Bioprocess Eng 2004;9:223–8. [5] Mikkola JP, Salmi T, Sjöholm R, Mäki-Arvela P. Hydrogenation of xylose to xylitol: three-phase catalysis by promoted raney nickel, catalyst deactivation and in situ sonochemical catalyst rejuvenation. Stud Surf Sci Catal 2000;130C:2027–32. [6] Nigam P, Singh D. Processes for fermentative production of xylitol – a sugar substitute. Process Biochem 1995;30:117–24. [7] Winkelhausen E, Kuzmanova S. Microbial conversion of d-xylose to xylitol. J Ferment Bioeng 1998;86:1–14. [8] Saha BC. Hemicellulose bioconversion. J Ind Microbiol Biotechnol 2003;30:279–91. [9] Salgado JM, Rodríguez N, Cortés S, Domínguez JM. Development of cost-effective media to increase the economic potential for larger-scale bioproduction of natural food additives by Lactobacillus rhamnosus, Debaryomyces hansenii and Aspergillus niger. J Agric Food Chem 2009;57:10414–28. [10] Yu J, Li Z, Ye Q, Yang Y, Chen S. Development of succinic acid production from corncob hydrolysate by Actinobacillus succinogenes. J Ind Microbiol Biotechnol 2010;37:1033–40. [11] Rivas BJM, Parajó JC. Bioconversion of posthydrolysed autohydrolysis liquors: an alternative for xylitol production from corn cobs. Enzyme Microb Technol 2002;31:431–8. [12] Beltrán FJ, García-Araya JF, Álvarez PM. Wine distillery wastewater degradation. 1. Oxidative treatment using ozone and its effect on the wastewater biodegradability. J Agric Food Chem 1999;47:3911–8. ˇ [13] Jirku˚ V, Masák J, Cejková A. Yeast cell attachment: a tool modulating wall composition and resistance to 5-bromo-6-azauracil. Enzyme Microb Technol 2000;26:808–11. [14] Vázquez D, Lage MA, Parajó JC, Vázquez G. Transformación de materiales lignocelulósicos: composición, fraccionamiento y aprovechamiento. Rev Agroquim Tecnol 1991;31:143–64. [15] Cruz JM, Domínguez JM, Domínguez H, Parajó JC. Preparation of fermentation media from agricultural wastes and their bioconversion into xylitol. Food Biotechnol 2000;13:79–97. [16] Domínguez JM. Xylitol production by free and immobilized Debaryomyces hansenii. Biotechnol Lett 1998;20:53–6. [17] Akhnazarova S, Kafarov V. Experiment optimization in chemistry and chemical engineering. Moscow: Mir Publisher; 1982. [18] Box GEP, Hunter WG, Hunter JS. Estadística para investigadores. Barcelona: Reverté; 1989. [19] Domínguez JM, Cruz JM, Roca E, Domínguez H, Parajó JC. Xylitol production from wood hydrolyzates by entrapped Debaryomyces hansenii and Candida guilliermondii cells. Appl Biochem Biotechnol 1999;81:119–30. [20] Carvalho W, Canilha L, Silva SS. Semi-continuous xylose-to-xylitol bioconversion by Ca-alginate entrapped yeast cells in a stirred tank reactor. Bioprocess Biosyst Eng 2008;31:493–8. [21] Santos JC, Mussatto SI, Dragone G, Converti A, Silva SS. Evaluation of porous glass and zeolite as cells carriers for xylitol production from sugarcane bagasse hydrolysate. Biochem Eng J 2005;23:1–9. [22] Carvalho W, Silva SS, Converti A, Vitolo M, Felipe MGA, Roberto IC, et al. Use of immobilized Candida yeast cells for xylitol production from sugarcane

[23]

[24]

[25]

[26] [27]

[28] [29] [30]

[31]

[32]

[33]

[34]

[35]

[36] [37]

[38]

[39]

[40]

[41]

[42] [43]

[44]

[45]

397

bagasse hydrolysate: cell immobilization conditions. Appl Biochem Biotechnol 2002;98–100:489–96. Carvalho W, Silva SS, Converti A, Vitolo M. Metabolic behavior of immobilized Candida guilliermondii cells during batch xylitol production from sugarcane bagasse acid hydrolyzate. Biotechnol Bioeng 2002;79:165–9. Carvalho W, Silva SS, Vitolo M, Felipe MGA, Mancilha IM. Improvement in xylitol production from sugarcane bagasse hydrolysate achieved by the use of a repeated-batch immobilized cell system. Z Naturforsch C 2002;57:109–12. Liouni M, Drichoutis P, Nerantzis ET. Studies of the mechanical properties and the fermentation behavior of double layer alginate–chitosan beads using Saccharomyces cerevisiae entrapped cells. World J Microb Biotechnol 2008;24:281–8. Klein J, Stock J, Vorlop K-D. Pore size and properties of spherical Ca-alginate biocatalysts. Eur J Appl Microbiol 1983;18:86–91. Gåserød O, Smidsrød O, Skjåk-Bræk G. Microcapsules of alginate–chitosan – I A quantitative study of the interaction between alginate and chitosan 1998;19:1815–25. Nickerson KW, Atkin AL, Hornby JM. Quorum sensing in dimorphic fungi: farnesol and beyond. Appl Environ Microbiol 2006;72:3805–13. Gancedo JM. Control of pseudohyphae formation in Saccharomyces cerevisiae. FEMS Microbiol Rev 2001;25:107–23. Cruz JM, Domínguez JM, Domínguez H, Parajó JC. Dimorphic behaviour of Debaryomyces hansenii grown on barley bran acid hydrolyzates. Biotechnol Lett 2000;22:605–10. Gori K, Knudsen PB, Nielsen KF, Arneborg N, Jespersen L. Alcohol-based quorum sensing plays a role in adhesion and sliding motility of the yeast Debaryomyces hansenii. FEMS Yeast Res 2011;11:643–52. Inal M, Yi˘gito˘glu M. Production of bioethanol by immobilized Saccharomyces cerevisiae onto modified sodium alginate gel. J Chem Technol Biotechnol 2011;86:1548–54. Ling H, Cheng K, Ge J, Ping W. Statistical optimization of xylitol production from corncob hemicellulose hydrolysate by Candida tropicalis HDY-02. New Biotechnol 2011;28:673–8. Wang L, Yang M, Fan X, Zhu XX, Yuan TQ. An environmentally friendly and efficient method for xylitol bioconversion with high-temperaturesteaming corncob hydrolysate by adapted Candida tropicalis. Process Biochem 2011;46:1619–26. Wang L, Wu D, Tang P, Fan X, Yuan Q. Xylitol production from corncob hydrolysate using polyurethane foam with immobilized Candida tropicalis. Carbohydr Polym 2012;90:1106–13. Leonard RH, Hajny GJ. Fermentation of wood sugars. Ind Eng Chem Res 1945;37:390–5. Parajó JC, Domínguez H, Domínguez JM. Biotechnological production of xylitol. Part 3: operation in culture media made from lignocellulose hydrolysates. Bioresour Technol 1998;66:25–40. Baek S-C, Kwon Y-J. Optimization of the pretreatment of rice straw hemicellulosic hydrolyzates for microbial production of xylitol. Biotechnol Bioprocess Eng 2007;12:404–9. Canilha L, Carvalho W, Felipe MDGA, Silva JBDA. Xylitol production from wheat straw hemicellulosic hydrolysate: hydrolysate detoxification and carbon source used for inoculum preparation. Braz J Microbiol 2008;39:333–6. Salgado JM, Rodríguez N, Cortés S, Domínguez JM. Effect of nutrient supplementation of crude or detoxified concentrated distilled grape marc hemicellulosic hydrolysates on the xylitol production by Debaryomyces hansenii. Prep Biochem Biotechnol 2012;42:1–14. Gírio FM, Roseiro JC, Sá-Machado P, Duarte-Reis AR, Amaral-Collac¸o MT. Effect of oxygen transfer rate on levels of key enzymes of xylose metabolism in Debaryomyces hansenii. Enzyme Microb Technol 1994;16:1074–8. Roberto IC, Mancilha IM, Sato S. Influence of kL a on bioconversion of rice straw hemicellulose hydrolyzate to xylitol. Bioprocess Biosyst Eng 1999;21:505–8. Preziosi-Belloy L, Nolleau V, Navarro JM. Xylitol production from aspenwood hemicellulose hydrolysate by Candida guilliermondii. Biotechnol Lett 2000;22:239–43. Granström T, Aristidou AA, Leisola M. Metabolic flux analysis of Candida tropicalis growing on xylose in an oxygen-limited chemostat. Metab Eng 2002;4:248–56. Vicente AA, Dluhy´ M, Ferreira EC, Teixeira JA. Modelling diffusion–reaction phenomena in yeast flocs of Saccharomyces cerevisiae. Bioprocess Biosyst Eng 1998;18:335–42.