Optimization of Salts Supplementation on Xylitol Production by

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Optimization of salts supplementation on xylitol production by Debaryomyces hansenii using synthetic medium or corncob hemicellulosic hydrolyzates and further scaled up Guadalupe Bustos Vázquez, Noelia Pérez Rodríguez, José Manuel Salgado, Ricardo Pinheiro de Souza Oliveira, and José Manuel Domínguez Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b01120 • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on June 3, 2017

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Industrial & Engineering Chemistry Research

Optimization of salts supplementation on xylitol production by Debaryomyces hansenii using synthetic medium or corncob hemicellulosic hydrolyzates and further scaled up

Guadalupe Bustos Vázquez1,2, Noelia Pérez-Rodríguez1, José Manuel Salgado1,3, Ricardo Pinheiro de Souza Oliveira4, José Manuel Domínguez1*

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Department of Chemical Engineering, Faculty of Sciences, University of Vigo (Campus Ourense), As Lagoas s/n, 32004 Ourense, SPAIN and Laboratory of Agro-food Biotechnology, CITI (University of Vigo)-Tecnópole, Technological Park of Galicia, San Cibrao das Viñas, 32900 Ourense, SPAIN.

2

Departamento de Biotecnología, Unidad Académica Multidisciplinaria Mante, Universidad Autónoma de Tamaulipas, Blvd. E.C. Glez, 1201, col. Jardín, 89840 Ciudad Mante, Tamaulipas, México

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CEB-Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710− 057 Braga, Portugal 4

Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, Av. Lineu Prestes 580, Bl 16, 05508-900, São Paulo, Brazil

Keywords:

xylitol,

salts,

Debaryomyces hansenii,

corncobs

hydrolyzates,

charcoal

detoxification Running title: Influence of salts on xylitol production

ACS Paragon Plus Environment

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ABSTRACT: The synergic effect of eight salts on D-xylose to xylitol bioconversion by Debaryomyces hansenii NRRL Y-7426 was optimized through a central composite design (CCD) in shaker flasks. Optima conditions were then assayed using detoxified corncob hemicellulosic hydrolyzates as carbon source, increasing the fermentative parameters to QP = 0.118 g/L·h and YP/S = 0.45 g/g from QP = 0.101 g/L·h and YP/S = 0.39 g/g (without supplementation). Finally, the process was successfully scaled up to a 50L Braun Biostat D50 fermentor, since the time of fermentation was reduced from 103h to 42.8h and the fermentation parameters increased to QP = 0.395 g/L·h and YP/S = 0.59 g/g using synthetic medium and QP = 0.236 g/L·h and YP/S = 0.39 g/g with detoxified hydrolyzates. All the results confirmed the positive influence of supplementing synthetic culture media or detoxified corncob hydrolyzates with salts for the development of D. hansenii.


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1. INTRODUCTION Microbial production of xylitol from lignocellulisic materials using xylose utilizing yeasts has attracted much attention in the last two decades, taking into account that xylitol (C5H12O5), a high-value polyol with a hydroxyl group attached to each carbon atom in its chain, possesses a high sweetening power although with 40% less calories than sucrose.1 The structure of xylitol does not feature an aldehyde or ketone functional group; therefore, xylitol does not cause a Maillard browning reaction when it is heated or used in baked food products.2 Furthermore, it has prebiotic effects, which can reduce blood glucose, triglyceride, and cholesterol level.3 Other interesting properties are its high cooling power inherent in its high endothermic heat, high solubility, low glycaemic rates, lack of carcinogenicity and cariostatic properties.1 Xylitol has been reported to possess antidiabetic potentials and reduce the accumulation of visceral fat and prevent nonesterified fatty acid (NEFA)-induced insulin resistance.4 Xylitol also prevents tooth decay and can help to promote oral health.2 Consequently, xylitol has been approved for food use in over 50 countries, reaching a 12 % share of the total polyol market,5 thus being largely used as an alternative sweetener in the food sector for the elaboration of sugar-free confections, candies, caramels, chocolates, ice creams, jellies, marmalades, yogurts, jams, bakery products, and drinks.6,7 It is also used as a substitute for sugar in dental and confectionery products to prevent dental caries,4 and in biomedicince to prevent ear inflammations and to stimulate murine hybridoma cell production and for parenteral nutrition.7 Xylitol can be formed as a metabolic intermediary product of D-xylose fermentation.8 However, the presence of other sugars in the culture medium, mainly hexoses, may influence the use of xylose.9 For instance, Lee et al.10 described the


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simultaneous utilization of xylose and fructose, but when D-glucose, D-mannose or Dgalactose were present in the medium, Candida guilliermondii NRC 5578 exhibited a sequential pattern of utilization so that glucose, mannose and galactose were firstly consumed and xylose utilization did not start until hexoses concentration was below a certain threshold. Nitrogen source is also a critical parameter for xylitol production by xylitol-producing-yeasts, considering that organic nitrogen sources such as yeast extract, malt extract, peptone, urea or casamino acids provide all the necessary minerals and vitamins for xylitol formation stimulating the oxidative step of the pentose phosphate pathway, however, higher concentrations than 15 g/L blocked the conversion of D-xylose to xylitol.11 Additionally, there is also a positive effect in the addition of vitamins on growth and for enhancing productivity in some yeasts. For example, the effect of biotin on xylitol production in C. guilliermondii FTI 20037 was favored from 0.002 g/L·h in the control to 0.009 or 0.044 g/L·h when the fermentation medium was supplemented with 0.05 or 0.25 µbiotin/L; while in Pachysolen tannophilus NRRL Y2460 ethanol production was favored instead of xylitol.12 However, the effect of salts addition has been scarcely studied. Consequently this work aims to study the synergic effect of salts concentration on xylitol production by response surface methodology (RSM) using a central composite design (CCD). Debaryomyces hansenii was selected to perform the transformation in the view that this metabolically versatile, non-pathogenic, osmotolerant and oleaginous microorganism represents an attractive target for fundamental and applied biotechnological research.13 Optimal conditions were then assayed to perform the process using detoxified corncob hemicellulosic hydrolyzates as carbon source. Finally, the process was scaled up from 250 mL Erlenmeyer flasks to a 50L Braun Biostat D50 fermentor using both synthetic and detoxified hydrolyzates media, in a two-step aeration fermentation. In the first


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stage, cell concentration was increased under more aerated conditions, meanwhile, in the second stage, aeration was reduced to stimulate the production of xylitol.

2. MATERIALS AND METHODS 2.1. Reagents. Culture media in this study were prepared using D(+) xylose (29013.237) purchased to BDH Prolabo GPR Rectapur, VWR (Leuven, Belgium) as carbon source, and the following nutrients: yeast extract (403687.1210), malt extract (403690.1210) and peptone (403695.1210), all of them supplied by Panreac Química (Barcelona, Spain), as well as glycerol (141339.1211) used for maintenance of microorganism in cryovials. Bacteriological agar (1800.00), employed to prepare slants tubes was acquired to Pronadisa CONDA (Madrid, Spain). The salts used to enrich fermentative media were FeSO4·7H2O (F7002) and Al2(SO4)3 (368458), purchased to Sigma Aldrich (St. Louis, MO, USA) and, MnSO4·H2O (141413.1210), ZnSO4·H2O (131788.1210), MgSO4·7H2O (141404.1210), CuSO4·5H2O (141270.1210), KH2PO4 (121509.1210) and NaCl (131659.1211) provided by Panreac Química. The reagents used for hydrolyzates preparation: H2SO4 (131058.1211), CaCO3 (141212.1211) and activated charcoal (211238.0914) were acquired from Panreac Química. Meanwhile HPLC analytical reagents and standards: D(+) glucose (49139), D(+)

xylose

(95729),

L(+)

arabinose

(A3131),

furfural

(185914),

5-

(Hydroxymethyl)furfural (H40807), D (+)-L (-) malic acid (M0875-100G), citric acid (C0759-500G), oxalic acid (75688) and xylitol (S X3375) were supplied by Sigma Aldrich (St. Louis, MO, USA). Additionally, ethanol 96 % v/v (131085.1611), formic acid 98% v/v (131030), acetic acid 96% (122703.1611); glycerol (141339.1211); and H2SO4 (131058.1211) used as mobile phase were purchased to Panreac Química


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(Barcelona, Spain). Finally L(+) tartaric acid (95308) and lactic acid (69775) were from Fluka Sigma- Aldrich Chemie GmbH, Steinheim Germany. 2.2. Microorganism and growth conditions. Debaryomyces hansenii NRRL Y-7426 was acquired from the National Center for Agricultural Utilization Research (Peoria, Illinois, USA). Freeze-dried cells were grown on a basal medium formulated with 30 g commercial xylose/L and the following nutrients: 3 g yeast extract/L, 3 g malt extract/L, and 5 g peptone/L (noted as YMP). Cells were maintained at -80ºC in cryovials containing 30% (v/v) glycerol and growth medium. Vials were transferred monthly into slants tubes including the growth medium and 20 g/L agar, and maintained at 5ºC. A loop full of a slant culture was transferred to 125 mL Erlenmeyer flasks containing 50 mL of the growth medium. Flasks were maintained under agitation of 200 rpm in a constant temperature incubator shaker (Optic Ivymen System, Comecta S.A., distributed by Scharlab, Madrid, Spain) at 30°C for 24h. Cell concentration in inoculum was measured 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. Concentration was adjusted by dilution with water to reach a final concentration in the fermentation broth of 0.10-0.12 g/L. 2.3. Corncobs and chemical characterization. Corncobs locally collected were dried at room temperature, milled to a particle size suitable for acid hydrolysis (< 1 mm), homogenized in a single lot, air-dried and stored. Moisture of samples was considered for both quantitative acid hydrolysis and further fractionation by prehydrolysis. The composition of the raw material was determined after Soxhlet extraction and subsequent quantitative acid hydrolysis in two-stages following standard


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procedures using a 72 wt % sulfuric acid treatment at 30ºC for 1h, followed by a 3 wt % sulfuric acid hydrolysis at 121ºC and 1h,14 and final analysis of the hydrolysis products by HPLC as described in the analytical section. 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: 31.2 ± 1.3; hemicelluloses: 33.8 ± 0.4; acetyl groups: 4.4 ± 0.6 and lignin: 20.9 ± 1.8. 2.4. Prehydrolysis of material and charcoal detoxification of hydrolyzates. Fifty grams of dried corncobs were hydrolyzed with dilute sulfuric acid in autoclave (Trade Raypa SL, Terrassa, Barcelona) using 1L Pyrex bottles, under optimized conditions (2 % H2SO4, 15 min, 130°C, and liquid:solid ratio of 8:1 g/g).15 The liquid phase was recovered by filtration and neutralized with CaCO3 after cooling to reach a final pH in the vicinity of 6.0. The CaSO4 precipitated was separated from the supernatant by filtration and discharged. Neutralized hydrolyzates were detoxified with activated powdered charcoal (powdered charcoal was previously activated with hot water and dried at room temperature) at a mass ratio of hydrolyzate:activated charcoal of 10 g/g at room temperature under stirring for one hour procedures.14 2.5. Experimental design and statistical analysis to optimize the addition of salts on xylitol production by D. hansenii. The synergic influence of eight salts (FeSO4·7H2O, MnSO4·H2O, ZnSO4·H2O, MgSO4·7H2O, Al2(SO4)3, CuSO4·5H2O, KH2PO4 and NaCl) was evaluated during the growth of D. hansenii on culture media formulated with 30 g/L pure xylose and YMP. Fermentations were conducted using 250 mL Erlenmeyer flasks containing 100 mL of culture medium. Flasks were maintained at 200 rpm and 30°C in a constant temperature incubator shaker (Optic Ivymen System, Comecta S.A., distributed by Scharlab, Madrid, Spain) for 72h.


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A central composite design (CCD) was proposed to maximize the concentration of xylitol. CCD contains an imbedded factorial or fractional factorial design with center points that is augmented with a group of star points that allow estimation of curvature. The selected CCD was a 2**(8-2) CCD containing eight independent variables in oneblock which encompasses 85 runs. The design was performed at five levels including 64 factorial experiments (coded to the usual ± 1 notation); sixteen star points (on the axis at a distance of α = ±2.8284 from the center) and five replicates at the center of the experimental domain for estimation of the pure error. This kind of designs allows the estimation of the significance of the parameters and their interactions. Experiments were randomized in order to maximize the effects of unexplained variability in the observed responses due to extraneous factors.16 Concentrations of salts were established on basis to the references listed in Table 1. This Table also provides information about the levels of independent variables and dimensionless coded variables definition (xi). For statistical calculations, the independent variables were coded as x1 (coded FeSO4·7H2O), x2 (coded MnSO4·H2O), x3 (coded ZnSO4·H2O), x4 (coded MgSO4·7H2O), x5 (coded Al2(SO4)3), x6 (coded CuSO4·5H2O), x7 (coded KH2PO4) and x8 (coded NaCl). The correspondence between coded and uncoded variables was fitted according to the linear equations also showed in Table 1, which were deduced from their respective variations limits. Meanwhile Table S1 shows the design matrix of the coded variables. The influence of salts concentration was studied on xylitol production after 72h fermentation and evaluated using the Statistic software package version 7.0 (Stat Soft, USA) at 95 % confidence level, considering the pure error to evaluate the significance of the effects.


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Data from the factorial design were subjected to a second-order multiple regression analysis using least squares regression methodology to obtain parameters of the mathematical model. The interrelationship between dependent and operational variables was fitted by a polynomial quadratic equation established by a model including linear, interaction and quadratic terms: y = β0 + ∑ βi·xi + ∑ βii·xi2 + ∑ βij·xi·xj where y represents the dependent variable (xylitol concentration after 72h fermentation); β denotes the regression coefficients (calculated from experimental data by multiple regression using the least-squares method): βo is the constant, βi is the linear coefficient, βij is the second-order coefficient and βii is the linear quadratic coefficient; and x denotes the independent variables (salts concentration). The response (xylitol concentration after 72h) was subjected to analysis of variance (ANOVA) and calculation of the coefficient of determination (R2) using the MiniTab (version 16.1) to statistically judge the reliability of the model. 2.6. Optimization of the operational conditions and validation of the model. The profile for predicted values and desirability option from the Statistic software package version 7.0 (Stat Soft, USA) was used for the optimization of the xylitol production, as well as for validation of the experimental model. 2.7. Influence of salts addition on fermentations carried out using culture media formulated from hemicellulosic corncob hydrolyzates. Additional shake flask fermentation experiments were carried out at 30°C under microaerophilic conditions in 250-mL Erlenmeyer flasks containing 100 mL of culture media placed in a orbital shaker (Optic Ivymen System, Comecta S.A., distributed by Scharlab, Madrid, Spain) at 100 rpm for 103h. The culture medium was prepared with detoxified hydrolyzates supplemented with YMP, and supplemented or not with the optima salts concentrations


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calculated in the experimental design. All media were sterilized in autoclave (Trade Raypa SL, Terrassa, Barcelona, Spain) at 100ºC for 1h. Samples (0.5 mL) were taken at given fermentation times to determine xylose uptake, xylitol production and cell growth. 2.8. Scale up of the process. Finally, in order to scale up the process, two culture media were prepared with pure xylose or detoxified hydrolyzates, supplemented with the nutrients reported before and the optima salt concentrations calculated in the experimental design. A loop full of a slant culture was transferred to 250 mL Erlenmeyer flasks containing 100 mL of the growth medium. Flasks were maintained under agitation of 200 rpm in a constant temperature incubator shaker (Optic Ivymen System, Comecta S.A., distributed by Scharlab, Madrid, Spain) at 30°C for 24h. Then, 1 mL of inoculum was transferred to 1L Erlenmeyer flasks containing 300 mL of the growth medium and cultivated under the same conditions. Finally, 1L portion of the preculture was added to a 50L Braun Biostat D50 fermentor (Melsungen, Germany) containing 30L working volume (previously sterilized inside the bioractor at 100ºC for 1h and pH adjusted to 5.5 with NaOH 5N) involving an initial biomass of 0.11 g/L. Fermentations were performed at 30ºC in a two-stage aeration process. The stirring rate was fixed at 200 rpm and filter-sterilized air was supplied to the bioreactor throughout the whole period of fermentations. During the first stage (24h), the airflow rate was maintained at 9 L/min. During the second stage (remaining period of time), the airflow rate was reduced to 3 L/min. Samples (45 mL) were withdrawn at given fermentation times for analysis. 2.9. Analytical methods. Samples (from prehydrolysis or fermentation) were centrifuged (EBA20, Hettich Zentrifugen, Germany) at 3421 × g at 4ºC for 10 min and filtered through 0.2 µm pore diameter cellulose acetate membranes. The liquid phase of


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samples was employed for analysis of glucose, xylose, arabinose, furfural, 5(Hydroxymethyl)furfural (HMF), acetic acid, formic acid, xylitol, glycerol, oxalic acid, citric acid, tartaric acid, lactic acid and ethanol analysis by High Performance Liquid Cromatography (HPLC) (Agilent, model 1200, Palo Alto, CA) using a refractive index detector with an Aminex HPX-87H ion exclusion column (Bio Rad 300 × 7.8 mm, 9 µ particles) with a guard column, eluted with 0.003 M sulfuric acid at a flow rate of 0.6 mL/min at 50 ºC. Five µL of conveniently diluted samples were injected. Calibration was done with standard solutions ranging between 0.5 and 5 g/L. The cells obtained after centrifugation were resuspended in an equal volume of distilled water to obtain a cell suspension. The culture growth was turbidimetrically monitored by optical density measurements at a wavelength of 600 nm using a UV-Vis Spectrophotometer (Libra S60-Biochrom, Cambridge, U.K.) and converted to dry cell weight at 105°C Binder – Model 53 ED (Tuttlingen, Germany).

3. RESULTS AND DISCUSSION 3.1. Optimization of salts concentration on xylitol production by experimental design. Experimental designs have been previously used to optimize medium composition in order to improve the parameters of fermentation.16 In our case, a 2**(8-2) central composite design was employed in order to examine the synergic influence of salts addition on xylitol production using 3% D(+)-xylose as carbon source supplemented with nutrients. Table S1 shows the design matrix of the coded independent variables together with the observed and predicted values. Results showed that observed and predicted values were similar, with the higher difference found in experiment 61 where the observed xylitol concentration was 13.4 g/L and the predicted value 14.4 g/L. The reliability of the model was examined with the plot of experimental


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and predicted values for xylitol production after 72h fermentation. As it can be seen in Figure 1 points in the plot form a fairly straight line, showing that the model is adequate to describe the production of xylitol by RSM. By applying multiple regression analysis on the experimental data, a secondorder polynomial equation was obtained. Table S2 displays the regression coefficients of the dependent variable that allowed obtaining a regression equation for xylitol produced after 72h as a function of salts concentration. Analysis of variance (ANOVA) was performed to examine whether the studied experimental factors, were significant in the performance of the proposed method. An effect was considered significant when it was above the standard error at the 95% confidence level (p < 0.05), which is denoted by the vertical line on the Pareto chart for xylitol concentration as a function of independent variables (Figure 2). Under these conditions, five of the salts studied showed significance in the lineal terms: x1 (FeSO4·7H2O), x2 (MnSO4·H2O), x3 (ZnSO4·H2O), x5 (Al2(SO4)3) and x8 (NaCl), being clear that x8 had the strongest positive significant effect (p < 0.05) on xylitol concentration, followed by x3 and x1, although in this case with negative influence. Four quadratic terms were also significant (p < 0.05): x32 (ZnSO4·H2O)2, x52 (Al2(SO4)3)2, x62 (CuSO4·5H2O)2 and x72 (KH2PO4)2. It is also noteworthy the significant effect of some interactions: x1·x4, x1·x7, x1·x8, x2·x5, x2·x7, x3·x4, x3·x5, x3·x6, x3·x7, x3·x8, x4·x6, x4·x7, x4·x8, x5·x6, x5·x7, x5·x8, x6·x7, x6·x8 and x7·x8. Only x4 (MgSO4·7H2O) showed not to be significant (p < 0.05) neither in linear nor in quadratic terms, meanwhile ZnSO4·H2O, Al2(SO4)3 and NaCl revealed significance both in linear and in quadratic terms.


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Consequently, the regression equation for xylitol concentration after 72h fermentation (y) as a function of the significant linear and quadratic terms and interactions can be presented as: y = 13.92939 - 0.44409·x1 - 0.35898·x2 + 0.56538·x3 – 0.4337·x32 + 0.39478·x5 0.38591·x52 – 0.51657·x62 - 0.27543·x72 + 0.81416·x8 + 0.31518·x1·x4 – 0.50226·x1·x7 – 0.53795·x1·x8 + 0.42183·x2·x5 -0.72682·x2·x7 + 0.65336·x3·x4 - 0.48308·x3·x5 + 0.33602·x3·x6 + 0.37406·x3·x7 + 0.38506·x3·x8 + 0.6257·x4·x6 + 0.80249·x4·x7 + 0.56931·x4·x8 – 0.49355·x5·x6 – 0.58583·x5·x7 – 1.13431·x5·x8 – 1.1136·x6·x7 – 0.64785·x6·x8 – 0.64511·x7·x8 Positive coefficients indicate that xylitol production is favored in the presence of high values of the respective variables within the range studied, while negative coefficients indicate an antagonistic effect. This means, for instance, that the individual addition of FeSO4·7H2O (x1) and MnSO4·H2O (x2) was detrimental for the production of xylitol; meanwhile the addition of ZnSO4·H2O (x3), Al2(SO4)3 (x5) and NaCl (x8) resulted positive. Meanwhile Figure 3 shows (as an example of the multiple possible combinations), the response surface models involving variables 1, 3 and 8. This kind of figures predicts the result of dependent variable (xylitol concentration after 72h fermentation) by three-dimensional responses. In each 3D surface plot, the remaining independent variables were kept constant in medium level while the two independent variables depicted varied in the selected range. In Figure 3a (x3 (ZnSO4·H2O) versus x8 (NaCl)), an optimal clear region was attained. By moving along the X and Y-axes, it can be observed that at higher value of NaCl and middle value of ZnSO4·H2O lead to maximum concentration of xylitol. Conversely, in Figure 3b (x1 (FeSO4·7H2O) versus x3 (ZnSO4·H2O)), these optimal conditions were achieved again at middle value of


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ZnSO4·H2O, but at the lowest value of FeSO4·7H2O, which is in agreement with the previous findings that the individual addition of this salt was detrimental. The analysis of variance (ANOVA) is showed in Table 2. ANOVA is a statistical technique that subdivides the total variation of a set of data into component associated to specific sources of variation and is important in determining the adequacy and significance of the quadratic model.16 Thus, the main effects, interaction effects, as well as quadratic effects were evaluated through this analysis. The goodness of the model was statistically judged by the coefficient of determination (R2). The value (0.938) demonstrated a satisfactory adjustment of the model as well as high correlation between observed and predicted values. The model could explain approximately 94% of the variability of xylitol concentration after 72h fermentation and only 6.16% of the total variation cannot be explained. The adjusted and predicted R2 were reasonable (0.8706 and 0. 0.7089 respectively) confirming the kindness of the model. After performing the F-test, it was also observed a good fit of the data to the model, since the F calculated (12.6156), considering the pure error, was higher than the value of F tabulated (5.673) at 95% confidence level. The computed model F-value of 13.84 and the p-value of 0.000 (p

Optimization of Salts Supplementation on Xylitol Production by

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